IMMUNOLOGY 9

Immune System
system (1.S) is a remarkable adaptive defence system that has evolved in vertebrates to
The Immune
protect them from invaders pathogenic microgranisms and cancer. It is able to generate enormous
variety ofcells. and molecules capable specifically recognizing and eliminating an apparently limitless
of

variety of foreign invaders. These cells and molecules act together in an exquisitely adaptable dynamic
network whose complexity rivalsthat of nervous system.
-
T 1885. Pasteur
administered the first vaccine to a human the rabies vaccine. The experimental
rk EmilVon Behring and Shibasaburo Kitasate in 1890 provided
of
the first insight into the mechanism
Rimmunity and earned noble prize in medicine in 1901. In 1930, single substance called an antibody
a
ns shown. Immunity may be Active or Passive and Innateor Adaptive.
Active and Passive Immunity
Aetive immunity is the adaptive immunity that is induced by natural exposure to a pathogen (natural)
orby vaccination (artificial). In this kind of immunity a person develops his own immune response to
omicrobe. Immunity resulting from transfer of antibodies or immune cells from an immune to a non
iomune individual is known as passive immunity. It may be naturally (involves the transfer of antibodies
from one host to another) or artificially (when antibodies or lymphocytes that have been produced
outside the host are introduced into a host) acquired.

Active Immunity Passive Immunity

Exposure to antigen No exposure of antigen
Immunity achieved by injecting antigens Immunity achieved by injecting Ab or
Ag reactive T-cells

Activation of immune system No immune system activation

Immune state develops over a period of weeks Immunity develops immediately·
Immunological memory develops No immunological memory develops

Innate versus Adaptive systems

A number of cell types and defense molecules are usually present at the site of invasion or migrate
to the site. These constitute the
first line of defense'. This is called the 'innate immune system'
since it is present at birth and changes, little throughout the life of the individual. The cells and
molecules of this innate system are mainly responsible for the first stages of expulsion of
the microbe
and may give rise toinflammation. Phagocytes are important cells
in the innate immune system
since they ingest and kill microbes.
The second line of defense is the 'adaptive
immune system' and this is brought into action while the
innate immune system is dealing with the microbe and especially if is unable to remove
it the invading
microbe. The key difference betweent the two systems is
that the adaptive system shows far more
specificity and remembers that a particular microbe has previously invaded
more rapid expulsion of
the body. This leads to a
the microbe on its second and third time of entry. The cells, molecules amd
characteristics of innate and adaptive immune systems are shown in Table.
 IMMUNOLOGY
9.2

Table : The innate and adaptive immune systems

Cells Molecules
Characteristics
Natural Immunity
Responds rapidly Phagocytes (PMNs and Cytokines
macrophages) Complement
Has some specificity
No memory Natural killer cells Acute phase proteins
Mast cells
Dendritic cells
Adaptive Immunity
Slow to start
Highly specific T and B cells Antibodies
Memory Cytokines

Innate Immunity
The non-specific component of immunity, also known as innate immunity (Latin: innasci-toas
be born
a
in) is not directed against any particular pathogen but is general defence mechanism the human
body must constantly defend itself from microbial invasion. This defence mechanism is active right
from the time a child is born (hence innate). The specificity of innate immunity is relatively low as it
lacks the ability to distinguish one microbe from another.
The Components of Innate Immunity
The principal components of innate immunity are
:

(a) mechanical and chemical barriers, (b) phagocytosis, (c) fever, (d) inflammation and
(e) acute-phase proteins.
(a)Mechanical Barriers
A mechanical or physical barrier refers to the various physical hindrances blocking the entry of the
microbes into the host body. Mechanical barriers include the skin and the mucous membrane.
These barriers act as the first line of defence against infection.
Skin
The outer layer of epidermis is coated with a tough protein called keratin that does not support
viral replication or penetration by bacteria.
Mucous Membrane
(a) the lavaging action of physiological fluids,for example tears and saliva, that assist in flushing
microbes from the body;
(b) thetrapping action of mucous-coated hair in the anterior chambers of the nose;
(c) the expulsive effects of coughing and sneezing,which protect the respiratory and gastrointestinal
tracts; and
(a) the cool temperature of the upper respiratory tract which inhibits replication by many viruses.
(6)Chemical Barriers
Acidic Gastric Secretions
The physiochemical environment in the stomach appears to be extremely inhospitable to invading
pathogens. The secretion of hydrochloric acid by gastric parietal cells maintains the pH ofthe stomach
at 2.0, which kills most microorganisms (except some resistant ones such as Hepatitis A virus,
Picornaviruses and typhoid bacilli).
 MMUNOLOGY
9.3
Skin
Low pHH of the
Sebum, secreted
) bythe sebaceous glands present
maintain inthe dermis, contains organic acids.These organic
the pH of the skin in the range of 3-5. This low pH
acids inhibits or retards the growth of
most microorganisms present
on the surface of
the skin.
Lysozyme
meis a hydrolytic enzyme
present in all mucous secretions, including tears,
retion. It can lyse Gram-positive bacteria by cleaving the peptidoglycan layer foundsaliva and nasal
in the bacterial
cell wall.
Gastric and Duodenal Enzymes
A large array of enzymes,
including proteases and lipases, digests a variety of structural and metabolic
chemical components of pathogens.
Antibodies and Inhibitors
The mucous secreted by gastric and intestinal cells usually
Specific inhibitors of viral infections(for example, sialic acid
contains IgA molecules as well as non
found in mucous inhibits attachment of
influenza virus particles to cells). Antibodies (IgA) are also found tears
protects the body surface against invading in and saliva. Secretory lgA
pathogenic microbes.
Interferons
The name interferons refers to a group of proteins produced by
virus-infected cells that induce a
generalized antiviral state in neighbouring un-infected cells, These proteins also augment
immunity. innate
Complenment Proteins
Complement proteins are a group of serum proteins that circulate in an inactive state
A variety of specific and non-specific mechanisms activate in the plasma.
these proteins. The activated forms of
these proteins damage the invading pathogen.
Antimicrobial Peptides
Alinsects and mammals, including humans, secrete a number of antimicrobial
defensins, for their protection. The human body is protected by um peptides, such as
protects the external surface of the body from microbial assault.l thick biofilm of defensins, that
(c) Phagocytosis

Phagocytosis of invading microorganisms is another important

innate defence mechanism.
When bacteria or other invading parasites penetrate
the skin or mucous membrane, phagocytes,
such as neutrophils, blood monocytes and tissue macro-phages,the surge towards the site of infection.
These phagocytes engulf the bacteria to form a large
intracellular vesicle, called phagosome,
containing the bacteria. Then, the involuntary guest trapped
fusing it with "granules" (lysosomes) found in the cytoplasm
within the phagOsome is destroyed by
of the phagocytes. These granules
discharge their contents (enzymes and reactive oxygen species)
inside the phagocytic vacuole, thereby
degrading bacteria.
the
d) Fever
Fever is the condition of an abnormally high body temperature,
rate and dry skin. It providesa non-specific defence accompanied by increased pulse
against disease. Fever is a physiological response
to infection.
Fever is beneficial to the host because
it inhibits the growth of temperature-sensitive pathogens.
Also, increased cell metabolism encourages
rapid tissue repair and phagocytosis.
e)Inflammation
Inflammation is the reaction of living an injury or an infection,
tissue to
characterized by heat (calor), redness (rubor), either Inflammation is
response of the body to swelling (tumor) and pain (dolor). It is a
injury. non-specific
 IMMUNOLOGY
9.4
as histamine,
of tissue products such
The process inflammation may be initiated by a variety cells
of a

bradykinin, serotonin andiprostaglandins released by numberof (such as mast cells andI basophils,
bvy damaged cells, chemicals released by invading
round in most tissues). The mediators released system
blood-clotting
meroorganisms, products ofthe complement svstem and reaction products the macrophages and
of
O
i8ger the process of inflammation, Manv ofthese substances strongly
activate
other cells of the phagocytic systerm.
Attack by Tissue Macrophages
Macrophages are already present in the tissues-the alveolar macrophages (in lungs), histocytes
(in subeutaneous tissue), Kupfer cels(in liver), microglia (in brain). However, tissue macrophages are
present in smallnumbers initially, In thecase of inflammation, these macrophages immediately proceed
towards the injury site to begin their phagocvtic actions. They serve as the first line of defence against
infection.
"The next step in inflammation is the redirection ofblood phagocytes towards the lesion site. This is
tacilitated by vasodilation of blood vessels and canillaries at the site of injury. The increase in the
diameter of blood vessels is brought about by chemical mediators like histamine and brady-kinins.
These mediators bind to the receptors on nearby capillaries and vessels, causing vasodilation. The
engorged capillaries are responsible for increased blood accumulation and the redness of the inflamed
tissue (erythema).
The increased permeability of the capillaries permits the influx of f1luid from the engorged capillaries
into the site of irritation or injury. The accumulation of fuid at the site of irritation results in tissue
swelling (oedemna).
Margination and Diapedesis
The products released from the inflamed area also cause phagocytes (now mainly neutrophils) to move
towards the inflamed area, a process facilitated by vasodilation and increased capillary permeability.
The emigration of phagocytes involves the adherence of the cells to the capillary walls,a process called
margination. The adhered phagocytes then pass from the blood vessels into tissue spaces through the
spaces between capillary endothelial cells. This process is termed diapedesis. Once in the tissue
spaces, the phagocytes migrate towards the injured tissue.
Once phagocytes engulf the invading bacteria and the necrotic tissue, many phagocytes eventually die.
However, some enzymes nay leak out into the extracellular environment from the neutrophils before
the phagosome closes. This process, termed sloppy eating, damages healthy cells. After a few days, a
cavity containing necrotic tissue, dead bacteria and dead phagocytes is formed at the site of inflammation.
This fluid mixture is often called pus. Ordinarily, pus formation continues until all infection is suppressed.
The blood-clotting system is also activated in the inflamed region which results in the deposition of
insoluble strands of fbrin. This seals off the injured area from the rest of the body, thereby preventing
the spread of infection. This pus, enclosed in a wall of fibrin, usually forms an opening on the surface of
the body, from where it empties out. The fibrin clot dissolves, tissue repair occurs and scar tissue is
formed.
Acute-Phase Proteins
Acute-phase proteins are a group of heterogeneous plasma proteins that are important in the innate
defence against microorganisms (mostly bacteria and protozoa) and in limiting the damage caused by
infection, trauma, malignancy and other diseases to tissues. In response to tissue insult, cells circulating
in the blood, such as macrophages and neutrophils, secrete a variety of cytokines that stimulate the
liver to produce acute-phase proteins. Some important acute-phase proteins include C-reactive complement
protein (CRP) components, mannose-binding metal protein (MBP), binding proteins and protease
inhibitors Acute-phase proteins function by stimulating phagocytosis (acting as opsonins), activating
the complement system and clumping the invading microbes.
IMMUNOLOGY
9.5
Table. Acute-phase proteins and their functions.
Protein Function
C-reactive protein Binds C polysaccharide of S. pneumoniae;
phosphatidyl-choline ofmicrobes, damaged tissueand activates complement binds
(C1)
pathway/cascade
Serum amyloid protein Binds DNA, activates complement (C1)
pathway/
cascade
Complement components C2,C3, C4, Chemotaxis and cell lysis
C5,C9, Factor B
Haptoglobin/ Haemopexin Binds iron and makes it unavailable for bacterial
growth
Ceruloplasmin Binds copper and rendersit unavailable for bacteria
Mannose-binding protein; Binds mannose and lipopolysaccharide on
Lipopolysaccharide binding protein bacterial surface(respectively), acts as opsonin,
activates complement pathway/cascade
Fibrinogen Blood coagulation
vonWillebrand factor involved in hemostais
Antitrypsin. Antichymotrypsin Proteinase inhibitor
Adaptive Immunity
Adaptive immunity is a more evolved and specific defence
mechanism. The characteristics of adaptive
immunity are exquisite antigenic specificity and the ability to "remember"
Adaptive immunity is activated only against invading different types of antigens.
foreign material and never against its own
molecules (except in autoimmune diseases). Thus,
non-self. Since it can differentiate between a it
has the ability todistinguish between self and
variety of different antigens, invading pathogens and
self-antigens, and induce different levels/types of immune response,
to as specific immunity. adaptive immunity is often referred

Cells of Immune System

An effective immune response is mediated by a
natural killer (NK) cells, eosinophils, basophils variety of cells including neutrophils, lympho-cytes,
and antigen-presenting cells.
Lymphocytes are mainly responsible for initiating
adaptive immune response
All lymphocytes are produced
in bone marrow stem cells by a process known as in the hunman body.
(A)B-Lymphocytes haematopoeisis,
lymphocytes originate in the bone marrow
B

Each B cell is genetically programmed to where they continue to differentiate and mature.
a unique antigen-binding
membrane. ThisB-cell receptor is actually a encode receptor on its
membrane-bound antibody molecule.
B cell (which has not previously
encountered comes When a naive
membrane-bound antibody, it multiplies antigen) in contact with an antigen via its
and differentiates into two types of cells--plasma
and memory cells. colls

Surface antibody
molecules T-cell receptor
B-cell
T-cell
 IMMUNOLOGY
9.6
numbers of soluble moleculos
Plasma cells lack B-cell receptors. However nlasmacells produce large c B-cell receptors
Called antibodies, B-cell receptors and antibodies differ conly at their terminal. to the cell
nave an additional transme mabrane segment at their terminal that anchors them blood and other
membrane. The antibody molecule is a large. polvfunctional glycoprotein found in
CiSSue fluids. One antibody molecule consists of two identical heavy chains and two identical light
to one beavy chain to forma heterodimer. Two Such heterodinmers
chains. Each light chain is linked a
are iinked by disulphide bonds toform tetrameric Y-shaped antibody molecule. The tips of the
wo armsof the Y-shaped molecule form the antigen-binding site. Thus, both the heavy chain and
the light chain form a part of the antigen-binding site.
Unlike plasma cells which last fora few davs. memory B cells have a longer life (sometimes upto
20 years) than naive B cells. In humans,B cells last for several months. Memory cells express
Some membranebound antibody molecules like their parent naive cells. Memory cells are functionally
inactive unless they are stimulated by the same antigen agaln.
(B) T-Lymphocytes
Like B lymphocytes, T lymphocytes too are produced in the bone marrow but they migrate to the
thymus to mature. During maturation,each T cell acquires a specific receptor on its membrane
termed as the T-cell receptor. A T-cell receptor does not recognize soluble antigens. It recognizes
antigens onlywhen they are associated with a protein complex called the major histoconmpatibility
complex (MHC).
MHCS are diverse transmembrane glycoproteins present on a variety of cells. These molecules
present (display) the antigens to the cells of the immune system. MHC molecules are so named
because they were first identified as "antigens" responsible for the acceptance or rejection of tissue
grafts (histocompatibility). There are three classes of MHHC proteins. Class I MHC molecules
are expressed on nearly all nucleated cells. They present antigenic determinants to a specific class
of effector T cells. Class II MHC molecules are expressed only by antigen-presenting cells. They
present antigens tothe other class of effector T cells. Class III MHC proteins are of diverse types
and are indirectly involved in the immune response.
There are several different groups ofT cells. One group interacts with antigen-class IIMHC molecule
complex, gets activated and starts secreting cytokines. This group of T cells is called T helper (T,)
cells. T cells activate B cells via cytokines and help them to divide, differentiate and secrete antibody
molecules. Another group of T cells is responsible for the destruction of virus-infected host cells,
intracellular pathogens harbouring host cells, tumour cells and cells of a foreign tissue graft. These
Tcells are called cytotoxic T cells (T). They recognize an antigen when it is associated with class I
MHCmolecules. It should be emphasized here that T cells recognize antigens only when they are
presented in association with MHC molecules. The receptor that recognizes an antigen-MHC complex
is present on the cell membrane of T cells and is termed T-cell receptor (TCR).
(C) Antigen-Presenting Cells
Any cell that is capable of processing and presenting antigens to the cells of the immune system may be
termed an antigen-presenting cell. However, this term is reserved for only those cells that display antigens
associated with class II MHC molecules. These antigen-presenting cells present antigens to T, cells.
Antigen-presenting cells include dendritic cells (interdigitating), macrophages and B lymphocytes.

Class II MHC molecule

and processed peptide
MMUNOLOGY 9.7
Antigen-presenting r cells take up antigens either by endocytosis or phagocytosis. The intern- alized
antigen is then degraded and processed. Small peptides derived from antigen processing are then
naved on the surface of the antigen-presenting cells in association with class II MHCmolccules.
eantigen-class II MHC complex is recognized by T, cells. This binding with T,, cell also activates
iren-presenting cells, which produces signal molecules (cytokines) that leads to the activation of
the T cell
which further augments the immune response.
Cells
Natural Killer
0)
Natural killer (NK) cells play an important role in immune surveillance and innate immunity.
nhese large granular cells, that have a natural "instinct" to kill tumour or virus-infected cells with
prior immunization, are called natural killer cells. It is believed that NK cells recognize cell
t
lost class I MHC molecules from their surface. Many surface changes occur on tunour
bat havesome
oells and virus-infected cells, including loss of class IMHC molecules. NK cells recognize an
damage these class-I-MHC-negative cells very effectively.
(E) Eosinophils
Eosinophils are a specialized group of leukocytes that are weak phagocytes exhibiting chemotaxi
Eosinophils are produced in large numbers in individuals with parasitic infections. Sometime when
a parasite is too big to be phagocytosed, (such as schistosomes or flatworms), they attack themselves
to these parasites and release their intracellular granular contents that contain sever toxic proteins
to kill the parasite.

F) Basophils and Mast Cells

Basophils circulating in the blood are very similar, though not identical, to the large mast cells
located outside many blood capillaries (mainly in tissues). These cells (basophils and mast cells)
have stored intracellular granules that contain a variety of molecules capable of triggering
inflammation upon their release. These mediators are released when the cells are activated. Basophils
and mast cells have bound antibody (IgE)molecules on their surface. The binding and cross-linking
ofthese antibody molecules by an allergen stimulates the release of pharmacologically active granules
that are stored in basophils/mast cells.These granules, when released inside the host body, elicit
symptoms of an allergic reaction.
(G) Mononuclear Phagocytes
The mononuclear phagocyte system comprises blood monocytes and tissue macrophages (histocytes).
Monocytes are incompletely differentiated cells that circulate in blood. These monocytes are
to as macrophages when they migrate through blood
referred
vessels and are sequestered into the
surrounding organs and connective tissues. Monocytes are derived from
bone marrow stem cells. Their major functions involve phagocytosis
the myeloid lineage of
and destruction of invading
pathogens. Moreover, these cells are also very effective at
presenting antigens to T lymphocytes.
(H) Neutrophils
A neutrophil is another important phagocyte
as polymorphonuclear that has a multilobed nucleus, and is often referred to
neutrophil (PMN). Neutrophils express F receptors for
that bind the F region of the antibody) and receptors antibodies (receptors
phagocytose the opsonized bacteria.
for complement proteins that help to
These cells are also involved in inflammation,
in response to chemical signals received
and migrate from blood vessels into inflamed
from the lesion site. However, after phagocytosingtissue
bacteria, neutrophils themselves die, 5-20
near the neutrophils probably by the action of a variety of enzymes released
during phagocytosis. Thus, neutrophils are short-lived lytic
cells.
Organs of Immune System
Anumber of morphologically
and Their Functions
and functionally diverse organs and tissues
This can be divided on the basis of have various functions in
development of immune response.
Secondary function into the primary and
the
lymphoid organs. The thymus marrow
and bone
Where maturation of
lymphocytes occurs. The lymph nodes,constitute the primary lymphoid organs
spleen and various mucosal associated
 9.8 IMMUNOLOGY

tissues (MALT) compose the secondary (or pheripheral) lymphoid organs, which trap antigen and provide
site for mature lymphocytestointeract withthatantigen. in addition, tertiary lymphoid tissues, which
normally contain few lymphoid cells, can import lymphoid cells during an intlammatory response.
Most prominent of these are cutaneous associated lvmphoid tissue. Once mature ymphocytes are
generated in the primary lymphoid organs,theycireulate in the blood and lymphatic system.
(A)Primary Lymphoid Organs.
Immature lymphocytes generated during hematopojesis mature and become committed toa particular
andgenic specificity within the primary ]vmphoid organs, Only after a lymphocyte has matured within
primary lymphoid organ is the cell immuno competent. In mammals, B cell maturation occurs in the
bone marrow and T cell maturation occurs
in the thymus.
1ymus :The thymus is a flat. bilobed organ situated above the heart. Each lobule is organized
mto wo compartments : the outer comnartment or cortex, is densely packed with immature T
Celis, called thymocytes, whereas the inner compartment or medulla is sparsely populated with
thymocyte.

er the progenitor T cells formed during hemotopoiesis enter the thymus, they are thought to
muibply rapidly within the cortex, The small subset of more mature thymocytes are then thought
mgrate from the cortex to the medullar where they continues to mature and finally leave the
us Via post capillary venules. Before leaving the thymus, when the developing thymocytes
eEexpress antigen binding receptors, they are subjected to a 2 step selection process, so that
y' cells recognizing antigenic peptides in the content of self MHC molecules are released from
the thymus.
(u) Bone Marrow : In birds a lympboid organ called the bursa of fabricius is the primary site of B cell
maturation. There is no bursa in mammals and no single counterpart to it as a primary lymphoid
Organ. lnstead, regions of the bone marrow and possibly of other lymphoid tissues serve as the
bursal equivalent' where B cell maturation occurs.
Stromal cell within the bone marrow interact directly with the B cells and secrete various cytokins
that are required for B cell developmental process. Similar to thymic selection process within the
bone marrow eliminates B cells with self reactive antibody receptors.
(B) Secondary Lymphoid Organs : Lymph nodes and spleen are the most highly, organized of
the
secondary lymphoid organs, in addition to lymphoid follicles, they possess distinct regions of T
cell and B cell activity and are surrounded by a fibrous capsule. Less organized
lymphoid tissue
collectively called mucosal associated lymphoid tissue (MALT) occurs in various body
sites.
(i) Lymph Nodes : Morphologically, a lymph nodes can be divided into 3 rougly
concentric regions,
the cortex, paracortex and medulla each of which provides a distinct micro environment. The
outer most layer, the cortex contains lymphocytes (mostly B cells), macrophages
and follicular
dendritic cells arranged in primary follicles. Intense B cell activation and differentiation into
plasma and memory B cells occurs in the germinal centres oflymph nodes. Beneath
the cortex is
the paracortex which is populated largely with Tlymphocytes and also contains interdigitating
dendritic cells thought to have migrated from tissues to the node. These interdigitating dendritic
cells express high levels of class II MHC molecules, which is necessary for
antigen presentation to
T, cells.
(ü) Spleen: The spleen is a large, ovoid secondary lymphoid organs. Unlike lymph nodes, which are
specialized to trap localized antigen from regional tissues spaces,
the spleen is adapted to filtering
blood and trapping blood borne antigens, and thus can respond to
systemic infections. Spleen is
compartmentalized into red pulp and white pulp, which are separated by a diffuse marginal zone.
The splenic red pulp consists of a network of sinusoids populated with macrophages
and numerous
red blood cells, it is the site where old and defective red blood cells are removed. The splenic
white pulp surrounds the arteries forming a periarteriolar lymphoid sheath (PALS) populated
mainly by Tlymphocytes. The marginal zone, located peripheral to PALS, is rich in B cells organized
intoprimary lymphoid follicles. The initial activation of Band T cells takes place in the T cell rich
PALS.
NMUNOLOGY
9.9
Mucosal Associated Lymphoid Tissue : The mucosa lining
(úi) genitourinary and other the alimentary, respiratory,
lumina anmd surfaces are constantly exposed to numerous antigens.
These areas
are endowed with a rich collection of lymphoid cells,
either specialized aggregates
the Payer's patches oT Scattered isolated lymplhoid follicles collectively called the mucosa
niated lymphoid tissue. Those lymphoid tissue present in gut are GALT and in bronchus are
BALT. MAIT contains lymphoid as well as phagocyte cells. Both B and T cells are present.
While the predominant immunoglobulin produced in the mucosa is secretory IgA, other
immunoglobulin classes, IgG, IgM, IgE are also formed.
Tertiary LymphoidTissues :The skin provides an important barrier to the enternal environment
and its large surface area makes this tissue important in nonspecific innate defences. The outer
epidermal layer of the skin is composed largely of specialized epithelial cells called keratinocytes.

Antigens
Classically, an antigen is defined as a
substance that has the
ability to induce the fornmation of specific antibodies,
) ability to react specifically with the generated antibodies, and not with other antibodies that are
also present in the system.
A more recent term
used is immunogen. An immunogen may be defined as any chemical substance
that either elicits the production of specific antibodies or binds to T-cell receptors. Thus, an immunogen
atimulates both B cells that form antibodies and T cells that mount an effective immune response. 1his
may be represented as
:

Immunogen + B cells Antibodies

Immunogen + T
cells Activated effector T-cells (for example, T,cyt Ty, etc.)
The complete antigen molecule is not immunogenic. Instead small regions of the antigen are antigenic
or immunogenic. These regions are termed
antigenic determinants or epitopes (Greek: epi non
self and topos-map or place).
Antigenic determinants, are regions of an antigen that can bind an antibody or an antigen-specific
membrane receptor on a lymphocyte. The size of an antigenic determinant usually ranges from 6 to 8
amino acids in protein antigens and 6 to & monosaccharide units (molecular weight: 750 Da) in the case
of polysaccharide antigens.
Antigenic determinants are normally well-separated spatially and twO separate antibody molecules can
bind to the same antigen molecule without influencing the binding of the other. Such determinantsare
called non-overlapping determinants. In other instances, antigenic determinants may be locnted so
close that the binding of one antibody to a determinant may sterically interfere with the binding of
another antibody molecule. In such cases, determinants are said to be overlapping.

Antigenic
determinant

Antigen
Fig: Schematic representation showing protein antigen and antigenio determinant.
 IMMUNOLOGY

9.10

General Properties of Antigen

Molecular are found to be non-
below which molecules
A limit
relative molecular mass of about 1,000 is the lowestare though better than 1000
Da weakly immunogenic induce a sufficiently
immunogenic. Molecules between 5,000--10,000 mass can generally
Da molecule. Immunogens of above 10,000 an immune
relative molecular are most effective in triggering
strong immune response, and molecules above 100,000
response.
Self or Foreign ce cannot act as
antigen
Generally, the body's own antigens are and hen
recognized as self as an
will be recognized immunogen
/immunogen. However, homologous antigens from another species between two
accordinglv Cenerolly fhe rreater the phylogenetic distance
td mll be dealt with disparity between them; and hence, stronger
Specles, the greater the structural (and benoe 9nigenic)
the immune response.
Chemical Complexity
response. Heteropolymers
ofone amino acd) fail to elicit an immune
ynthetic homopolymers (polvmerare as they are structurally complex. A complex
the best immunogens
pOlymers differentamino acids)
of
are themselves non-antigenic) associated with polysaccharides is called aipopolysaccharide.
O pds (that
This is a chemically complex molecule that is strongly immunogenic.
Route of Entry
as it is commonly called, refers to the route taken by
1ne route, or portal of entry (Latin: porta-gate) response to an antigen varies
Une pathogen (or antigen) to enter the bodv of the host. The immune
or
according to the portal of entry of that antigen. Protein antigens that enter subcutaneously
ntradermally are usually immunogenic as these antigens are taken up by the antigen-presenting cells
(Langerhans cells) and transported to lymph nodes, where the immune response occurs. In contrast,
large doses of protein immunogen, administered intravenously, often induce specific irresponsiveness.
Such irresponsiveness could be due to tolerance induction in lymphocytes or suppression of reactive
T-cell clones.

B-cell and T-cell Epitopes

The mechanism of recognition of antigens by T-cells and B-cells is basically different. Studies have
revealed that B and T-cells recognize different antigenic determinants on the same antigen molecule.
Properties of B-cell Epitopes
The properties of B-cell epitopes are as under :
generally composed ofhydrophilic amino acids;
located on the surface of thenative protein so thatit is topographically accessible to the antibody or
antibody-like B-cell receptor;
can contain linear or conformational epitopes;
tend to be located on the flexible region of the immunogen, thus maximizing its easy binding with
the antibody that might not otherwise react with it, ifit was rigid; and
may contain overlapping and non-overlapping determinants. Bovine serum antigen (BSA) has 25
overlapping antigens on its surface.
Some determinants induce a more pronounced immune response than other epitopes in a given animal.
Such epitopes are called immunodominant. These are usually those epitopes that project distally from
the central mass of the immunogen.
MMUNOLOGY 9.11

Properties of T-Cell Epitopes

properties of T-cell epitopes are as follows:
The
Oncessing of an antigen is required before it can be presented to a T cell. This processing yields
peptides which bind to class I or II MHC molecules and this complex is then presented to T
cells.
meellepitopes are always presented together with MHO. T-cell receptors do not recognize any
epitopes that are presented alone.
Antigens recognized by 'T cells have two regions--epitope, that interacts with T-cell receptor, and
,
agretope, that interacts with the MHC molecule. Interactions between the epitope and T-cell receptor,
ond the agretope and MHC are purely non-covalent.
Roitopes recognized by'T cells are often internal and usually sequential.

Superantigens
Recently, a class of protein antigens with "super" antigenic properties has been characterized and
named superantigens. Superantigens bind simultaneously to T-cell receptors and class II MHC
molecules. This non-specific binding activates into the lymphoproliferative phase in T cells and
induces pronounced eytokine production by T cells in vivo, thereby causing a variety of pathological
consequences such as fever, malaise, diarrhoea, etc. Unlike conventional antigens, superantigens
are not internalized and degraded by antigen-presenting cells. Instead, they bind directlyto class II
MHC molecules outside of the antigen-binding cleft.

Haptens
The term hapten (Greek; aptein-to grasp or fasten) was introduced by Ehrlich who considered the
antigen (he was studying the diphtheria toxin then)to be a distinct molecular
entity, containing two
active groups-haptophore, the group for binding
and toxophore, the group for toxicity.
Ahapten can be defined as any substance of molecularweight less
than 1,000 Da, that, though incapable
of stimulating antibody formation by itself,
of
is able to react with pre-formed antibody molecules. Examples
haptensinclude 2,4 dinitriphenol p-aminobenzenearsonate, monosaccharides,
biologically important substances such as sugars,
amino acids, etc. Many
amino acids, drugs, peptide hormones, etc. function
as haptens. A
large number of chemicals such as dinitrophenol (DNP),
classified as hapten.
and drugs such as penicillin, are
A hapten becomes
immunogenic when it is attached to an immunogenic
protein, yielding an immunogenic
hapten-carrier conjugate.
carrier molecule such as a
Consider the case of DNP. DNP a
is hapten and is unable to induce
test animal, alone. However, antibody formation when injected in
when DNP is linked to an immunogenic
serum albumin (BSA), carrier molecule such as bovine
it results in the formation
of anti-DNP antibodies.
Although anti-hapten
antibodies are usually predominant,
produce antibodies, not animals immunized with such a
only against hapten, but also conjugate
Moreover, antibodies are against epitopes of the carrier molecule.
also formed against neoantigenic
carrier (BSA) and hapten determinants formed by parts of
molecules. both

Adjuvants
Adjuvants (Latin: adjuvare--to
help) are substances which
With it, accelerate, when mixed with antigen and
enhance and prolong the immunogenicity injected
chemical composition
of the antigen. Adjuvants are of the antigen without altering the
agents that enhance the immune response
 IMMUNOLOGY
9.12
or is low
are usually used when either the concentration the antigen its immunogenicity
of

response manifold.
in both cases, the addition of adiuvant to the antigen augments the immune
Ghemicals commonly used as adiuvantsare alum (aluminium potassium sulphate), Freund'g
complete and incomplete adjuvants.
Freund's incomplete adiuvant is an emulsion of oilin water (aqueous solution of mnineral oil and
mannide monooleate which acts as emulsifer). Freund's complete adjuvant contains heat-killed
myco-bacterium in ojl-and-water emulsion, Freund's complete adjuvant, though a highly effective
adjuvant, is never used in human vaccines.
For the vaccination of human beings. the most commonly used adjuvants are still common ones
Such as aluminium hydroxide, aluminium phosphate or alum precipitate.

Major Histocompatibility Complex and Antigen Processing and

Presentation
Although molecules coded for by the MHC were originally identified based on their role in
uransplant rejection, they actually evolved to present foreign antigens to T cells. Two classes
(Class 1 and ii) of MHC genes, closely linked on chromosome 6 in humans, code for human
leukocyte antigen (HLA)which are the molecules critical to antigen presentation. MHC molecules
are important in determing the ability of an animal to mount an immune response. Because
tnere are atleast 20 MHC genes and atleast 50 alleles for each genes, it is virtually impossible
TOr any two people to have matching sets of MHCmarkers on their cells, thus the MHC is a
biochemical finger print unigue to each individual. The MHC contains 3 groups of genes known
as class I, II, III.
Class I and II gene code for integral membrane proteins, whereas class III gene codes for serum
proteins T lymphocytes recognize surface antigen only when associated with class I, II MHC
molecules. Two main classes of MHC molecules mark cells as 'self. Class I MHC molecules are
located on all nucleated cells, almost every cell of the body. Class II MHCmolecules are restricted
to macrophages and B lymphocytes. They play a important role in interaction between cells of
immune system. Class Iregion isencoded by DP, DQ and DR. Since there is a pair of chromosome
6 in diploid cell, an individual has two a genes and two b genes. These alleles are
codominant
which means product of both the alleles are expressed in cells.
Class I Gene Products
These are glycoproteins found at the surface of all nucleated cells. Class I protein is a single
polypeptide chain associated with smaller proteins called p,-microglobulin which is invariant
and is not coded by MHC. The proteins specified by different alleles differ in their amino acid
composition at amino terminal ends of molecule. Class I proteins restrict the activity of cytotoxic
T cells. For example T cytotoxic lymphocytes is specific for measles virus, will kill cells infected
with that virus only. If the T cell and the virus infected cells have same. Class I antigens also
acts as variability factor what the means is that the T cells must recognize it
self MHC proteins
inorder to react against the non self (viral proteins).
Class II Gene Products
These are also glycoproteins found on restricted range of cells, such as antigen
B cells. Class II proteins consist of 2 dissimilar polypeptides
presenting cells,
called a and 3 subunits both of
which are encoded within the MHC. Most of the differences between class II proteins coded by a
different alleles lie in the amino acid composition of the B chain.
MMUNOLoGY
9.13

Peptide in Peptide in bindingthe

Class Closs N groove of class l
binding groove
I heavy chaln molecule
d dass

a chain u chain chain

B

p-2 micro globulin

Cell
Cell(APC)

Fig. MHC class I and class

II molecules binding peptide.
The polymorphic regions of MHC class I and II are the peptide binding domains of these molecules and
-
bind peptides ranging from 9
-10(for MHC class I) and 12 20 amino acid residues (for MHC class II)
MHC class II molecules are expressed on B cells, dendritic cells and macrophages, efficient APCs for
the activation of CD+ helper T cells.

Anting Processing and Presentation

In order for a foreign protein antigen tobe recognized by a T cell, it must be degraded intosmall
antigenic peptides that form complexes with class I or class II MHC molecules. This conversion of
proteins into MH associated peptide fragments is called antigen processing and presentation. Whether
a particular antigen will be processed and presented together with class I or class II MHC molecules
anpears to be determined by
the route that the antigen takes to enter a cell.
1. Endogenous antigens:Itis produced withinthe host cell itself. Two common examples are viral
protein sythesized with virus infected host cells and unique proteins sythesized by cancerous cells.
Endogenous antigens are degraded into peptide fragments that bind to class I MHC molecules
within the endoplasmic reticulum. The peptide class IMHC complex is then transported to
membrane. Since all nucleated cells express class I MHC molecules,all cells the cell
producing endogenous
antigen use this route. T cells displaying CD8 recognize antigen associated
molecules and thus are class I MHC restricted. These cytotoxic T
with class I MHC
- cells attack and kil cells displaying
the antigen MHC class Icomplex for which their receptors are specific.
2. Exogenous antigen :
It is produced outside of the host cell and enters the cell by endocytosis or
phagocytosis. APCs cells (macrophages, dendriticcells
and B cells) degrade ingested exogenous
antigen into peptide fragments within the endocytic processing
class II MHCmolecules are expressed within pathway.Experiment suggest that
the endocytic processing pathway and
produced by degradation of antigen in this that peptides
pathway bind to the cleft within the class II MHC
molecules. The MHCmolecules bearing
the peptide are then exported to the cell surface,
expression of MHCclass II molecules is
limited to APCs, presentation of enogenous Since
MHC complexes is limited-to these
cells. T cells displaying CD4 recognize peptide class II
class II MHC molecules and
thus are said to be class II MHC restricted. antigen combined with
function as T helper cells. These cells generally

Antibody Structure, Function And

Antibodies - Immunoglobulins
Diversity
Antibodies or immunoglobulins
presence of antigens. (Ig) are produced by B lymphocytes
Immunoglobulins constitute 20-25 percent (plasma cells) in response to the
physiological and antigenic of the
difference five classes of immunoglobulins total
serum proteins. Based on
IgG, IgA, IgM, IgD and IgE.
have been recognized
 9.14 IMMUNOLOGY

Structure of Antibody:
An antibody molecule consists of two identical 1ight protein chains and two identical 'heavy' protein
Cnains, all held together by both hvdrogen bonding and precisely localized disulfide linkages. The N
vermnal regions of the light (L) and heavv (H) chains together form the antiogen recognition site of
each antibody what is called F, fragment. Thesites that recognize and bind antigens consist of three
omplementary determining regions (CDRS) that lie within the variable (V, and V,) regions at the N
ermnal
to ,ends of the two heavy and two light chains. There are five different kinds of H chains (referred
as u, Y, & and chains), which determine the class of antibody (IgM, IgD, IgG, IgE and IgA,
respectively).

NH, NH,
NH2 Fab NH,

HOOG COOH

3)
Inter Chain disulphide bond

Inter Chain di sulphide bond

HOOC COOH

Fig. Structure of Immunoglobulin

There are also two different kinds of chains - Kand ,each with a MW of 23 KD. Each antibody unit
L

can have only K or A, L chains but not both. In addition to the variable regions (V,,, ),
V, each chain
contains one constant region, or domain (C, )and each H chain has three constant regions, or domains
(C,, Coy Cua). Most of the antibody molecule (the C terminal three quarters of the H chain and the
terminal half of the L chain) are constant regions of the antibody nolecule and are the same for all
C

antibodies of the same class and subclass. The terminal half of the H chain the F,region (fragment
C

that crystallized) serves other functions i.e., combines with complement, is cytophilic. Carbohydrates
are also present on antibodies on the F, portion of H chain when antibodies are digested with the
protolytic enzyme papain, three fragments are released:
Two identical (F.) fragments, and one F, fragment.
Antibody Classes
Different antibody classes with different biological activities have evolved to deal with antigens
(e.g. microbes) with different properties and which enter the body at diferent sites - through the skin,
the gastrointestinal or the genitourinary tract.
IgG
They have a MW of 150 KD are found in vascular and entravascular spaces as well as in secretions.
IgG is the most abundant immunoglobulin in the blood and is the only antibody class to cross the
placenta to provide passive humoral immunity to the developing fetus. There are different subclasses
ofIgGl designated
IgG,IgG2, IgG3, IgG4.
IgG3 is most effective complement activator.
MMUNOLOGY
9.15

IgA
immunoglobulin is present in the serum as a 170 kD,
This
It is
four polypeptide (two L
and
chain protein. the major immunoglobulin present as colostrum,
two H).
in external secretions
where it exists as a 420 kD dimer. In addition to k or chain and
such
milk and saliva
I
the IgA heavy chain
(o«) it also contains
two other polypeptide chains secretory component (SC) J chain (joining chain).
and
Most IgA is synthesized locally by plasma cell in mammary and salivary glands, and along the respiratory,
gastrointestinal and genitourinary tracts. It
is then transported through epithelial cells to the lumen.
Bte antibody is a first line of defense against microbial invaders at mucusal surfaces.
IgM
It accounts for 5-10%% of the total serum immunoglobulin. IgM is the first antibody produced by, and
exDressed on the surfaceof a B cell. It acts as an antigen receptor for these cells, and is also present as
a solublemolecule
in the blood. On the B cell surface this molecule is expressed as a four chain unit
two uH chain and two L chain. In the blood, IgM is composed of fve four chain units held together by
disulfide bridges at the carboxy terminal endsof the u chain J chain is also associated with IgM in the
blood. Because of its size (900 kD), IgM is found primarily in the intravascular space. As IgM is the first
antibody produced an immune response. Although IgM antibodies usually have low affinity binding
in
sites for antigen, they have 10combining sites per molecule which can synergize with each other on
the same molecule when it binds to a microbe. Thus, the overall tightness of binding of an IgM molecule
to a microbe (avidity) is quite high, making antibodies of this class very effective in removal of the
microbe.
IgD
IgD is present in low quantities in the circulation (0.3 mg/ml in adult serum). Its primary function is that
of an antigen receptor on B lymphocytes. B cells thus can express both IgM and IgD and both are specific
for the same antigen when IgM and IgD expressed on a B cell interact with an antigen for which they are
specific, the antigen isinternalized, and processed and presented to helper T cells which trigger the B
cells to proliferate and differentiate into plasma cells, thus initiating the development of a humoral
immune response.
IgE
IgE is present in serum at very low levels (nanograms per milliliter),
but plays a significant role in
enhancing acute inflammation, in protection from injection by worms, and in allergic reactions.
Antibody
mediated allergy is predominantly associated with IgE. Antigen reintroduced into a perviously
sensitized
individual binds to antigen specific IgE on 'armed' most cells and triggers release of
active agents (e.g. histamine). It is involved in immediate
the pharmacologically
hypersensitivity syndromes such as hay
fever and asthma.
Ichain

L chain

H chain

Allergic
B cell
responses
Surface Enhances acute
Receptor
Inflammation
IgD IgF
IgM
H chain
Circulatory Ig A
L
chain
Secretory Ig A J chain
Fig. IgA Immunoglobulim
 IMMUNOLOGY
9.16
antibody. The term
affinity alludes to the strength of
:
Affinity Strength binding
of of antigen to antibodies.
the
determination and monovalent
bond between antigen
interaction between a
large antigen with multiple epitopes and polyvalent
Avidity:Overall strength of

antibody molecule such as IgM.

Allotypes
segregate within the species. IfIg expressing
These are genetic markers on immunoglobulin (Ig) that
Igs do not express that allotype, an immune
m

a
particular allotype is injected | into an individual whose
types, they are inherited in mendelionfashion
response with develop against the allotype. Like blood
but are usually of no functional consequence.
Idiotypes
of antibodies and
These are unique antigenic determinants associated with antigen binding sites
seguences which determine their specficities.
the result of the different amino acid
Functions of Antibodies
a
Antibody alone can neutralize viruses and toxins if it binds tightly to, and blocks, part of the toxin
Virus critical to its biological activity, Similary, antibodies
can inhibit microbes from colonizing mue
areas and in some cases may induce programmed cell death (apoptosis).
IgG or IgM antibodies can, on binding to antigen, activate the classical pathway of comple-ment leading
to lysis of the cell on which the antigen is located or to attraction of immune cells (chemotoxis) which
phagocytose the antigen expressing cells.
A variety of effectorcells have receptor for the F, region of antibodies. Phagocytes (PMNs, macrophages
and cosinophils) utilize their F, receptors (F R)for IgG (FerR)or IgA (FaR) to enhance phagocytosis
of antibody opsonized microbes. In addition these F,R can mediate killing of cells through antibody
dependent cellular cytotoxicity (ADCC). Binding of antigens to IgE already bound toF, receptors for
IgE on mast cells results in degranulation and subsequent enhancement of the acute inflammatorv
response.

T- Cells :Receptors and Activation

Tlymphocytes are produced in the thymus from cirulating T cell precursors derived from HSC
(Henoepoietic stem cell) in the bone marrow. These T coll presursors differentiate within the thymus
into mature T cells expressing molecules important to T cell function, such as the T cell receptor, CD4
and CD8. The thymus thus generates large numbers of functionally mature antigen specific T cells
which then migrate tothe peripheral lymphoid tissues to mediate protection against invading microbes.

CD4°
CD4*,CD8* CD4'.CD8* Periphery
TCR
LSC CD8*
CD3
Lymphocyte Double
stem cells Positive

Fig. Developmentof CD4 and CDS' T cell in the thymus.

Generation of T cell diversity
Each of the very large number of T cells produced in the thymus has only one specificity, coded for by
its antigen receptor. Millions of "T cells, each with receptor specificfor different antigens, are generated
by gene rearrangement from multiple (inherited) germ line genes. The T cell receptor consists of two
polypeptide chains:
a and ß or y and 8.
IMMUNOLOGY
9.17
member of the Ig superfamily and thus has a uniform domain structure produced by
Each chain is
a

Sadhain disulfide bonding. Unlike most proteins produced in the body, each polypeptide chain of
the
receptor is coded for by several different genes.
cell
T

LSC

TCR
B
T

T,

Fig. Development of aß and y6 T cells.

The genes for the CR a, ß and chains are coded by three unlinked gene groups,
y

genes lie within whereas the chain

the a chain genes. Each gene group consists of multiplegene segments which code
VD andJgene segments (ß and ô genes) or V and gene segments for
region genes. In the germ line the V and
J (a and y genes) as
well as Constant
non coding DNA J (a chain)or V,D and J (B chain)segments are separated by
During T cell differentiation these V,D and J segments are
DNA. During "T cell differentiation separated by non coding
these V, D and J segments are rearranged to
gene. As in form a complete V
thecase of lgs rearrangements, the eXpression of a V-C (constant region) and the T cell
encludes further rearrangement (allelic
exclusion) and the T cell thus becomes committed to
expression of a single V-C a chain combination and a the
single V-C B chain combination, which together
constitute an antigenic binding site and determine
the specificity of the T cell.
T Cell Receptor (TCR)
for Antigen :
The TCR for antigen is only found on
the T cell membrane and is composed of two polypeptide chains,
a and ß (50 and 39 kDa). Each of these glycoproteins
is made up of constant and variable regions like
those of Ig and together the a and ß variable
regions compose a T cell antigen binding site. Some T
cells, whose function is not clear, express a TCR
consisting of y and Schains.

Alpha chain Beta chain

Variable region

Constant region
|-S-S

Lipid layer

Fig. T cell antigen receptor aß dimer.

TCell Receptor Complex
"The 'T cell receptor complex
consists of the antigen receptor, the af or y dimer ass0ciated with
&

Several other polypeptides important in T cell signalling and recognition. In particular, the TCR is
associated with CD3, a signaling complex which is itself composed of
several polypeptides including
 IMMUNOLOGY
9.18
cells expressing the y &
T
Y.8 and e an,a B- CD3 complex.
chains. Together
these molecules form a T cell
augen receptor form a y 8-CD3 complex. Twoother molecules on T cells also play role in
a on Th cells: but als0 a molecule that can bind to the
ecogntion of antigen. CD4is not iust markermolecule restricts these Th cells to recognizing only
non polymorphic region of MHC class II This "T cells binds the
CD8 on cytolytic
Pepudes presented on MHC class II molecules Similarly,cells to recognizing only cells presenting
.
honpolymorphic region MHCclass restricting
of these killer T
peptide in MHC class I molecules.
Selection of the T Cell
that evnrose a TR hot hinds weakly to self MHC are positively selected.
1n the thymus, those T cells
1nose that express a TCR that binds strongly to se)f MHC are negatively selected. Thus, the T cells
asurmve and mature are those that recognize modifed self MHO molecules-self MHC molecules
plus foreign peptides.
T Suppressor Cells (TS)
e are
Th cells which promotes immune response. It is clear that some T cells help to
suppress the
humoral and the cell mediated branches of the immune system but there is also a regulating set (1S)
of T cells whose job is to prevent helpers from getting out of hand.
Two Types of T Helper Cells
GD4 cells can differentiate down at least two pathways to become inflammatory T cells (Thl) or Th2
cells which help B cells. Thl cells are involved in mediating inflammatory responses (through the
activation of macrophages),while Th2 are involved in the induction of humoral immunity. Thl cells
produce cytokines that act on macrophages (IFNY and TNFa) whilet Th2 cells are involved mainly in
B
cell differentiation and maturation (via IL-4, IL5, IL-6).
T Cell Activation
Initial recognition of processedantigen by T cells is via the T cell antigen receptor. Accessory molecules
further link the APC and the T cell leading to a stronger cell interaction. For e.g. CD4 binds to the
constant region domain of class II MHC molecules whilst CD8 binds to class I MHCmolecules. Other
ligand-receptor pairs such as intracellular adhesion molecules (ICAMS) and lymphocyte function
associated antigens (LFA) are also important.
Co Stimulatory Molecules
Full activation of antigen specific T cells requires two signals-one signal coming via the TCR and the
other signal through engagement of co-stimulatory molecules. T cells receiving one signal via their
TCR are turned off (become anergic), whilst those receiving two signals e.g. via T cell CD28
binding to
B7 on the APC induce T cell lymphokine production and T cell proliferation.
Super-Antigens and Th Cell Activation
Some protein products of bacteria and viruses produce proteins known as super-antigens
that bind to
lateral surfaces of the MHC class II molecules and the V region of the B submit of the TCR. These
antigens are not processed into peptides but are able to bind to a specific family of TCR. Among the
bacterial super antigens are the staphylococcal enterotoxins (SE) that cause common food poisoning
and the toxic shock syndrome toxin (TSST).
Activation of Cytotoxic T Cells
CD8- Tc cells also require activation to produce functional CTLs
containing granules and perforsin.
This is mediated through attachment of their TCR to MHC classI peptide complexes on
case, however, it appears that, the second signal can APCs. In this
only be supplied by APCs that have been conditioned
by Th cells. As with Th activation, cytokines produced by Th cells and APCs are
important for 1e
activation.
NUNOLOGY
TLymphocyte, Activation 9.19

cell to fulfill itsfunction it has to convey a

a T
orderr for signal from the cell 8urface via the
In thenucleus to give rise to a specific genetranscription. cytosol
into at This signal transduction is brought
several molecules the cell surface. The key players about
by phosphorylate (kinases) and desphosphorylate
in this amplification process are enzymes
which (phosphatases) particular amino acids
them. in a sequential fashion leading eventually
production of functional
to activation of specific transcriptionthus activating
factors in the
pucleusandi proteins. Initiation of
the processisthrough
-
asSOciated kinase (lymphocyte kinase Ick) by CD45 activation of a CD4
(a phosphatase), one of its
functions being to
phosphorylate tyrosines of the immuno stimulatory - based
tyrosine activation motifs (ITAM) on the
of CD3. This, in turn activates
zetachain another protein tyrosine kinase ZAP
70 which activates an
enzyme responsible for activating the phosphatidyl inositol
pathway and further down stream events
leading to the activation
of transcription
factors regulating production of IL-2 and other
proteins.
important

B-Cells: Maturation and Activation

The generation mature B cell first occurs in
of
the embryo and continuess throughout life. Before birth,
ke volk sac, fetal liver, and fetal bone marrow are the major, sites of B-cell
maturation, generation of
mature B cells occurs in the bone marrow.
Dandenitor B Cells Proliferate in Bone Marrow
B cell development begins as lymphoid stem
cells differtiate into the earliest distinctive
B.lineage cell-the progenitorB cell (pro B cell)
which expresses a transmembrane tyrosine phosphatose
called CD45R. Pro Bcells proliferate
within the bone marrow. Proliferation and differentiation of pro B
cells into precursor B cells (pre-B cells)
requires the micro environment provided by the bone marrow
stromal cells. The stromal cells play two
important roles: they interact directly with pro B and pre B
cells, and they secrete various cytokines (IL-7) that support
the developmental process.

LSC
Antibody gene
J
Pro Bcell rearrangements
heavy chain

K and a
Pro B cell right chain
Pro B cell
receptor
Lsig M
Immature
B cell

mature B
cell M
sig
Ig Alg G sig D

Memory cell

Plasma cells

Fig: Life History of B-Cell.

B
cell maturation depends on rearrangement
of the lg DNA in the lymphoid stem cells.
in the procell stage is a heavy chain D,, to J gene rearrangement
B First to occur
rearrangement. If the first heavy this is followed by a V,,to D,, J
chain rearrangement is not productive, then v,, - D,, - Jy
 IMMUNOLOGY

9.20
upon completion ofB
heavy chain rearrangement,
chromosoms cell into an immature B cel)
rearrangement continues on the otherContinued development of a pre exclusion, only one light chain
the cell is classified as a pre B cell. arrangement, Because of alletic mIgM on the cellsurface.
requiresa productive light chain gene of a cell. Immature cells express
B
B

isotype is expressed on the membrane

Cell Receptor Complex (BCR)
B

association withtwo other polypeptides, Iga and

in
The BCR complex consist of the antigen receptor are also requiredfor assembly and expression
Ig,
Igp. Iga and Igß are signaling molecules for the BCR and associate with the BCR complex
CD32, CD19, and CD81,
coreceptors, CD21,
coreceptors are in ked through an antigen
of Ig. B
cell including one or more
BCR 9nd of the
eSpecially when both the on which molecules are ligated, signaling by the IgJtr
npiement /antibodycomplex, Depending
Igß complex is enhanced or inhibited.
Cell Activation and Proliferation
B

proliferation and differentiation.occur in the

After export of cells from the bone marrow, activation,
B

called humoral immune

ey and requireonantigen. This type ofresponse mediated by antibodies is two different routes
esponse. Depending the nature of the antigen. cell activation proceeds by
B

one dependent upon T, cells, the other not.

Thymus Independent (T-I) Antigens
of T-cells, can also induce a response from
R
dependent antigens, without the interferance of polysaccharide of protein. Multiple
hoe T-independent antigens contain the repeating subunits
bondsare formed with B cells by these antigens.
Thymus Dependent (T-D) Antigens
The Tdependent antigen consists of bacteria, some of proteins, RBC ete, The B cells need the help of antigen
presenting cells (APC) andspecialized helper'T cells to produce antibodies against T dependent antigens.
e ceIs recognize antigenic peptides on the surface of antigen specific B cells because oncethe B cell can
capture antigen specifically via membraneIg and associate it with class II MHC molecules triggered
Via the TCR, Th cells express CD40L which triggers B cell activation via its CD40 surface receptor.
Activated B cells reciprocally co-stimulateT cells viaCD28, which produce IL- 2, IL- 4 and IL- 5. As a
result, both the T cells and B cells clonally expand and differentiate.
Macrophage
(APC)

APC
Contain selfantigen (microbial antigen digested)

T
APC– Helper cells

B cells

Plasma cell

Antibodies
MMUNOLOGY
9.21
CD79 a/bm
transmit the first signals following cell
molecules B

intracytoplasmic tails. Co receptors of cell interaction with antigen through ITAMof

B
their receptor complex((CD21, CD19 and C8Sl) modulate
signals CD21 binds to C3d ifit is
these initial bound to
cell activation. Cross linking of the
specific antigen and provides an additional
positive signal in
B

membrane receptor antibodies on cells by T-I

B

antigens induces clustering of co-receptors leading to

multiple signals enhancing activation of kinase
networks andl of IgM producing B cells. B cells primed a
CD40L binds to CD40 on by T-D antigen receive a second signal when
the Th cell the cell. Together with cytokines such as 1 L-2, 1 L- 4 ete, this
B

signaling induces proliferation and differentiation of

the cells into plasma cells or memory cells.
B

Polyclonal Antibody
Antibodies derived from different B
cell lines.They are a mixture of immunoglobulin molecules secreted
against a specific. antigen each recognizing a
different epitope.
Production:
These antibodies are typically produced
by immunization of suitable mammal, such as a mouse, rabbit
orgoat. Larger
mammals as as
often preferred the amount of serum that can be
An antigen is injected into collected is greater.
the mammal. This induces the B lymphocyte to produce IgG immunoglobulins
snecific for the antigen.
This polyclonal IgG is polyclonal purified from mammal's serum.

Monoclonal Antibodies
Ifa specific lymphocyte, after isolation and culture vitro,
in becomes capable of producing a single type of
antibody which bears specificity against specific
antigen,it is known as 'monoclonal antibody'. Due to
presence of desired
immunity, monoclonal antibodies are used in the diagnosis of the
with antibody secreting cells are diseases. Major difficulties
that they cannot be maintained in culture. But myeloma
marrow tumour cells cells (bone
due to cancer) grow indefinitely to produce a huge quantity
and also produce immunoglobulins in of identical cells (clones)
the same amount. The immunoglobulins are infact monoclonal
antibodies.
() Hybridoma : Cesar Milstein of Argentina tried to culture
myeloma cells. In 1973, in collaboration
with Cotton, he succeeded to fuse rat and mouse myeloma
cells with the result of production of
hybrid cells. The hybrid cells secreted the immunoglobulins
which consisted of several types of
polypeptides. In 1974, George Kohler and Milstein successfully
isolated clones of cells from the
fusion of two parental cell lines i.e. between lymphocyte
from spleen of mice immunized with red
-blood cells from sheep and myeloma cells. These cells were
maintained in vitro and produced
antibodies. The antibodies for specific antigens immunized
1975). Moreover, the hybrid cells maintained
the myeloma cells (Kohler and Milstein,
the character of lymphocyte to secrete antibodies,
and of myeloma cells to multiply in culture. These hybrid cell
lines (cell clones) are known as
hybridomas which are capable of producing unlimited supply of
antibodies. For the discovery of
production of monoclonal antibodies Kohler and Milstein along
with Neils Jerne were awarded
Nobel Prize in 1984, in physiology and medicine.
The cells growing on culture and producing sufficient amount
of monoclonal antibodies can be frozen.
The cells are collected through centrifugation and resuspended a
at density of 10" cells per ml freezing
medium in cold at 0°C. Samples (0.7-1.0 ml) poured in
sterile glass vials are sealed and stored at 80°C
 IMMUNOLOGY
9.22
Bone mArrow tumour
Rat
Cuture
Immunize to produce myeloma cells
antbodies
Myeloma culture
Spleen cells

Fuslon

Mbture of spleen colls,

myeloma& hybridoma cells
Testing antibodies to select hybridoma
cells for the desired antibody
Individual hybridoma cells

Cloning

Clones of hybridoma cells

Screening of hybridoma cells
for desired antibody
Cloning
Clones of hybridoma calls
Injection of hybrldorma cells to rat
to develop tumour that will also
produce antibodies, colection of blood
serum containing antibodies. tiKsis
Immunoglobulin

Application of antibodies to
immunize animals against diseases
Fig. Outline of production of monoclonal antibodies.
cases:
Uses: Monoclonal antibodies are useful in the following
groups, cancer, pregnancy, allergies, viral diseases and certain hormones.
) In diagnosis ofABO blood

Antibody Diversity/Antibody Genes one for chains .

groups encoding immunoglobulins-one for chains,
K

There are three unlinked gene

a different chromosome. Within each of these gene groups
on the
one for H-chains, each on
and recombine at the level ofDNA to yield
a
chromosome there are multiple coding regions (exons) which
V region for the
binding site. In
a
mature B cell or plasma cell, the DNA encoding the
of a continuous uninterrupted nucleotide
sequence. In contrast,
H-chain of a
specific antibody, consists exons,
non B cell) for this region exists in distinct DNA segments,
V

the DNA in germ line cell (or

a

of noncoding DNA. The exons found in the

area encoding the V
separated from each other by regions
region of the H-chain are: segment (encoding
V
approximately the first 102 aminc acids),D segment
remaining 14 or so amino acids in the V
(encoding 2-4 amino acids), and J segment (encoding the
approximately 95 amino acids) and J segment
region). For L-chains there are only V (encoding the first gene group,
exons. In each there are from 30-65 functional
(encoding the remaining 13 or so amino acids) on the chromosome and there
V
segment genes. The D and J regions are between the V and Cregions
encode the binding site of
are multiple different genes for each. Thus, DNA segments that ultimately
on chromosome to form a DNA sequence
antibodies have to be moved over distances (translocated) the
encoding the V region.
are 3' to the V genes, but separated
The DNA sequences encoding the C region of the L-and H-chains gene group usually
from them by unused J segment genes and noncoding DNA. Furthermore, each
gene group has
has one functional gene segment for each class and subclass. Thus, the H-chain
nine functional C region genes, one each encoding 8, yl, y2, y3, y4, al, a2. For the L-chain
u, ,
MMUNOLOGY
9.23
groups, there is one geme segment encoding
gene Cregions, theC region of k
L-chains, but four encoding
L-chain
Table. Genes for human immunoglobulins
Ig polypeptide Chromosome
H-chain 14
K-chain 2
-chain 22

Gene
rearrangement
During its development, single a B
cell randomly selects one one and one J (for H-chains),
V, D

one V and one

(for J
L-chains) for rearrarngement (translocation). Gene segments encoding a
and
of the region V
are moved adjacent to other gene segments encoding
portion
a gene the rest of the
V region to create segment encoding the entire region, with the intervening DNA
V

removed. More specifically, the H-chain gene group is

segment genes adjacent
the first to rearrange, initially moving onea
of several
D

to one of
several J segment genes. This creates
DJ combination, which encodes the C terminal part of the H-chain V region. A V segment gene
then rearranges to become contiguous with the DJ segment, creating a DNA sequence (VDJ)
encoding a complete H-chain V region (Fig). This VIDJ combination is 5' to the group of H-chain
Cregion genes, of which the closest one encodes the chain. A primary mRNA transcript is then
made from VDJ through the C
region gene, after which the intervening message between
VDJ and the C region gene is spliced out to create an mRNA for a complete u H-chain.
After the H-chain has successfully completed its rearrangerment, one of the V region gene segments
in either the 2 or K gene groups (but not both) translocates next to a J segment gene to crente a
gene (VJ) encoding a complete L-chain V region (Fig). For k chains, the DNA sequences encoding
the C region of the L-chains are 3' to the V genes, but separated from them by unused J segment
genes and noncoding DNA (Fig.), For 2, chains, since the J segment genes are ench nssocinted
,
with a different C. gene, translocation of a gene segment to a J gene segment results in a
V

V region next to a particular C gene (e.g. CA2 ). It is important to repent that in eaclh B cell, only
oneof two L-chain gene groups will be used. A primary mRNA transcript is then made from VJ.

V
segments D segments eegments
J

Germ line
DNA CH genos
DNAspllced DNA spllced
out out
Noncoding DNA

Other
B cell DNA S'
CH genes
Functlonal
V-DJ gene

Fig. In the germline, and therefore in a celldestined tobecomeaB

H-chain genes and translocation.
cell, the H-chain gene loci contains many V segment genes. In a developing B cell, one of these V
segments recombines with one of many D segments, which hasalready recombined withone of several
J segments, to produce a functional DJ gene. In each B cell, the rearranged gene is transcribed,
spliced and translated into an H-chain protein.
 9.24 IMMUNOLOGY

(a) V segments JDegnents

Germ llne
DNA
DNA Bplleed out
B
cell DNA
AC
Primery RNA
Cx
trenscript
RNA spliced out

ve b3 Cx mRNA

VK
C*K chain

(6)
V
segnents and C region segments
J

Germ line
vv|v| DNA
DNA epliced out

Tvva cazcDNA B cell

va c Primary RNA
transcript
RNA spliced out -

va acaz mRNA
Va caz A
chain

rearrangement ofthe H.
Fig. L-chain genes and translocation. During differentiation ofa B cell, and after
groups rearrange. In particular;,either (a) a germ line Vxgene combines
chain genes,one of thetwo rchain or (b) agerm line VAgene combines with one of the J
with a J segment gene to form a VJ combination; gene is then transcribed
segment Chgene combinations to form a VJ CA combination. The rearranged sequences
noncoding spliced out to form
intervening
transcript which then has the
intoa primary RNA translated
mRNA. This is then into light chain porotein.
through the L-chain C region gene, after which the intervening message between VJ and the Cregion
gene is spliced out to create an mRNA for a complete Lchain.
of two recombination-activating
genes, RAG-land
Gene rearrangement in B cells requires the products enzymes break
developing lymphocytes. These
RAG -2, which appear to be only expressed together in
are thus critical to the generation of diversity.
and rejoin the DNA during translocation and
Allelicexclusion a
segments, the cell is committed to the expression of
After successful rearrangement of the Ig DNA
L-chain and excludes other H- and
particular V region for its H-chain and particular V region as its
a for
process is referred to allelic exclusion and is unique to B and
Lrchain V region rearrangements.This
occurs on the first chromosome the process will
Tcell antigen receptors. If aberrant rearrangement
an
process stops,
continue, i.e.,the process does not stop if the cell does not get it right the first time. The
however, if the cell gets it right or runs out of chromosomes to
rearrange. In fact, following successtul
MMUNOLOGY 9.25

gene
rearrangement. on one chromosome there is active suppression of further rearrangement of
VH segments. Similarly, following successful VL gene rearrangement
other VH gene there is active
suppression rfurther rearrangement
the of of other Vand J gene segments.
B cell makes L-chains all of which
Thus, each
contain a region encoded by the same VJ region
V

sequence and H-chains all of which contain Vregion encoded by the same VDJ sequence. Each B cell
a

express antibodies on itS surface, all of which have exactly the samne specificity. This cell
ailltherefore are committed to express and produce antibodies with these regions.
its progeny V

and all
of
H L
Synthesis and assembly of and chains
Rr successful rearrangement of both L- and H-chain DNA, L- and H-chain mRNA is produced and
nslated into L- and H-polypeptide chains that combine in the ER to form an antibody molecule,
which is transported to the plasma membrane as the antigen specific receptor for that cell. Since the
B

gene encoding the H-chain also contains coding sequences for transmembrane domain, the H-chain
a

produced contains a terminal amino acid sequence which anchors the antibody in the plasma
C

embrane. In plasma cells, the part of the mRNA encoding the H-chain transmembrane domain
important for its membrane expression on B cells is spliced out. Thus, the antibody produced by a
nlasma cell does not become associated with the membrane, but rather is secreted.

B
cell DNA vDu Cu C6

Primary
transcrípt |vDuCu Cs

VDJCu:CS Cacs
Ditferentlal cleavage
and splicing out

mRNA

S
chain chain

L chain

lgM IgD

of IgM and IgD on a mature B cell.

Fig. Expression

Differential splicing and class switching

As indicated above, the first antibody produced by a B cell is of the IgM class. Soon thereafter the B cell
produces both an IgM and an IgD antibody, each having the same V regions and thus the same specificity.
This is the result of the differential cleavage and splicing of the primary transcript. In particular, a
primary transcript is made which includes information from the VDJ region through the Ca region
-
Pig). This transcript is differentially spliced to yield two mRNAs one for an IgM H-chain and the
other for an IgD H-chain. In a mature B cell both aretranslated and expressedon the B coll surfaco
with L-chain.
 IMMUNOLOGY
9.26
Bcells expressing lgM and lgD on their surface are capable ofswitehing to other H-chain classes, Thie
isotype (elass) switching requires stimulation of the B cellby 'T cells and cytokines. These signala
induce translocation of VDJ and its insertion 5' to another constant region gene (Fig). Class switch i
guided by repetitive DNA sequences 5' to the C region genes and occurs when these switch regionS
recombine. The intervening DNA is cut out and the resulting DNA in the B cell which has class switched
and in plasma cells derived from this B cell, no longer contains Cu, Cô or other intervening H-chain n

C region genes. Aprimary transcript is made and the RNA between the VDJ coding region and the
H-chain coding region is spliced out to give an mRNA for the new H-chain.
B cell DNA

C8
DNASplced out Switçh regions

Cu lCy3

Switch region recombination

and looping out
VDJ
Caic
B cell DNA atter class switch

Primary transcript VDJ

mRNA vDuCr

IgG1 H chain polypeptide

C
region region

Fig. Class switching.

Ways of creating diversity

is created by several antigen
Ig diversity (the generation of antibodies with different specificities)
independent mechanisms. In addition, in B cells that have been stimulated by antigen and received T
may increase the affinity ofthe antibody produced by
cell help, Ig genes undergo mutational events that
the B cell Overall, diversity isgenerated by:
Antigen independent events
V and J gen.
at the DNA level as a result of multiple germ line D and J and
V,
segments,
• at the DNA level as a result of random combination of V, D and J segments C, V and J
• at the DNA level as a result of imprecise joining of V, D andJ
segments,
• at the protein level as a result of random selection and pairing of different
combinations of L- and
H-chain V regions indifferent B cells.
Antigen dependent events
• at the DNA level as a result of somatic mutation in the V region, which
may create higher affinity
antibody binding sites.
will make up the V region genes occurs in an
Although rearrangement of the gene segments that
events occur in a vast number of
ordered fashion, they are chosen at random in each B cell, As these are generated.
cells, the result is that millions of B cells, each with a different antigen specificity,
Additional diversity is created during recombination ofV and J(L-chain) and V,D andJ
(H-chain) gene
segments due to imprecise joining of the different gene segments making up the V region. That is for
occur with all three
example, although translocation of a V gene segment to a J gene segment could
ANUNOLOGY 9.27
V
of the
nucleotides of the last codon segment joining with all three nucleotides of the first codon of
segment, it is also possible that one or two nucleotides at the 3 end of the V
segment could
the J one or two nucleotides of J segment. a
replacethe firstoccurs can the Such difference in the position at which
recombination change the amino acid I sequence in the antigen binding area of the resulting
and thus change its specificity.
Vregion of the antibody,
Furthermore, after antigen stimulation of the cell, the DNA of its L- and H-chain regions becomes
B V

particularly susceptible to somatic mutation. This results in changes in the nucleotides of the DNA
and thus
s corresponding changes
in the amino acid sequence ofthe V regions of the antibody exxpressed
Bcell. As result, the cell may have different specificity and not bindto or be stimulated by
a B a
bythe
Dyriginal antigen. However, it often happens that at least some mutations result in amino acid
changes which increase the tightness of binding of the antibody on the cell to its antigen. These
B
B

C'ts willcompete morea efficiently for antigen than the original B cell, and will differentiate into
plasma cells producing higher affinity antibody (affinity maturation).
nvorsity is also generated as a result of the fact that any L-chain can interact with any H-chain to
create a unique binding site. Thus, for example, an L-chain with a particular VJ combination for its
tinding site could be produced by many different B cells and interact with the different H-chains (i.e.
:ferent in their VH region) generated in each of these B cells to create many different specificities.
In sum,almost unlimited diversity is created from a limited number ofV regions. The diversity almost
ortainly exceeds the amount of diversity needed to bind the immunogens of microbes. Moreover, the
vast majority of the different B cells generated will never encounter antigen to which they can bind,
ond thus will not be stimulated to further development. And yet, such apparent wastefulness is justifed
bv the fact that this mechanism of creation of diversity ensures that there are cells and thus antibodies
B

Teactive with virtually all antigens that will be encountered. When an antigen to which this antibody
binds is encountered, the B cell is triggered to divide and to give rise to a clone of cells,each one of
which makes, at least initially, the originally displayed antibody molecule.
Antigen selection of B cells for stimulation
During an antibody response to an antigen, the overall affinity of the antibodies produced increases
with time. This is partly because, as the B cell numbers increase during an immune response, antigen
becomes limiting and there is competition for antigen among B cells. Those expressing higher affinity
antibody compete more successfully for antigen and clonally expand, contributing a higher proportion
to the antibody pool. Thus,the affinity of antibody produced in the secondary immune response is
higher and thus more efficient at effector functions than that produced in the primary response.
In addition and of particular importance, B cells already stimulated by antigen and T cells undergo
somatic mutation in the V regions of their H and L genes, which results in affinity maturation the
generation ofB cells expressing antibody with higher affinity for the antigen. These B cells contribute
significantly to the pool of antibodies in the circulation. Typical antibodies have binding constants of
104-7 M-, After successive immunization with limiting antigen they are usually 10%- M- but may be as

high as 1012 M-1,

Cytokines
Cytokines are small molecules, secreted by cells in response to a stimulus. They may have an effect on
the cell that produces them and are critical to signaling between cells, with each cytokine often inducing
several different biological effects. Many different cells release cytokines, but each cell type releases
only certain of these molecules. As a group, cytokines induce growth, differentiation, chemotaxis,
activation, and/or enhanced cytotoxicity. Moreover, it is not uncommon for some cytokines to have
similar activities and for many cytokines, some with opposing activities, to be released by a particular
stimulus. Thus, the resulting biological effect is a factor of the sum of all of these activities.
Cytokine nomenclature
To some extent cytokinescan be grouped by the cell populations that secrete them. Monokines are
eytokines secreted by cells of the myeloid series (monocytes, macrophages) and lymphokines are
 9.28 IMMUNOLOGY

ytokines secreted primarily by lymphocytes, although some cytokines are produced by both lymphocte
and myeloid colls. The term interleukin (|L) is often used to describe cytokines produced by leukocte
although some cytokines labeled as IL are also produced by other cell populations. A group of smal
heparin binding cytokines, chemokines, has recently been recognized which direct cell migration, and
may also activate cells in response to infectious agents or tissue damage. Interferons are produced by
a variety of cells in response to viral infection. In addition, a variety of other less easily categorized
cytokines exist which have critical immunologic activities.
Interferons
These molecules can be divided into two groups, type I IFNs(IFNa and IFNB) anctype I TFN (IFNY)
also called immune IFN. IFNa and IFNB are produced in response to viral infection (and probably to
double-stranded viral RNA)by many diferent cells. They inhibit viral replication in uninfected cells,
inhibit cell proliferation, increase the lytic activity of NK cells and modulate the cellular expression of
MHCmolecules. The receptor for both interferon a and interferon is the same and found on most
nucleated cells. Binding of interferons a or B to this receptor inhibits viral replication in that cell as a
result ofblocking translation ofviral proteins and induction of the synthesis of inhibitoryproteins. In
addition these interferons induce increased expression of MHC class I and other components of the
class I processing and presentation pathway which leads to induction of antigen specific CD8 CTL
responses against virally infected cels. Induction of MHC class I is also important for protection of
uninfected cells from killing by NK cells.
Lymphokines
A variety of cytokines are produced by lymphocytes and lymphocyte subsets many of which are growth
factors for ]ymphocytes or which influence the nature of the immune response. IL-2 is made by T cells
as a critical autocrine growth factor that is required for proliferation of cells, especially Th0 and Thl
T

cells andCTL. On activation of these T cells as a result of the interaction. of their antigen receptor
on for and
secretion
complexes with antigenic peptide in MHC molecules APCs, the T cells make IL-2
at the same time IL-2 receptors with which to bind and be stimulated by the relensed lL-2. the
In
absence of IL-2and/or its receptor, many antigen specific T cells do not expand, severely compromising
immune responses.
cell types
IL-3 is a cytokine that appears to be involved in the growth and differentiaton ofavariety of by
as a result of its synergistic activity with other cytokines in hematopoiesis. IL-4 is produced Th2
cells) and Beells, and
cells and mast cells. It is a growth and differentiation factor for T (primarily Th2 to this nctivity,
lgE nntibodies. Closely related
inparticular for B cell class switch to the production
of
response as it can induce the
IL-4 has an important role in influencing the nature of the immune responses.
of"Thl Thus, IL-4
development of Th2cells from Th0 cells and can inhibit the development B cell and its subsequent plasma cells
not only is involved in B cellgrowth, butit can also influence the to
IL-5 is also produced by Th2 cells and mast cells and important
is
toproduce IgE antibody. Iike IL-4, a role in eosinophil growth and diflerentiation.
B cell activation and production of lgA antibody. It also has
It induces Th2 responses
IL-10, which is produced by Th2 cells and MØ, is involved in cell activation.
B
by suppressing MØuctivity
andinhibits Thl responses, perhaps by enhancing IL-4 production and/or
and production IL-12, a Thl-stimulatory cytokine.
of

Monokines
This group of cytokines has many different local and
systemic activities that are critical to immune
cytokines, are important mediators of
defense. In addition, these molecules, also called pro-inflammatory
ingestion of grann negative
inflammation. In particular, as result of an appropriate stimulus, including
a

IL-8, IL12 and '"NFa. IL-1, TNFuand

bacteria and subsequent activation by LPS, MØ secreteIL-1, IL-6,
lymphocyte activation, which
IL-6 have activities which include: (a) increasing body temperature and
responses; (b) mobilization of neutrophils for
decrease pathogen replication and increase specific immune complement activation
phagocytosis; (c) induction release acute phase proteins (CRP,MBP) and thus
of of

and opsonization.
MMUNOLoGY
9.29
Representative lymphokines and monokines
Table:
Cytokine Produced by Activity
MØ, epithelial cells Activates vascular endothelium: tissue destruction;
IL-l
effector cell access: fever: lymphocyte activation; increased
mobilization
of PMNs; induction of acute phase proteins (CRP,
T cells
MBP)
IL-2 Proliferation of T and NK cells
IL-3 T cells, thymic cells Proliferation, differentiation of
hematopoieticcells
IL-4 Th2 cells, mast cells| B cell activation, proliferation,
IgE response; induces Th2 and
inhibits Thl responses
IL-5 Th2 cells, mast cells| Eosinophil growth, differentiation; B
cell activation, lgA
response
IL-6 T cells, MØ Lymphocyte activation; fever
IL-8 MØ, MØ, Fb, Kr Increases tissue access for, and chemotaxis of PMNs
IL-10 Th2 cells, MØ cell activation; suppression of MO activity: induces Th2 and
B

inhibits Thl responses

IL-12 B cells, MØ Induces Thl and inhibits Th2 responses: activates NK cells
IFN-y T cells, NK cells MØ and PMN activation: induces Thi and inhibits Th2
responses
TNFa MØ, T cells Activates vascular endothelium; fever; shock; increases vascular
permeability; mobilization of metabolites
Monocytes (Mo), macrophages (MØ), endothelial cells (En),
fibroblasts (Fb), keratinocytes (Kr),
neutrophils (PMNs), chondrocytes (Co)
Table: Cytokine effects on Thl and Th2 immune responses
Enhance Inhibit Cytokines produced

IL-4 Th1 Th1 Memory cells

IL-12 IL-10 IFNy Help for CTL and lgG
IFNy
IL-2 antibody responses
T,0
IL4
IL-10 Th2 Th2 Memory cells
IL4 Help for lgA and lgE
IL-5 antibody responses
IL-6
IL-10
IL-13

IL-l also activates vascular endothelium (in preparation for neutrophil chemo
production of IL-6. IL-8 increases access for, and chemotaxis of,
taxis) and induces systemic
neutrophils. It also activates binding by
integrins, which facilitates neutrophil binding to endothelial cells
IL-1, TNFa.also activates vascular endothelium and migration into tissues. Like
and is able to increase vascular permeability. It activates
MØ and induces their production of nitric oxide (NO).
Although produced by monocytes and MØ,
TNFa is also produced by some T cells. Finally, IL-12, which
is also produced by B cells, activates
NK cells which then produce IFNy, a cytokine
Thl cells (Table).
important to inducing differentiation of Th0 cells to
 IMMUNOLOGY
9.30

Chemokines
This group of more than 50 small, closely related cytokines (mol. wt 8-10 kDa) are primarily involved
in chemoattraction of leukocytes (lymphocytes, monocytes and neutrophils) (Table). They are made by
monocytes/macrophages, but also by other cels including endothelial cells.platelets, neutrophils, T
cells, keratinocytes and fibroblasts. Chemokines can be divided into four different groups based on
unique aspects of their amino acid sequence, and in particular the position of conserved ysteine
residues. One group has two adjacent cysteines (CC), a second has two cysteines separated by another
amino acid (CXC), another has one cysteine, and the last has two cysteines separated by three other are
amino acids. For the most part, CC chemokines such as monocyte chemotactic protein (MCP-1)
chemotactic for monocytes, inducing them to migrate into tissues and become macrophages, whereas
CXCchemokines such as IL-8 are chemotactic for neutrophils inducing them to leave the blood and
can be
migrate into tissues. Some of these chemokines also are chemotactic for T cells. Chemokines
or not only
produced by many cells in response to an infectious process to physical damage. "They
mav also activate the cell for dealing with the
direct a cell to the source of infection /damage, but
infectious agent or damage.
Table. Representative chemokinte
Classl Name Source Chemoattractant for Activation of
CXC (%) IL-8 Mo, MØ, Naive T cells, PMNs PMNs
Fb, Kr
NAP-2 Platelets Neutrophils
MIP-1ß Mo, MØ, CD8T cells
En, PMNs
CC(B) MCP-1 Mo, M. Memory T cells, Mo Monocytes
Fb, Kr
Rantes T cells Memory Th cells
Lymphotactin Lymphocy tes
C(y)
CX,C(6) Fractalkine Lymphocytes, monocytes,
NK cells
with the characteristic feature that they
Receptors for chemokines are all integral membrane proteins molecules are coupled to
span the membrane seven times (seven transmembrane glycoproteins). These
as moiety of thereceptor. Although most
G(guanine nucleoside binding) proteins which act the signaling on
receptors can bind more than one type of chemokine, they are usually distributed only
of these
chemokines to have selective activity.
particular cell populations, permitting different to
chemokines, for example IL-8 and MCP-1, have been shown to work by first binding
Some of these
or on the extracellular matrix.On this solid surface they then
proteoglycan molecules on endothelial cells passage and directing them to migrate downa chemokine
bind blood neutrophils or monocytes, slowing their
Although the role that each plays in immune
concentration gradient toward the source the chemokine.
of

these molccules are potent agents for

defense and pathology is still being clarified, it is evident that
tissue damage as well as for controlling
activating and directing effector cell populations the site of
to
leukocyte migration in tissues.

Other Cytokines
defense, several are particularly noteworthy
Ofthe many other cytokines which are important immune
to
(GM-CSF), granulocyte CSF (G-CSF) and
(Table). Agroup of CSFs, including granulocyte-monocyte CSF
monocyte CSF (M-CSF) are cytokines which drive the development, differentiation
and expansion of
cells of the myeloid series. GM-CSF induces expansion ofmyeloid progenitor
cells and their commitment
specific commitment
to the monocyte/MØ and granulocyte lineage, after which G-CSF and M-CSF induce
MUNOLOGY 9.31
granulocyte or monocyte lineage, respcctively, andthentheir subsequent expansion. These factors,
totheespecially GCSE, are important clinical tools in a number of
diseasc situations as they are used to
and Imyeloid effector cell populations critical defense
expand to against pathogens(Topic G3).
a variety of cells including monocytes, MØ, T cells
TGFBis produccd by andchondrocytes, and plays an
importantrrole in suppressing immune responses, it can inhibit activation of MØand growth ofB and
as
Tcells. TNFB (ymphotoxin) is
a
molecule which is cytotoxic to a variety of cell types, including ineffectual
MØ.
chronically infected .Thereleased pathogen is then killed by less compromised MØ.
cytokines
Table. Other
Cytokine Produced by Activity
GM-CSF MØ, T cells Stimulates growth, differentiation & activation of
granulocytes, Mo, MØ
GOSF Mo, Fb, En Stimulates PMN development
M-CSF Fb Stimulates Mo, MØ development
TGF-B Mb, T cells, Co inhibits cell growth and inflammation
TNFB lymphotoxin) T cells cytotoxic to T, B and other cells
Cytokine treatment
doveral cytokines which enhance immune responses, including IL-2, IFNy and IFNa have been approved
tor treatment of certain tumors. .G-CSF is extremely useful in treating cancer patients with low PMN
cnunts resulting from chemotherapy or irradiation.
Table. Cytokines and their inhibitors as tools in the clinic.
Cytokine treatment Patient group
For infection
IFNa Some infections
IFNY Treatment of patients with CGD
G-CSF Treatment of patients with low granulocyte counts (e.g. resulting
from chemotherapy or irradiation)
For tumors
IL-2 Renalcell carcinoma (20% of patients have partial responses)has
been used together with tumor infiltrating lymphocytes for some
tumors
IFNa Hairy cell leukemia
Cytokine inhibitors (anti-inflammatory)
Anti-TNFa Some success in treating RA
Soluble cytokine Some success in treating RA
receptors e.g. IL1, TNFa
Cytokine receptor targeting
Antibodies to cytokine receptors or soluble cytokine receptors themselves used to treat autoimmune
diseases where pro-inflammatory cytokines are involved in maintenance of chronie inflamnation e.g.
TNFa in RA.

Molecules of The Innate Immunity

Complement System
The complement system (C.S.) is the primary numeral (plasma) mediator of antigon (Ag) and antibody
(Ab) reaction. The system consist of 20 chemically and immunologically distinct plasma proteins found
in blood plasma along with blood clotting fibrinolysis and kinin formation, complement forms one ofthe
triggered enzyme system.Synthesized by hepatocytes and monocytes, these proteins help (complement)
antibody responses and have a wide spectrum of activities including a pivotal role in innate defense
 9.32 IMMUNOLOGY
mechanism. Comnplement may be activated by either the classical pathway (through antibody) or the
alternative pathway (innate). The two pathways converge at a point from whichn final commonor
pathway ensues, the terminate at the end point of complement activation, which is cytolysis
cytotoxicity. There are other non complement enzymes of serumor ccllular origin which can activate
the complement system in midway, e.g. trypsin like enzymes such as plasmin. The most abundant.
and pivotal component of complement system is C3. This is present at a concentration of 1.2 mg/ml in
plasma and has mol.wt of 195 kD.

Classical C.P.
C.C.P. isactivated by antigen antibody binding. Among theIgG subclasses IgG3 has been known to
become most active, followed by IgG1 and IgG2, IgM is the most efficient immnunoglobulin activator of
the C.C.P. Activation occurs by binding of first complement Cl to a site located in CH2 region of
immunoglobulin.
The formation of an Ag-Ab complex induces conformational changes in the F. portion of IgM molecule
that expose a binding site for the Cl component of the C.s. C1 in serum is a complex consisting of Clq
and two molecules each of theClr and Cls, held together ina complex (Clqr,s,) stabilized by Ca* ions.
C1 normally present as a complete entity. Individual proteins of Cl are found only in the diseased
is
state. Clq is unique as its structure is similar to collagen. Clg consists of 18polypeptide chains of 3
distinct type that are organized into structure consisting of 6 globular proteins connected by fibrinular
strand to a central structure. Cl is able to bind 6 IgG molecules. Once Clg binds to Ig molecule Clr, a,
B globulin acquires the ability to enzymatically activate Cls. Cls once activated acquires proteolytic
activity, and the initial phase of complement pathway is activated. The earlier reactants including Ag,
Ab and Clq or Clr are not necessary for the progression of complement reaction, Activated C1s mediates
the next phase of complement cascade. Cls cleaves C4 yielding C4a and C4b. It also cleaves C2, initiate
the formation of the key enzyme C4b2a on the activator surface. C4b contains a labile binding site which
binds with activation in a transient manner. C2a also has a labile binding site which allows it to bind to
C4b. Mg* ions required for the formation of C4b2a complex. Activated C4b2a is a proteolytic enzyme
that assumes the role of continuingthe classical complement reaction. Once it has been formed, earlier
reactant are no longer required. C4b2a is termed as C3 convertase thus activates C3. The fragment
from C4 cleavage, C4a, is an anaphylatonin, or mediator of inflammation.
Cleavage of C3 results in formation of C3a and C8b. C3a is biologically coating peptide, the attachment
of C3b to membrane in the incinity of C4b2a molecule lead to generation of last enzyme of classical
pathway CAb2a3b. Called C5 convertase. The C3b component of this complex binds C5 and alters its
conformation, so that the C4b2a component can leave C5 into C5a which diffuses away, and C5b which
attaches to C6 and initiates formation of the membrane attack complex described later.
Alternative Pathway
The alternate pathway was originally deseribed as properdin system, consist of a group proteins involved
in resistance to infection. There were thought to be similar to but distinct from complement. The
properdin system was found to be involved in destruction of certainbacterin, neutralizing of virus and
lysis of erythrocytes. This system does not seem to require specific Ab. Several factors involved in the
system has now been isolated and identified. These includes properdin, the Y, globulin, factor a has
been identifed as C3. Factor B, a ß, protein is similar to but distinct from C2. Factor D and a globulin
and Factor I and HBglobulin. The properdin system is now known as alternative C.P., because of its
major role in resistance to infection it is considered to be the pathway of most, important to man.
Initial requirement for activation in the presence of C3b, is continuously generated. This occurs
following cleavage of the trioester bond in C3 thus forming C3* (active) which reacts with factor B and
D to generate an enzyme able to cleave C3a and C3b. It is possible that C3 in circulation is also cleaved
by enzymes of coagulation system, the enzyme zymogen a yeast polysaccharide also cause C3 activation
in the presence of Mg?* ions. The most of newly generated C3b remains in the fluid phase, some binds
to cellular surfaces. In either cases this C3b is rapidly inactivated by controlled proteins factor I and
NWUNOLOGY
9.33

This surface
bound, protected C3b interact with factor in presence ofMg? ions to
B
form C3bB with
addition factor D, which acts on bound factor to cleave it yielding Bb, and enzyme (C3hBb
of B

the
generated. This:surface bound enzyme is also called the amplifving C3 convertase and is able to cleave
is
very large
amount of C3
this results in accumulation of C3b which undergoes lyclic amplification of
component. This is.also called as a positive feeback mechanism which
critical1C3b enhances C3b formation.

Aternative pathway Classical pathway

(microbe alone)
( antibodymediated )

Convertases

Activation leads to

Inflammation Lysis
Enhanced
phagocytosis
Fig. Complement aystem

Lectin Pathway:
Itorginates with hst proteins binding microbial surfaces. Lectins are proteins that recognize and bind
to specific carbohydrates. The lactin pathway like alternative pathway does not depend on antibody for
its activation. However, the mechanism is more like the classical pathway. because after initintion it
proceeds, through the action of C4 and C2,to produce a C5 convertase.
Three Complement Pathways Converge at the Membrane Attack Complex
:

The terminal sequence of complement activation involves Cab, C6, C7, C8 and C9, which interact
sequentially toform a macromolecular structure called the membrane attack complex (MAC), This
complex forms a large channel through the membrane of the target cell, ennbling ions and mall
molecules to diffuse freely ncroSs the membranc.
Functions : The major functions of complement system are
:

) Initiation of inflammation as a result of the stimulation of degranulation ofmost cells by C3a and
C5a (anaphylectoxins).
ü) Attraction, by C5a, of neutrophils to the site of mierobial attack fehemotoxis). (IHi)
-
(ü) Enhancement of the attachment of the microbe to the phagocyte PMNs attracted to a site of
complement activation by Côa bind to C3b enhancing their internalization of the nmicrobe
(opsonization).
(iv) Killing ofthemicrobe by themembrane attack complex' which through C9 produces 'pores' in the
target cell membrane leading to lysis of the microbe.
Regulation : Complement components rapidly lose binding capacity afer activation, liniting their
membrane damaging ability to the immediate vicinity of the activation site. The conplenent system is
also tightly regulated by inhibitor /regulatory proteins which inclue Cl inhibitor, Factor 1, CAb bindng
protein, Factor H, decay accelerating factor (DAF), membrane cofactor protein (MCI) and CD59(protecting
These molecules protect host cells from destruction or damage at different stages of the complement
cascade.
 IMMUNOLOGY
9.34

Acute Phase Proteins against microbeos

group plasma proteins importantr in innate defense
of

These are a heterogeneous malignancy and other diseases. They include C reactive
limiting tissue damage caused by infection, trauma, mannose binding protein (MBP).
A
(SAA) and
protein (CRP), serum amyloid protein
usually as the result of microbial stimulus.
a

Acute phase proteins are mainly produced in the liver,

rapidly. These proteins are produced
t
Production is at a low level but on stimulation increases macrophages
response to the cytokines IL-1, IL-6. TNFa. IFNy that are released by activated
NK cells.
Interferons
Interferons (IFNs) are proteins involved in protection origins against viral infections. The two kinds
mediate a range of different
interferons type I and type II, have different cellular but alsoand are signaling molecules between
activities. These molecules interfere with viral replication
cells.
are produced by many different cells in
Type I: IFNs consist ofIFN-alpha (IFNa) andbeta (IFNB) IFNB inhibit protein synthesis in
the body and interfere with viral replication. Both IFNa and B form the
virally infected cells by preventing mRNAs translation and DNA replication. IFNa and
part of innate immune response.
Type II: immune interferon IFNy (gamma), has no structural homology with type I responses. is mainly
produced by the Thl (T-helper 1) cells and NK cells and functions to regulate Thl
increase phagocytic mechanisms and increase antigen presentation.
Table: Interferons
IFN-a/B IFN-y
Chromosomal location 12

Origin All nucleated cell, NK cells and Thl, y S and

especially fibroblasts, CD 8T cells
macrophages and dendritic cells
Induced by Viruses, cytokines some Antigen stimulated
intracellular bacteria and protozoans T cells
Functions Antiviral, increases MHC Antiviral, increases MHC
class I expression, I and II expression,
inhibites cells proliferation activates macrophages

Hypersensitive Reactions
An immune response mobilizes a battery of effector molecules that act to remove antigen by various
mechanisms. Generally, these effector molecules induce a localized inflammatory response that
eliminates antigen without extensively damaging the host's tissue. Under certain circumstances,
however, this inflammatory response can have deleterious effects, resulting in significant tissue
damage or even death. This inappropriate immune response is termed hypersensitivity or allergy.
Classification
Hypersensitivity reactions occur at different times after coming into contact with the offending
antigens, within a few minutes (i e. immediate), minute to hours (intermediate) or after many
hours (delayed). Generally, the delayed responses are mediated by the cellular components of the
immune system (i.e. T cells) whilst the former are the result of the humoral arm of
response which incldces antibodies the immune
and the complement system. The original classification by Gell
and Coombs was into four main types, a fifth has since been added.
immune system components which contribute to tissue damage.
Tablel summarizes the main
than one of these mechanisms can contribute to any one It should be stressed that more
particular disease process.
INMUNOLOGY

Classification ofhypersensitivities
Table 1. 9.35

ofappearance Type
ime (immediate) Immune mechanism
9-30 min
lgE antibodies (enhancement acute
(eytotoxic) I of inflammatory response)
5-8 h Antibody and complement
9.8 h (immune complex) III Antibodylantigen complexes
24-72 h (delayed) IV
T cell mediated (can be granulomatous)
(Stimulatory) V Antibody mediated
IgG-mediated Type I Hypersensitivity
common type of : Allergy
This most hypersensitivity
is mediated by IgE
ne sting) clinical situations. Some individuals (atopic)
and causes
a l
mild (hayfever) to life threatening
Skin testscan be used to test for sensitivity to have genetic predisposition to make high levels of
allergens,
Sensitization Phase
Sensitization to a particular antigen
is dependent on stimulation of IgE antibody
requires CD4+ Th2 cells to induce class production. This
B cell growth and differentiation.
switching of antigen specific cells and to secrete IL-4
B
for
BEfectorphase-lgE -mediated mast cell
ToE antibodies
degranulation
produced following initial contact with the specific
mast cells and basophils. Cross-linking antigen, bind to IgE receptors on
by antigen of the IgE and the receptors with which
pharmacological mediators (e.g. histamine)it
results in rapid degranulation and release of is associated
inflammation (anaphylaxis). In the case of systemic causing local
nroduced requiring treatment with adrenaline release, systemic anaphylactic reactions are
(epinephrine) to restore blood pressure.
Common antigens causing type IHypersensitivity
These include grass and tree pollens, insect venomns, nuts,
drugs and animal dander. Fungal and worm
antigens also induce this type of hypersensitivity.
Drugsand immunotherapy (desensitization)
Drugs used to counteract Type I hypersensitivity inhibit production or
(nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and release of inflammatory mediators
and cromolyn) or Inhibit the action of inflammatory mediators which indomethacin, glucocorticoids
then relieve symptoms (benadryl,
dramamine, glucocorticoids). Epinephrine is used to counteract mediator effects
pressure and bronchospasm. The aim of such as low blood
desensitization is to induce an IgG immune response and/or
divert the immune response away from production of IgE. This approach has becen
a used successfully
foronly few allergens (e.g. bee venom).
B/plasma cellproduces
allergen-specific lgE

IgE antibody binds

to lgE Fc receptor (FceR|)
and 'ans' the mast cell

Granules
Allergen

IgE crosslinking
Degranulation
FigIgEmediated mast celldegranulation. Allergen binds to and erosslinks cytophilte (cell oound) IgE,
ašgnaling FeelH to trigger mast cell activation and degranulation with release of histamine, leukotrienes, ete.
 9.36 IMMUNOLOGY

IgG and Igm-mediated Type II Hypersensitivity

Antibody (IgM or IgG) directed mainly to cellular antigens (e.g. on erythrocytes) or surface autoantigens
can cause damage through opsonization, lysis or antibody dependent cellular cytotoxicity. Also called
cytotoxic hypersensitivity.
Rhesus incompatibility
Pregnant mothers who are rhesus D (RhD) antigen positive can respond to RhD antigen inherited from
the father. Sensitization occurs either through prior blood transfusion with RhD+ erythrocytes or at
parturition when fetal erythrocytes pass into the maternal circulation.During subsequent pregnancies,
small numbers of fetal erythrocytes stimulate a memory response with the result that lgG antibodies
to RhD antigen pass across the placenta and destroy the fetal erythrocytes (hemolytic disease of the
new-born).
Transfusion reactions
Natural antibodies (isohemagglutinins) to major blood group antigens (A, B) bind to transfused
now rare due to blood
erythrocytes carrying the target antigens resulting in massive hemolysis. This is
group typing.
Autoantigens
Antibodies to a variety of self antigens such as basement membranes oflung and kidney (Goodpasture's
syndrome), the acetylcholine receptor (Myasthenia gravis) and erythrocytes (hemolytic anemia) can
result in tissue damaging reactions.
Drugs
Drugs such as penicillin can attach to erythrocytes and cause IgG-mediated damage to erythrocytes.
Stimulatory hypersensitivity
a
A variant of type II hypersensitivity (sometimes called type V), this results in binding to receptor and
acting as the natural ligand e.g., in Graves disease antibodies are present which react with the thyroid
stimulating receptor.

Complement
activation

Target cell

-F. receptor

NK cells
Target cell

Immune-complex Mediated Type IIIHypersensitivity

as as
Immune complexes can form to serum products well mnicrobial and self
antigens, either in local
damage.
sites or systemically, leading to phagocytic and complement mediated
Mechanisms of type III hypersensitivity
enzymes from neutrophils.
Tissue damage is caused mainly by complement activation and release of lytic
Local damage (Arthus reaction) can be seen in pulmonary disease resulting from inhaled antigen.
Systemic antibody complexes with microbial or autoantigens result in immune complex deposition in
blood vessels (vasculitis)or in the renal vessels (glomeruli) of the kidneys leading to glomerulonephritis.
IMMUNOL0GY 9.37

Diseases associated with type III hypersensitivity

Pulmonary diseases
result from inhalation of bacterial spores
fancier's
(Farmer's lung) or avian serum/fecal
proteins (bird disease). Systemic disease can occur from streptococcallinfections (streptococcal
nephritis,), autoimmune complexes (e.g. systemic lupus erythematosus
(SLE)) or drugs (e.g. penicillin)
made in animals.
or antisera

Immune complexes

Actvate Crosslnk
complement Fc recoptors
C4a,
C3a, C5a Mast cell
C' fixaton
Intarnmatory mediators
Chemotaxis

PMN
Phagocytosis
Blood vessel

Fig. The Arthus reaction.Small immune complexes in the skin directly trigger Fe receptors and activate
complement resulting essentially in an aeute inflammatory response mediated through mast cells. Small
immune compleres can also lodge in blood vessels and induce vasculitis or glomerulonephritis in the kidney.

Delayed-Type Type IV Hypersensitivity

This occurs from 24 h after contact with an antigen and is mediated by T cells together with dendritic
cells, macrophages and cytokines. The persistent presence of the antigen e.g. chronic mycobacterial
infections, results in granulomas. Skin contact with a number of small molecules (chemicals and plant
molecules)can also result in delayed hypersensitivity.
The tuberculin reaction
This is a recall' response to purified mycobacterial antigens and is used as the basis of a skin test for
an immune response (not necessarily curative) to TB.
The production of granulomas
The inability to kill all mycobacteria in macrophages by T cells often results in a chronic stimulation of
the mycobacterial specific T-cells. The cytokines produced are responsible for 'walling off the
macrophages containing the persistent antigens and thus the production of granulomas. This also
occurs as a response to shistosomula worms and is seen in some clinical conditions with, as yet,
undefined antigens.
Contact sensitivity
Contact with a number of small molecular weight chemicals (e.g. nickel in a watch strap buckle) and
molecules from some plants (poison ivy) can penetrate trie skin, bind to self proteins and induce a
specific CD4+ T cell response. The resulting cytokines induce a local redness and swelling which
usually disappears on removal of the antigen.
 IMMUNOLOGY
9.38
49-72 hars
TUberculln O hours

Skin

Dendrtic )
Dermls
çell..
T cells. .

macrophages,
Processing flud

Presentation Cynokines

Toell
Blood vessel
(delaved-tvpe kypersensitivity). Tuberculin protein
introduced into the
Fig. 1. The tuberculinr reaction molecules. Cytokines produced
processed and presen ted by dendritic cells to T cells via MHC class II to enter the site of injection
dermis
by the is n
T cells alter local endothelial cell adhesion molecules altoun
macrophage products result in cdema
(Auid) and suelling. A
and develop into macrophages. T cells and injection.
a swelling which is maximal at 48-72 h after
positive skin test shows up as firm red

Autoimmunity
several autoimmune diseases, Some of theseAutoimmune diseases are caused by
Human beings suffer from are cause tissue destruction. iseases are
autoantibodies, while others due to T cells that
or systemic. In case of organ-specific diseases, the immune response is directed
either organ-specific or organ. As a result, the manifestations are largely limited
to a target antigen unique to a single gland or blockage/overstimulation of the normal function
organ; the manifestation may be tissue damage
to that occurs when lymphocytes or antibodies bind to cell-membrane
of the organ. Direct cellular damage organ. Gradually, the
cellular lysis and/or an inflammatory response in the affected
antigens causing a in function of that
by connective tissue leading to decrease
damaged cellular structure is replaced are anaemias, insulin-dependent diabetes mellitus, etc.
organ. Examples of such diseases autoimmune
e.g., disease, the antibodies bind to hormone receptors and activate them.
In some diseases, e.g., Grave's receptors.
some myasthenia gravis, antibody binding blocks the activation ofthe
while in others,
diseases, the immune response is directed towards a broad range of
In case of systemic autoimmune a number of organsand tissues. These diseases reflect
a general
target antigens and, as a result, involves widesprend,
regulation that results in hyperactive T cells and B cells. Tissue damage is
defect in immune autoantibodies, e.g., systemiclupus erythematus, or
e.g.,multiple sclerosis and
is caused by both T cells,
complexes, e.g., rheumatoid arthritis.
by accumulation of immune
Causes ofautoimmune disease.
many possible ways, some of which are as follows.
Autoimmune diseases could arise in for clonal
sequestered from circulation will not be employed in the thymus
(1) A tissue antigen that is into circulation due to
When such an antigen is released
deletion of T cells reactive to this antigen. or a viral or bacterial infection, it may induce
autoantibody
trauma to tissues caused by an accident
formation. are identical or similar to normal
of bacteria and viruses have antigenic determinants that
(2) Anumber
Infection by such pathogens
may initiate autoimmunity.
host cell components.
bacteria can induce nonspecific polyclonal B cells that produce IgM in the
(3) A number of viruses and to self-antigens and, thereby,
cause
could activate B cells reactive
absence of TH,cells.Such infections
autoimmunity.
do not express them could lead to autoimmunity. Such an
(4) Expression of MHC II in cells that normally
inappropriate expression of MHC II is induced by certain
agents like IFN-y.
(MMUNOLOGY

Thble
Some
autoimmune diseases of human 9.39
beings
Autoimmune disease
Self-antigen
Organ-specific autoimmune Immune response
Autoimmune haemolytic : anaemia
RBC membrane
diseases
proteins
Grave's disease Thyroid-stimulating Autoantibodies
hormone receptor Autoantibody (stimulation
Insulin-dependent diabetes of the receptor)
Pancreatic beta cells
mellitus Tp; cells*, autoantibody
Myasthenia gravis Acetylcholine receptors
Autoantibody (blockage of
Spontaneous infertility the receptor)
Sperm
Autoantibodies
Systemic autoimmune diseases
Multiple sclerosis Brain
Rheumatoid arthritis Connective tissue,IgG Tn, andT, cells, autoantibodies
Autoantibodies, immune
Scleroderma complexes
Nuclei, heart, lungs, kidney,
Autoantibodies
gastrointestinal tract
Systemic lupus erythematus DNA, nuclear protein,
RBC Autoantibodies, immune
and platelet membranes complexes
s=T cells involved in delayed type hypersensitivity.
Treatment ofAutoimmune Diseases
The current therapies provide relief by nonspecific
suppression of the immune system. Some
agents like cyclosporin A are somewhat selective in
that they inhibit only antigen-activated T
cells. More directed approaches are still at experimental level; some
of these are briefly summarise
below.
1. T cells specific forthe concerned antigen are injected into individuals to immunize them
against these T cells (T cell vaccination).
2. A synthetic peptide differing by only one (or few) amino acid
may be used as therapy. The synthetic peptide competes from the autoantigenic peptide
with the autoantigenic peptide for
MHCmolecules (peptide blockade ofMHC molecules).
3. Monoclonal antibodies may be directed against the following: (1) CD4 (depletes all T,, cells),
(2)the a. subunit of IL-2 receptor, which is expressed only by antigen-activated T,, cells (blocks
antigen-activated T,, cells), (3) specific T cellreceptor, and (4) the specific allelic variant of the
MHC molecule that is associated with the autoimmune disease.
4. Oral administration of the concerned autoantigen way induce tolerance to the antigen.

Transplantation Immunology
Introduction
Transplantation is the process of taking tissues or organs (or even cells) and placing them into
the same or different individual. The tissues, organs or cells that are transferred from one individual
to another are called grafts. The individual who donates the graft is called donor and individual
who receives the graft is referred to as recipient. Clinical transplantation is usually performed
to overcome a functional or anatomical deficit in an individual.
The transplantation of kidney,
heart, liver, cornea, lungs, pancreas and bone marrow is now performed worldwide.
 IMMUNOLOGY
9.40

Autologous graft (autograft): These are graft transplants from one region to another on the
same individual.
Syngenic graft (syngraft)/Msograft: Isograft involves the transfer of graft between geneticaly
identical (syngenic) individuals of the same species. This is possible between genetically ientical
twins or genetically identical mice.
Allogeneic graft (or allograft): A graft transplanted between two genetically non-identical
individuals of the same species is called allograft.
Xenogeneic graft (or xenograft): These are graft transplants between individuals of different
species.

Vaccines
A vaccine can be defined as a preparation of bacterial, viral
or other pathogenic agents or of their
isolated antigens which is administeredwith the objective of stimulating a recipient's protective
occurs an
immumity. After primary exposure of antigen to immunocompetent lymphocytes, there
response. Primnary
initial but slightly delayed immune response called primary immune exposure. There are two main
immune response peaks on approximately the 14th day of antigen
are activated;
outcomes of primary response first, specific immuno-competent cells (B and cells)
: T
are formed. Subsequent exp0Sure to the
and second, and the more important one, memory cells more heightened immune
same antigen stimulates these memory cells which results in a rapid and
response. Secondary response usually occurs within two to three days. It must also be mentioned
while a secondary response
that a primary immune response elicits the formation of IgMisantibodies which secondary
protects the host with higher-affinity IgG antibodies. It the rapidity with against any potential
response occursupon secondary exposure to antigen that protects the host
threat by repeated exposure to pathogen.
or its component that can induce secondary or adap-tive
Thus, a vaccine is basically an antigen severe complications of infections by reinforcing or
immunity in the host. It aims to prevent
memory.
broadening the defences by introducing immunological

Secondary response

Primary response
titer

Antibody

First exposure of antigen,

memory cells formed
Second exposure of antigen,
stimulation of memory cells

IgG

IgM

7 14
Time (days)
Fig. Primary and secondary immune responses.
MUNOLOGY 9.41
Vaccines
Typesof
several types ofvaccines that are currently and conventionally used. These include natural
There are
vaccine, live attenuated vaccine, inactivated vaccine, toxoid vaccine, polysaccharide vaccine,
live
recombinant antigen vaccine, live vector vaccine and a more recent DNA vaccine.
Types of common vaccines for humans.
Table

Type Examples of Vaccines used or Explored

vaccines Cowpox (vaccinia) virus used for immunization against smallpox
Natural live
(historic importance, now redundant); simian rotavirus is used
in vaccinations against gastroenteritis.
vaccines BCG; Sabin polio vaccine; MMR*; varicella- zoster vaccine (for
Iive attenuated
chickenpox), yellow fever vaccine
Inactivated vaccines Salk polio vaccine, rabies vaccine, pertussis vaccine
Toxoid vaccines Dipthcria vaecine;tetanus vaccine
Polysaccharide vaccines Hib vaccine; Vaccine for pneumococcal pneumonia;
meningococcal meningitis
Recombinant antigen vaccines HBs Ag vaccine
Vral vector vaccines An HIV vaccine containing enu. gag and, pol genes in canary
pox virus - vector (in clinical trials): animal model is
experimented with hepatitis B antigen; influenza antigens
inserted in vaccinia virus vector
Bacterial vecto vaccines Attenuated strain of Salmonella typhimurium (Ty2la) is explored
as vector for cholera vaccine
DNA vaccines Rabies vaccine; Influenza vaccine; HIV vaccine-are currently
being explored
Not:'Mumps-Measels-Rubella Vaccine;*Haemophilus influenza type b; ^Recombinant hapatitis B V-irus
NaturalLive Vaccines
These preparations include natural non-pathogernic organisms, but which still induce specific immunily.
The problems of these vaccines reside in their ability (albeit hidden) to mutate and convert into forms
that could be pathogenic to human hosts.
Live Attenuated Vaccines
Attenuation (Latin: attenuare-to weaken) refers to the weakening of the pathogenic bacteria or virus by
making it less virulent without altering its immunogenicity. Microorganisms are attenuated or weakened
so
that they do not cause any disease. Attenuation can be achieved by growing pathogenic microorganisms
(bacteria or virus) for a long period of time in a foreign host such as embryonated eggs or tissue culture
cells. The second and more famous attenuation was successfully carried out by two scientists Albert
Calmette and I.Camille Guerin in 1921 (the first being carried out by Louis Pasteur himselfin 1880 on
bacteria of fowl cholera). Calmette and Guerin grew the bovine strain of Mycobacterium tuberculosis
(called M. bovis) for several years on a medium containing increasing concentration of bile. This in
vitro cell culture of pathogenicM. bovis changedit into a less virulent and more suitable form ofbacilli
known as BCG (Bacillus Calmette-Guerin) a commonly used vaccine against tuberculosis. Attenuated
viruses are also used as vaccine, the vaccines of polio, yellow fever and measles viruses being the most
successful.
The process of attenuation involves growing microbes (bacteria or viruses) under abnormal in vitro
conditions, be it high bile concentration or passage through foreign cell/tissue such as embryonated
eggs. Theseabnormal environmental conditions select those mutant cells that are able to survive and
multiply under these conditions. These microbes are then harvested and used as vaccine.
 IMMUNOLOGY
9.42

Inactívated Vaccines s..

is to inactivate the whole pathogen and then
Another commonly used method for vaccine production chernicallwL

it for vaccination. The inactivation could be

achieved by modifying the antigen (pathogen)
killed bacteria or viruses are then usedi
formaldehyde treatment or physicallyby heat treatment. The
vaccines.
Toxoid Vaccines
on the production ot exotoxin. Exotoxins
The virulence or some pathogenic bacteria depends primarily pathogens
are cytotoxicmicrobial poisons (proteinic in nature) produced by living cells.Some bacterial symptoms
characteristic
such as diphtheria and tetanus bacilli produce exotoxins that induce several
associated with these infections.
so if these exotoxins can be
In other words, the harmful part of these bacteria is their exotoxin,
occur. These exotoxins are isolated and chemically modified
neutralized by the body the disease will not
is lost. These non-toxic yet
(usually with formaldehyde) so that their toxicity (but not immunogenicity)
immunogenic derivatives of exotoxin, or toxoids, are commonly used in vaccines.
Polysaccharide Vaccines
to system. Therefore, the surface
The surface of bacteria is the first thing that is expOsed the immune
antigens can serve as an excellent vaccine. Moreover,
some bacteria have resistance to phagocytic
example, the
invasion because these cells have capsular polysaccharides that resist phagocytosis (for The
polysaccharide capsules of S. pneumoniae, Haemophilus influenzae and Klebsiella pneumoniae).
components marks them
coating of these capsular polysaccharides with antibodies and/or complement way
paved the for synthesizing vaccines
for destruction by phagocytosis. These observations have
against bacterial-purified polysaccharides.
Recombinant Antigen Vaccines can
With the advent recombinant DNA technology, virtually any gene-encoding immunogenic protein
of
or even mammalian cells, using recombinant DNA
be introduced and expressed in yeast, bacterial endogenously is
technology. These cells are then cultured in the laboratory and the protein produced
are usually surface
harvested. The genes that are selected for making recombinant antigen vaccine
genes coding for various surface antigens have been
antigens (mostly glycoproteins). A number of' Yeasts have emerged as better
a
successfully cloned in bacterial, yeast and mammalian cells cultures.
process carbohydrate molecules on the protein
choice for making these surface antigens ns they add and
care is needed in comparison with
in their Golgi bodies in a manner more similar to mammals, and less
use is hepatitis B vaccine.
mammalian cells. One such vaccine approved for human
LiveVector Vaccines a
live vector vaccines, the desired gene coding for target antigens of the virulent pathogen is put into
In orally) to the vaccinee.
vector (atlenuated bacteria or virus) and then this vector is infected (or administereda
it serves as source of the antigen,
This vector slowly replicates inside the inoculated individual and a response. A number
strong immune
delivering a large amount ofantigen into the system and provoking
vectors are the vaccinia virus (the
oforganisms have been used as vectors. The most commonly used viral
smallpox vaccine virus), adenovirus and
canary pox virus (that infects cells butdocs not replicate in humans),

Viral Vector Vaccines

genetically engineered recombinant
The most commonly used viralvector is the vaccinia virus, though
viral vectors such as pox viruses or adenovirus
are also deliberated upon.
Genesthat have tihe potential to induce protective immunity (such
as coat antigens) are inserted into
a genome (about
attenuated live virus. Vaccinia, a commonly used virus has large, double-stranded genes can
a be
187,000 kB and approximately 200 genes). Because ofits large size, large number of
to cells. The
replaced with foreign (antigen) genes without causing any loss of their capacity infect host
or gene in vitro.
disadvantage is, because of the large size, it becomes difficult to manipulate insert the
MWUNOLOGY
9.43
simple but Jong method is used to prepare the vaccinia virus containing a
So a foreign gene. The
gene (that encodes the antigen) is first inserted into a plasmid vector. The plasmid vector is
foreign
transfected in tissue culture cells.
then
These tissue
culture cells are then infected with the vaccinia virus. Inside the tissue culture cells, the
vaccinia virus replicates in the cytosol where it undergo homologous recombination with plasmid
containing antigen gene. This recombination event causes the antigen gene (which had the vaccinia
promoter associated with it) to get transferred to
virus the vaccinia virus genome. This foreign gene is
usuallyinserted into the viral tk (thymidine kinase) gene inactivating it. The viruses that show
tk-negative phenotype are selected.
This genetically manipulated vaccinia virus expresses a high level of gene product (foreign antigen)
andthis serves as a potent source of immunogen inside the host. Effective live vaccines based on this
method have been developed for rabies in animals. Experimental viral vector vaccines generated by
this method include those that express influenza virus haemaglutinin, malaria protein, hepatitis B
surface antigen, herpes simplex virus proteins, among others, An HIV vaccine that carries HIV enu,
gas, protease, part of pol genes in canary pox are in clinical trials,
Bacterial Vector Vaccine
Like the live viral vectors, some attenuated bacterial strains have been engineered to carry genes of
virulent pathogens. The DNA encoding thc antigenic determinants is inserted into the attenuated
bacterial genome. The bacteria then express the antigen along with its own protein. The production
and expression of antigen by the bacterial vector inside the host body stimulates the immune system.
An attenuated strain of Salmonella typhimurium (Ty2la), the causative agent of food poisoning, is
being explored as a vector and is currently in human trials.
DNA Vaccine
The DNA vaccine (or more properly DNA-bascd vaccine) represents a new class of vaccines in which
there is a deliberate introduction of a DNA plasmid usually into the muscle cell of the recipient. The
plasmid contains a protein-coding gene (of antigen) that gets expressed in the cell, leading to both
humoral and cell-mediated immune responses. The plasmid DNA can be introduced into the muscle
cell either by infection or by bombarding the skin with DNA-coated gold particles with a fine airgun
(gene-gun).Currently, attempts are also underway to introduce DNA intothe nasal tissue via nasal
drops. It should be noted that once inside the cells of the recipient, the plasmid does not replicate, but
only expresses itself, that is, protein is produced. Usually bacterial plasmid is used and a gene coding
the antigen is inserted into the control of a mammalian promoter and this chimeric plasmid is then
introduced into the recipient. The recipient cell then expresses the foreign antigenic protein coded by
the introduced DNA in the host body. The immune system then responds to the antigen as to any
other antigen entering the body.

Immuno Deficiency Diseases

Immunodeficiencies occur when one or more components of the immune system is defective. Most of
immunodeficiency diseases are inherited.
Acquired immunodeficiencies are caused due to malnutrition or by agents, such as drugs or irradiation
that damage lymphocytes,or infection with measles or HIV. Immunodeficiencies may be primary and
secondary. Primary immuno deficiency diseases are disorders caused by a defect in one or more genes
involved in the development or function of the immune system. By contrast, a secondary immune
deficiency disease is one in which the immune system is compromised by factors from the outside,
such as viruses, radiation treatments or chemotherapy.
 9.44 IMMUNOLOGY

Example ofprimary immuno deficiency diseases:

Severe combined immunodeficiency (SCID)
SCID is a genetic disorder which is characterized by a very low number of circulating lymphocytes.
Both arms (B cells and T cels) of the adaptive immune system become non-functional. As such patients
make neither specific T-cell dependent antibody responses nor cell-mediated immune responses, and
thus cannot develop immunological menmory. Several different defects can lead to the SCID phenotype.
In X-linked SCID, which is the commonest form of SCID. T cells fail to develop because of defect in the
genes code for several cytokine receptors, including those for the interleukins IL-2, IL-4, IL-7, IL-9,
and IL-15. The autosomally inherited SCID occurs due to adenosine deaminase deficiency. Adenosine
deaminase catalyzes conversion of adenosine to inosine, and its deficiency results in accumnulation of
adenosine, which interferes with purine metabolism which result in an accumulation of nucleotide
metabolites that are particularly toxic to developing T cells.
Chediak-Higashi syndrome
Chediak-Higashi syndrome is an autosomal recessive disease. It is characterized by recurrent bacterial
1ntections, lack of skin and eye pigment. Phagocytes from patients with this immune defect contain
giant granules but do not have the ability to kill bacteria. With time, patients develop massive infiltrates
Ot lymphocytes and macrophages in the liver, spleen, and lymph nodes. The molecular basis of the
detect is amutation in a protein involved in the regulation of intracellular trafficking. The mutation
impairs the targeting of proteins to lysosomes.
DiGeorge syndrome
DiGeorge syndrome, or congenital thymic aplasia, is not hereditary but occurs sporadically and is
result of a deletion in chromosome 22. The syndrome is caused by defective migration of fetal neural
crest cells into the third and fourth pharyngeal pouch. DiGeorge syndrome in its most severe form is
the complete absence of a thymus. This developmental defect causes immunodeficiency along with
hypoparathyroidism, and congenital heart disease. The immune defect includes a profound depression
of Tcell numbers and absence of T-cell responses. Although B cells are present in normal numbers,
afected individuals do not produce antibody in response to immunization with specific antigens.
Failures of host defense mechanisms
The propagation of a pathogen depends on its ability to multiply in a host. Hence pathogens must
therefore grow without activating an immune response. The most successful pathogens persist either
because they do not elicit an immune response, or because they evade the response once it has occurred.
Pathogens have developed various strategies for avoiding destruction by the immune system. Some of
strategies are mentioned below:
1. One way in which a pathogenic agent can evade immune response is by altering its antigens. There
are three ways in which antigenic variation can occur. First, many pathogenic agents exist in a
,
wide variety of antigenic types. For example, there are many types of Streptococcus pneumoniae.
Each type differs from the others in the structure ofits polysaccharide capsule. The different types
are distinguished by serological tests and so are often known as serotypes. Infection with one
serotype of Streptococcus pneumoniae can lead to type-specific imnmunity, which protects against
reinfection with that type but not with adifferent serotype.
2. A second, more dynamic mechanism of antigenic variation arises due to antigenic drift and
antigenic shift. Antigenic drift is caused by point mutations in the genes encoding epitopes.
For example in influenza virus new variant arises with mutations in genes encoding the major
surface proteins hemagglutinin and neuraminidase. Thus individuals who were previously
infected with, and hence are immune to, the old variant are thus susceptible to the new variant.
Periodically influenza viruses also show an antigenic shift through reassortment of their
segmented genome with another influenza virus, changing their surface antigens radically.
Such antigenic shift variants are not recognized by individuals immune to influenza.
MMUNOLOGY

The third mechanism of antigenic 9.45

variation involves programmed
the pathogen. For example, African trypanosomes which
3. rearrangements in the DNA of
major surface antigen cause sleeping
the repeatedly withina single sickness in humans changes
a single type
of glycoprotein,
the variant--specific infected host. Thetrypanosome
glycoprotein (VSG), which elicits an is coated with
The trypanosome genome, however, contains antibody response.
distinct antigenic properties. Only one of about 1000 VSG genes,
each encoding a
protein with
he changed by gene rearrangement.these expressed at any one time. The VSG gene expressed
is
So, by having own system of gene
that can change the VSG protein produced, trypanosomestheir rearrangemernt
capable of generating many distinct keep one step ahead of an system
antibodies by gene rearrangement. immune
Some pathogens have evolved
4. specialized mechanisms
nochanisms. For instance, some bacteria to resist destruction by host defense
Lave evolved means of that are engulfed in the normal way by macrophages
avoiding destruction by these phagocytes.
tuberculosis is taken up by macrophages For example, Mycobacterium
rotecting itself from the bactericidal actions but prevents the fusion of the phagosome with the lysosome,
of the lysosomal contents.
Immunological Techniques
hen there is both sufficient Ag and Ab, the combination of Ag and Ab proceeds
formed which are insoluble in water until large aggregates
and precipitate. The extent to which a lattice forms depends on
the relative amount of Ag and Ab present.
Tattice formation, and precipitation are
the basis for several qualitative quantitative assays for Ag
a Ab.
These assaysare done in semisolid gels into which holes are cut and Ag lor Ab and diffusion occurs
for
until Ag and Ab are at equivalence and precipitate.
Immunodiffusion
Two types of immunodiffusion
Ag. to compare antigens, or to
reactions can be used to determine relative concentrations of Abs or
determine the relative purity of an Ag preparation. They are radial
immunodiffusion (the mancini method) and double immunodiffusion
sre carried out in a semisold (the ouchterlong method); both
a
medium such as agar. In radial immunodiffusion, an antigen sample is
nlaced in well and allowed to diffuse into agar containing a
suitable dilution of an antiserum. As the
antigen diffuses into agar, the region of equivalence is
established anda ring ofprecipitation, percipitin
ring forms around the well. The area of
the precipitin ring is proportional to the concentration of
antigen. By comparing the area of the precipitin ring with a
antigen sample can be obtained. In the ouchterlong, method both Ag
standard curve, the concentration ofthe
wells toward each other, thereby establishing a and Ab diffuse radially from
concentration gradient. As equivalence is reached, a
visible line of precipitation, a precipitin line forms.

Ag)

(Ag
(a)
Double Diffusion Ab diffusion Precipitin lines
(Ag Ag

(Ag
(6) Single radial diffusion
diffusion -Precipitin ring
Ab -Containing
agar
 IMMUNOOGY
9.46

Trough containng
Ab
Ag application point
diffusion Precipitin
(c) Immuno
electrophoresis arcs
Electrophoretically
separated proteins

Ab containing agar

Rocket-Immuno
electrophoresis Precipitin
(d)
electrophoresis rockets

(Ag) (Ag) (Ag

(e)
Ag) Ab containing Precipitin
gel rockets
Crossed transfer 2nd
Immuno dimension
electro electro
phoresis phoresis

(+)
Ag application
lst dimension point
clectrophoresis
Fig. Diagrammatic representation of immunoprecipitation techniques in agar
Immunoelectrophoresis Combines Electrophoresis and Double Immunodiffusion
In immunoelectrophoresis, a mixture of proteins containing the antigen are first separated bv aras
gel electrophoresis.Antibody is then allowed to diffuse through the gel from a through cut in the ga
parallel to the direction of electrophoresis. Precipitin arcs are then formed where antibody meets
antigen at equivalence. A disadvantage of this technique is the relatively poor resolution of antisen
mixtures using agar gel electrophoresis, but it can be useful for detecting precipitating antibodies
Clinically, the technique is carried out on samples of patient's serum, or spinal
fluid to detect abnormalities in concentrations of antigens/or the presenceconcentrated urine
of abnormal proteins relative
to normal control samples analysed at the same time.
Rocket immunoelectrophoresis is an adaption of single radial immunodiffusion
electrophoretic migration of antigen from wells cut in antibody containing and involves
gel. At equivalence, rocket
shaped precipitin lines are formed, the area under which is proportional to
The technique can be used to determine antigen concentrations the antigen concentration.
protein levels) by reference to a standard curve as
in unknown samples (e.g. serum
in RID, or to investigate immunochemical
relationships between different samples if these are placed in adjacent
wells close together.
In crossed immunoelectrophoresis, proteins are first separated agar gel electrophoresis, after
which they are electrophoresed into an antibody containing gel by
first electrophoresis. The technique can be used for analysis serum
at right angles to the direction of the
of proteins.
RIA and ELISA:
The presence of Ab to a particular Ag in the serum of a patient can
radioimmunoassay (RIA) or enzyme linked immuno-absorbent assavs be determined using very sensitive
particular value in demonstrating Ab to Ags to infectious agents, e.g. (ELISA). Such assays are
o

virus, bacteria ete.

MMUNOLOGY
9.47
Radioimmunoassay
present time. one ofthe most common uses of solid scintillation counting is inn certain radioimmunoassay
the
RIA)procedures,
At The RIAtechnique, developed
measure trace amounts of any in 1950s by S.A. Bersoni and R.S. Yalow, is a tracer procedure
Lsed to substance with antigenic properties. Depending on the specific
activity of the labeled compound, quantities of antigenthat are as low as 10 picograms (i.e., 10 millionths of
microgram) may be detected. Because many different kinds of
a chemical substances are antigenic (i.e. they
an immune response), they can be reliably quantitated using of
can elicit the RIA procedure. The sensitivity
RIA technique equals or surpasses all chromatographic and spectrophotometric methods currently in
the has been especially
use. The technique useful for assaying hormones.
Inthe
RIA technique, a sample containing the Ag to be quantified is mixed with a second sample
containing redioisotopically labelled Ag of very high specificactivity. To the mixture is added antiserum
containing antibody previously prepared against limiting
the antigen. The amount of Ab aadded must, bewhich
so that antigen is always in excess. The resulting Ag-Ab reaction produces complex in the
amounttof radioisotope present is inversly proportional to quantity of unlabeled antigen in
the original
the sample being ssayed. In the final stage of RIA, the Ag-Ab complex is separated from free
Ag by

precipitation. The redioisotopes used are H, 1251. The first RIA described was for insulin.
Labeled AgS Unlabeled AgS Ags (in antiserum)

Mixture

Ag-Ab complex

Precipitate Complex
and determine cpm
Fig. Radioimmunoassay

Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay, commonly known as ELISA (or EIA), is similar in principle to
RIA but depends on an enzyme rather than a radioactive label. An enzyme conjugated with an antibody
reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a
chromogenic substrate. A number of enzymes have been employed for ELISA, including alkaline
phosphatase, horseradish peroxidase, and B-galactosidase. These assays approach the sensitivity of
RIAs and have the advantage of being safer and less costly.
Variants of ELISA:Anumber of variations of ELISAhave been developed, allowing qualitative detection
or quantitative measurement of either antigen or antibody. Each type of ELISA can be used qualitatively to
detect the presence of antibody or antigen. Alternatively, a standard curve based on known concentrations
of antibody or antigen is prepared, from which the unknown concentration of a sample can be determined.
 IMMUNOLOGY
9.48
can or quantitatively determined with an indirect ELISA., Serum or
Indirect ELISA: Antibody be detected
d well
antibody (Ab,) is added to an antigen-coated microtiterpresence
Some other sample containing primary away, the
to well. After any free Ab, is washed
allowed to react with the antigen attached the an enzyme conjugated secondary anti-1sotype antibo
antibody bound to the antigen is detected by adding Ab, then is washed away, and a subStrate for the enzvme
(Ab,), which binds to the primary antibody. Any
free spectrophotometrio
amount of coloredreaction product that forms is measured by speCialized
is added. The wells of a 96-well plate in seconds.
measure the absorbance of all the
plate readers, which can human
Indirect ELISA is the method of choice to detect the presence of serum antibodies against envelope and
of AIDS. In this assay, recombinant
immunodeficiency virus (HIV), the causative agent infected with
Core proteins ofHIVare adsorbed
as solid-phase antigens to microtiter wells. Individuals
on these viral proteins. Generally, serum antibodies to
HIV will produce serum antibodies to epitopes
HIV can be detected by indirect ELISA within 6 weeks ofinfection.
or measured by a sandwich ELISA. In this technique, the antibody
Sandwich ELISA: Antigen can be detected containing antigen is added and allowed
(rather than the antigen) is immobilized a microtiter well. A sample
on
to react with the immobilized antibody Aferthe well is washed,a second enzyme-linked
antibody specific for
a
different epitope on the antigen is added and allowed to react with the bound
antigen. After any free second
colored reaction product is measured.
antibody is removed by washing, substrate is added. and the
Competitive ELISA: Another variation of measuring amounts of antigen is competitive ELISA. In
a antigen. "The antigen
this technique, antibody is first incubated in solution with sample containing more antigen present in the
antibody mixture is then added to an antigen-coated microtiter well. The of an enzyme
sample, the less free antibody wil1l be available to bind to the antigen-coated well. Additioncan be used to
conjugated secondary antibody (Ab,) specific for the isotype as of the primary antibody
an
determine the amount of primary antibody bound to the well in indirect ELISA.

(a)
wAsh wash vash

Antigen Add specfc Add enyne Add subsrate (S)

coated well antibody to be conjugated and measure
mased Secondary color
antibody
(6)

wash WAsh wash

Antbody Add antlzen Add enzyrie Add ubstrate

coated well to be mAsured conjugted and measure
secondary antbody color

wash waxh
Incubate
ancibody with
antigen to be Add Ag-Ab Add entyme Add sutbstrate
measured mbxture to conjugated and mcasure
ancigen-coated welI secondary color
antibody
Fig, Variations in theenzyme-linked immunosorbent assay (ELISA)technique allow determination of
antibody or antigen. Each asaay can be used qualitatively, or quantitatively by comparison with standard
curves prepared with known concentration of antibody or antigen. (a) whereas antigen can be determined
with a sandwich ELISA (b) competitive ELISA (c) In the competitive ELISA, which is an inhibition-type
assay, the concentration ofantigen is inversely proportional to the color produced. Antibody can be determined
with an indirect ELISA.
MMUNOLOGY
9.49

Cancer
Malignant Growth
which have
escaped normal control regulating its growth and division,
Cells
cells which invade adjacent tissues and may producing a clone of dividing
daughter
which undergo rapid, abnormal
interfere with their activities are called cancer
cells
cells. The and uncontrolled growth at the cost of remaining cells
neoplastic cells. These proliferating cells form a mass of undifferentiated cells. This cells mass
arecalled
called tumour or neoplasm or cancerous growth.
is are characterized by three properties
Cancer cells
diminished or unrestricted control of growth
() invasion of local
tissues and
spread of metastasis, to other parts of the body.
(m)
Tumours
are classified as benign or
malignant. Their study is known as oncology.
The tumours
A. Benign Tumours
Tumours that remains localized in a specific area at the site of origin, forming a single mass enclosed in
a capsule, are called benign tumours. Usuallythese are not fatal.
Benign tumours can be removed effectively with surgery. They usually donot reappear.
B. Malignant Tumours
The malignant tumours are cancerous. Their cells are poorly differentiated with high rate of mitosis,
nuclear polymorphism and abnormal mitosis. Malignant tumours contain rapidly growing, actively mon8
olls that tend to invade other tissues. The movements of malignant cells from their original site to other
body areas is called 'metastasis'. When metastasizing cells gain access to blood vessel, the circulating
blood carries those cells throughout the body. When they lodge at distant body site, the cells continue to
rOw, forming new tumours. The rate of cell production in malignant cancerous tissue exceeds the rate of
ell death and leads to tumour formation. These outgrow their blood supply and the rapidly increasing
number of cells compress the small blood vessels. This is called ischaemic necrosis. Many malignant
tumours grow progressively and relentlessly. The growth rate fluctuates greatly in different tumours.
Cancers or maligmant tumours are uncapsulated and invasive. These show excessive growth and infiltration
into adjacent tissues. Most cancer cells show tiny pseudopods indicative of their invasive nature. The
malignant tumours are usually poorly differentiated. Their cells may exhibit different degree of resemnblance
to the tissue cells from which these have originated. The partial loss of differentiation is termed as dysplasia
and complete loss, so that the tumour no longer resembles the tissue of its origin is called anaplasia.
Cancer cells can enter the cell cycle repeatedly and in this way they are immortal.
Cancer can be caused by physical, chemical or biogenic agents. There agents damage or alter DNA, SO
that canceris truly a disease of the genome. Initiation and promotion are well-established phenomenon
in chemicalcareinogenesis. Initiation probably damage DNA, but the mechanism involved in promotion
remain to be clarified. Phorbolesters are much studied promoting agents, they activate protein kinase
C, which exerts a veriety of effects.

Characteristics of Cancer Cells

) Cancer cells have abnormally enlarged nuclei containing abnormal number of chromosomes.
(ü) Nucleolus displays hypertrophy.
(üi) Endoplasmic reticulum and ribosomes are more abundant as compared to normal cells.
(io) Cytoskeleton undergoes deploymerization and disintegration, and the cells become independent
and their movements uncoordinated.
(U) Cytoskeleton
undergoes deploymerization and disintegration, and the cells become independent
and their movements uncoordinated.
(vi) Adhesiveness decreases.
 IMMUNOLOGY
9.50
ofproteolvtic enzymswhich act on inert
serum protein, plasminogen and fores
(n) Secrete large amounts
removes many extrinsic proteinsfrom cell surface and signals the
cell to divide
plasmin which
(vii) Negative surface charge increase. a
is depressed in tumour cells. Despite normal
(4) Warburg (1920) suggested that aerobic respiration normalglucose requirement and produee
OXYgen uptake, cancer cells tend to use several times the on
a the liver, which must use ATP to ct
lactic acid under aerobic conditions. This places burden
rid of lactate.
(*) They have ability to induce local blood vessel formation (angiogenesis). lt is a
required to bring
not contained in capsule.
nutrients and oxygen to a cancerous tumour whose growth is
causes neighbouring blood vessels to branch into the
Cancer cells release a growth factor. that
cancerous tissue.
(i) They have ability to spread (metastasis)
(xii) They loss cell cycle control.
are other
(au) Heritability, transplantability, genetic mutability and loss of density dependent inhibition
characteristics of cancer cells.
Types of Cancers
groups :

About 200 diferent types of can cers have been identified. These are broadly classified into following
() Carcinoma : It includes tumours of brain, breast, skin, cervical region. These are derived from
epithelial tissue, originating either from ectoderm or endoderm. These occur as solid tumours.
are the
located in the nervous tissue on body surface or in the associated glands. Carcinoma
commonest cancer and may be
-Squamous carcinoma--cancer of epithelial tissue
-Adeno Carcinoma-cancer of glands.
(iü) Sarcoma : Sarcoma are cancers (tumours) of connective tissue, cartilage, bone and muscles which
are mesodermal origin. Sarcoma are named according to the tissue in which these are formed :
Fibrosarcoma- Sarcoma ofibrous connective tissue
Liposarcoma--Cancer of Adiposetissue
Chondrosarcoma--Cancer of cartilagecells (chondroblast cells)
Osteosarcoma--Cancer of Bone
Leiomyo Sarcoma--Cancer of smooth muscles.
(iüi) Lymphoma : Lymph nodes, bone marrow, liver & spleen produce excessive lymphocytes. Hodgkin's
disease and mycosis fungoides' are examples oflymphoma.
(iv) Leukaemias : These include the neoplastic proliferation of leukocytes (white blood corpuscles
WBC) precursors in the bone marrow and are described as mycloproliferative disorders :These lead
to increase in the number ofleukocytes that infiltrate intovarious other tissues and organs, producing
enlargement of organs or tumours and can be
Monocytic leukaemia
Megakaryocytic leukaemia
Erythraemic myclosis
Promyelocytic leukaemia
» Lymphoblastic leukaemia
Myeloblastic leukaemia in white blood cell cancer
Melanomas -Cancer of pigment cells
Gilomas--Cancer ofglial cells of brain
Glomangioma--A benign tumour that develops from an arteriovenous glomus (cluster of
blood
cells) of the skin.
IMMUNOLOGY 9.31

Causes of Cancer
process of conversion of a normal cell to malignancy is called carcinogenesis and the agents which
The
causethis. are called carcinogens. Two major classes of genes contribute to causing cancer. Oncogenes
activiated to cause cancer. Tumour suppressors, which normally hold mitosis in check, must
must be
inactivated or removed to eleminate control of the cell cycle and initiate cancer.
be

Oncogenes
Oncogenes are genes that normally activate cell division in specific situations. When an oncogene activates
cell division in
the wrong time or place, cancer may result. Oncogenes are not alien tothe cell, they are
mal,essential genes that have undergone a mutation. In its normal non-mutated state, itis a proto
oncogerne, a gene that cantransformed into on oncogene.
be

The src
gene is now known to be an oncogene. It is possible that the src gene is under the control ofa
on.
riral promotor that has turned it
he oncogenes most frequently involved in human cancers belong to the rasgene family. An alteration
only a single nucleotide pair is sufficient to convert a normally functioning ras proto-oncogene to an
oncogene. The ras K oncogene is found in about 25% of lung cancers, 50% of colon cancer and 90% ot
ancreatic cancer. The ras N oncogene is associated with leukaemias and lymphomas and both ras
oncogenes are frequently found in thyroid cancers.
Activation of proto-oncogenes to oncogenes is achieved by at least five mechanisms (promnoter and
enhancer insertion, chromosomal translocation, gene amplification, point mutation).

Tumour Suppressor Genes

Tumour suppressor gene is so called because it prevents cancer from occurring. Researchers have
identified about a half a dozen tumour suppressor genes. When these gene malfunction, a tumour
results. Tumour suppressor genes are now recognized as key players in the genesis of cancer. The effect
of oncogenes on cell growth has been compared to putting one's foot on the accelerator of
an automobile,
whereas the action of tumour suppressor genes resembles taking one's foot off the brakes. Important
tumour suppressor genes include RBI and P 53, both of which are nuclear phosphoproteins and probably
affect the transcription of genes involved in regulating events in the cell cycle. A genetic model for
colorectal cancer invokes the interplay of tumour suppressor genes and the K-ras oncogene. Mutations
in mismatch repair genes have been found to be associated with hereditary nonpolyposis colon cancer,
and loss of responsiveness to the growth inhibitory effect of TGF B appears important at least in
development of this type of tumour. A number of cancer susceptibility genes have beon isolated. They
include RB 1, P 53, BRCA 1, BRCA 2. Tumour progression reflects an instability of the tumour genome,
probably due toat least in part to defects in DNA repair systems, activation to additional oncogenes, and
inactivation to additional tumour suppressor genes.
The RB tumour suppressor gene was discovered by Alfred Knudson. RB tumour-suppressor gene
has now been found to malfunction in cancers of breast, prostate, bladder. Loss of RB tumour suppressor
gene through chromosomal deletion is particularly frequent in a type of lung cancer called small cell
lung carcinoma.
It is now known that signaling proteins often regulate the transcription of genes whose products are
needed for cell division, and this appears to be true of the RB protein. When a particular factor attaches
to a receptor, the RB protein is activated. An active RB protein turns off the expression of a proto
oncogene, whose product initiates cell division. Translocations of genes may occur as result of mistakes
during mitosis, or they may be caused by carcinogens or viral infection.
 IMMUNOLOGY
9.52
more frcquently mutated in human
tumour-suppressor gene is callcd 53, a gene that is an a transcription
Another major gene. boen found that the P 53 protein acts
any other known It has
CAncers than on the exDression of agene called WAF or Clp 1.The producta
1

factor and as such is involved in turning P 53 protein. When

gene is a cell cycle inhibitor. A number of cell proteins combine with the
of this to tell whether it is activating P 53
or is being activated by P 53.
protein comines with P 53, it is hard
Thisintricate situation is now being unraveled.
gene is a top priority in biomedical research so far, the
Discovering the normal function of the P 53 genes, and in several
is cell's expression of other
normal form of gene is implicated in cellcycle control,
open the door to understanding many types of cancers.
metabolicprocesses. Understanding P 53 will
Carcinogens
cancer, other possible causes include
oncogenes and tumour-supperessors play a part in causing
wie
certain chemicals, nutrient deficiencies, and radiations.
in culture. Benzene is
Direct carcinogens cause cancer when thev are applied fbroblast cells growing
to
an example for this.
Procarcinogens are safe outside the body, but inside thev
are metabolized to produce intermediate
to
compounds that cause cancer. They include certain organicdyes. cigarettes, nitrites nitrates used
&
preserve processed meats.
Promotersare chemicals that make other carcinogens more powerful. They include alcohol, certain
hormones and various chemicals in cigrette smoke.
Polycyclic hydrocarbons produce cancer at the site of local application. Aromatic amines like 2
naphthylamine and benzidine cause cancer of urinary bladder. A20-dyes cause liver cancer and amino
fluorenes also cause cancer of liver and urinary bladder. Alkylating agents like mustard gas,
methylnitrosourea and nitrosamine and various inorganic compounds like asbestos fibres, arsenic,
berylium, nickel, cadmium, and chromates are all carcinogenic.
Radiations
X-ray, a, and y rays and ultraviolet light all induce tumours in man and animals. While passing
B

through the tissue, these release energy which alters various macromolecules of the cell including
nucleic acids. This causes increased rate of mutations in the irradiated cells.
Diagnosis And Treatment of Cancer
The traditional medical methods for treating cancers are quite direct and aggressive. Malignant neoplasm
are removed surgically, burned with radiation, or poisoned chemically.
Chemotherapy uses toxic chemicals to kill rapidly dividing cells. Many of these drugs are altered forms
of
thenucleotides found in DNA. Magnetic Resonance Imaging (MRI) uses an elcctromagnet to detect
tumours. Position Emission Tomography (PET) can reveal the altered metabolic activity of cancersand
differentiate them from normal tissue. Therefore, the early diagnosis is the most important factor.
Depending on the site, diagnosis is made by various means of which biopsy is the most important one.
Other are use of devices for visualization of hollow organs, roentgenography including computerized
axial tomography (CAT), mammography, ultrasound, cytology such as Papanicolaou test', and palpation
for lumps. Some of these techniques and devices will demonstrate an increase in the size or change in
the shape of an organ, but such alterations may be due to either a benign or malignant growth. Colon
cancer can be spotted early with an at home hemoccult (hidden blood) test. Most women have a yearly
Papsmear to detect Cervical cancer.
MMUNOLOGY
9.53

of SSomeImportant
Tumour Viruses
List
Members
Class
viruses
A) DNA Polyoma virus, SV 40 virus, human papilloma
1. Papoviruses virus (e.g. HPV-16)
2. Adenoviruses Adenoviruses 12, 18 & 31
3. Herpes virus Epstein-Barr-virus, Herpes Simplex virus type-2 (HSV-2), Luck's
disease virus, Marck's disease virus
hepadnovirus
4. hepatitis B virus
(B) RNA viruses
1. Retroviruses type C Murine sarcoma and Leukaemia viruses, Avian sarcoma
&Leukaemia viruses
Human T-ccll leukaemia viruses I & II
2. Retrovirus type B Mouse mammary tumour virus
Some Differences Between Oncogenes and Tumour Suppressor Genes
Oncogenes Tumour Suppressor Genes
1. Mutation in one of the two alleles Mutations in both alleles or a mutation in one
sufficient for activity, act dominant to followed by a loss of or reduction to
wild type homozygosity in the second
2. "Gain of function of a protein that Loss of function of a protein
signals cells division
3. Mutation arises in somatic tissues, not Mutation present in germ cell (can be
inherited inherited) or somatic cells
4. Some tissue preference Strong tissue preference (e.g. effect of RB
II gene in retina)
New
Some and DevelopingCancer Therapies
Therapy Example or Underlying Principle
1. Anti-angiogenesis Angiostatin, endostatin & other molecules
2. Antisense therapy (measurement of Directed against key oncogenes
protease specific antigen (PSA) is useful
to monitor prostatic cancer recurrence
or persistance after surgency). Increase
of PSA shows metastases.
3 Apotosis promoters Develop agents that stimulate apoptosis selectively
in tumour cells.
4. Cell cycle inhibitors Develop agents capable of selectively inhibiting key
steps of the cell cycle of cancer cells.
5. Differentiation stimulators All trans-retinoic
acid in the treatment of acute
promyelocytic leukaemia.
6
Gene Therapy Replace key tumour suppressor proteins (e.g. p 53,
p RB) or mutant repair enzyme.
 9.54
MMUNOLOGY
Therapy
Example or Underlying Principle
7. Farnesy/transferase inhibitors RASproteins are attached to surface membranes
via farmesyl, inhibiting its attachment inhibits
8. Glycosylation inhibitors function of ras proteins
Alter patterns ofsugar on cellssurface glycoproteins
9. Metastasis Inhibitors in order to inhibit metastasis.
Inhibitors of various proteases (e.g. metallo
10. Monoclonal Antibodies proteases) may inhibit metastasis
Directed against oncogene encoded receptors or
adhesion molecules on the surface of tumour cells
11. Prevention against the receptor
Aspirin to help prevent colorectal cancer. Anti
smoking measures.
12. Protein kinase Inhibitors
Many tumour cells have elevated amounts of
13.Ribozymes various protein kinases involved in signaling etc.
Can degrade RNA molecules (e.g.specific m RNAs)
14. Telomerase inhibitors that are relatively unique to tumour cells.
May act on tumour cells in which telomerase is
active
15. Vaccines
Inject peptides from relatively "tumour- specifie
proteins to boost the T-cell Immune response

OBJECTIVE TYPE QUESTIONS

1, Which of the following cells secretes IL-1? 6. By humoral immunity, the antibodies can
(a) Monocytes (6) Leucocyte destroy the target cell by which of the following
(c) Stromalcells (d) Both (a) & (b) mechanism?
2. The characteristic features of multiple (a) Classicalcomplement pathway
myeloma is (6) Agglutination
(a) Paraproteinemia (b) Proteinuria (c) Opsinization
(c) Lytic bone lesions (d) Allof these (d) All of these
3. Immunoglobulin's are secreted by which cells 7. How many heavychain domains are present
(a) plasma cells inthe lgE molecule?
(6) T-lymphocytes (a) 5 (b) 2
(c) T-cytotoxic cells (c) 4 (d) 1

(d) None 8. Halflife of lgG molecule is

4. Which method is used to detect the HIV (a) 6 days (b) 20 days
ANTIBODY? (c) 41 days (d) 10days
(a) ELISA (6) RIA 9. Which ofthe following lg fixes most cells and
(c) Immunofluorence (d) (a) and (c) basophils?.
5. The features of neutrophils is/are (a) IgM (6) lgA
(a) Active glycolysis (c) IgE
(6) Contain certain unique enzymes 10. Which of the following additional unit is
(c) Fight against, viruses present in the lgA molecule?
(a) 'S' piece (b) J' piece
(d) (a) and (b)
(c) 'S and J' piece (d) None
MMUNOLoGY

The producer the cells lymphokines

of 9.55
11. 20. T-helper cells carry which determinants on
(a) T-cytotoxic cells
the cellsurface?
(6) B-cell (a) CD8
Tcells when stimulated by antigens (6) CD12
(c) CD4 (d) A, B
(d) Both (a) & (b)
21. In which type of electrophoresis the precipi
Which type of variation is individually
12.
specific to each Ig molecule? tation arcs are formed?
(a) Isotopic (6) Idiotypic Where the Ag-Ab molecules are 1:1%.
(c) Allotypic (d) None (a) PAGE
The immunity which shows delayed type of
(6) SDS-PAGE
13.
hypersensitivity is (c) Immunoelectrophoresis
(a) Cell mediated immunity (d) All of these
(6) Humoral immunity
22. The use of Western blot technique is used to
(c) Antibody immunity find the
(d) ADCC (a) Size of specific proteins
14. In which condition IgM level in blood is in (6) Quantitative specific protein molecules
creased considerably with a monoclonal peak?
(c) Shape of the specific proteins
(a) Waldenstroms macroglobulinemia
(d) Both (a) & (b)
(6) Heavy chain disease
(c) Bence-Jones proteinuria
23. Which proteins are helpful to determine the
shape and flexibility of the red blood cells?
(d) Both (a) and (b)
(a) Spectrin (6) Ankyrin
15. How many sub units does the component C,
(c) Actin (d) Both (a) and (6)
has?
(a) 3 (b) 2 24. Universal donor blood group is
(c) 4 (d) 12 (a) A (6) AB
16. On which of the following loci the MHC genes (c) B (d) 0*
are present? 25. GaNAc is a
(a) A, C, B, D and DR (a) Immunodominant sugar
(6) A, C, B, D and DR (6) Immunosupress sugar
(c) B, A, C, B and DR
(c) Tumor marken
(d) Both (a) and (b) (d) None of theso
17. The functions of IFN's is
26. What amount of T- Lymphocytes and B-cells
(a) Tand B cell proliferation are present in peripheral blood?
(6) Proliferation of macrophages
(a) T-80% B-20% (6) T-20% B-80%o
(c) Antiviral
(c) T60% B-40% (d) T-5% B-95%
(d) Both (b) & (c)
27. The receptor used by the B-Cell is
18. IgG molecule is divided into which sequence
(a) Immunoglobulin (6) Protein
of the heavy chain constant region?
(a) Amino acid sequence (c)
Nucleic acids Lipids (cd)

(6) Globin sequence 28. Antigen processing and presentation are

(c) Protein sequencer processes that occur
(d) Heavy chain sequence (a) Outside the cell
19. Indirect ELISA technique is used to detect (6) Within the cell
(a) Antibody (6) DNA (c) Within receptors
(c) RNA (d) Both (a) and (b) (d) Within plasmamembrane
 IMMUNOLOGY
9.56
component of
29. Antigen processing and presentation are 36. B cells are an essential
processes result in (a) Innate immunity
(a) Proteolysis (6) Adaptive immunity
(6) Association of the fragments with MHC (c) Both A and B
molecules (d) None of these
(c)
Expression of the peptide-MHC molecules of
37. I. The path leading to the association
(d) All of these protein fragments with MHC molecules
30. Dendritic cells, ingest antigens
by differs for class Iand class II MHC,
(a) Pinocytosis II, MHCclass II molecules present degradation
(6) Phagocytosis products derived from intracellular
(c) Both (a) and (b) (endogenous) proteins in the cytosol.
(d) Doesnot ingest antigens III. MHC class I molecules present
31. The antigens that can polyclonally activate fragments derived from extracellular
an
(exogenous) proteins that are located in
T cells
intracellular compartment.
(a) Polyantigens
Which of the above statements are true about
(6) Polyclonal antigens MHC ?
(c) Supreantigens (6)II, III
(a) I, II
(d) Superantigens (c) I (d) I, II, III
32. An individual does not want functional T cells 38. I. Binding of peptide within the MHC is not
in the periphery because of covalent, there are many factors while
(a) Damages of healthy, normal tissues help stabilize the immunological synapse.
(6) Damage of healthy, normal organs II. Class I MHC molecules are composed of
(c) Damage of healthy, normal receptors two polypeptide chains, a long a chain and
a short B chain called Bl-microglobulin.
(d) Damage of healthy, normal MHCs
III. The a chain of MHC class I has four
33. If a self reactive B cell does get to the
regions.
periphery, it will not be activated due to
Which of the above statements are true?
(a) Lack of T cell help
(a) I, II (6) II, III
(6) Lack of cytotoxic cells help
(c) I,
III (d) I, II,III
(c) Lack of macropnages help
39. The CD3 complex is composed of
(d) None of these
(a) One Y, one S, two and 2 chains
34. A given T cell is restricted to recognize a
(6) Two Y, one 8, two e and 2 chains
peptide antigen only when it is bound to
(c) One Y, two 8, two and2 chains
(a) SelfMHC
(6) Anti-MHC (d) One Y, one ö, one e and 2 chains
(c) Auto-MHC 40. Immature cells are produced in the bone
B

marrow with an exception of

(d) None of these
(a) Horse (6) Rabbit
35. CD4 binding to Class II MCH,which ensures
(c) Drosophila (d) Pig
that
(a) Tecells can interact with target cells 41. When the B-cell fails in the maturation
process, it will die by a mechanism called
(6) CD2binding to LFA-3
(a) Senescence (6) Phagocytosis
(c) LFA-1 binding to ICAM-1
(e) Pinocytosis (d) Apoptosis
(d) The cells only interact with APCs
MUNOLoGY
9.57
Immunoglobulin which is expressed on Bl cell
CANCER
is (6) IgA 48. Blood cancer is excess production of
a) IgG (d) IgD
leucocytes. It is
(c) IgM Or
B
cells recognizetheir cognate antigen in
43. I.
processed form Blood cancer is also known as
its
(a) Leucopenia (6) Leucoderma
1
Tcells recognize their cognate antigen in
a native form (c) Leucocytosis (d) Leukemia
I1. critical difference between B cells and
A 49. Bones become fragile in
Tcells is how each lymphocyte recognizes (a) Gout (6) Osteoporosis
its antigen. (c) Arthritis (d) None of these
Among the above statements how many
V.
50. Which is not cancer
of
themare true about B and T- cell? (a) Leukaemia (6) Glaucoma
la) I,II (b) II,III
(c) Carcinoma (d) Sarcoma
(c) I (d) III
51. Alteration of which genes leads to cancer
4. Lipids and nucleic acids are antigenic when (a) Cell proliferation gene (proto-oncogenes)
combined with
(a) ATP and NADPH
(6) Tumor suppressor gene
(c) Tumor virus gene
(6) Magnesium and calcium
(c) Proteins and polysaccharides (d) All of these

(d) Enzymes
52. It causes cancer of vagina
(a) Cadmium oxide (6) Diethylsibestrol
A5, A class of antigens which cause non-specific
activation of T-cells is (c) Afflatoxin (d) Vinyl chloride
(a)
Superantigen (6) Allergen 53. Which one of the following not a
is property
(c)
Epitope (d) Tolerogen of cancerous cells whereas the remaining
46. Medulla is surrounded by cortex
except three are
(a) Spleen (a) They compete with normal cells for vital
(6) Adenoids
(c) Hilum (d) Peyer's patches
nutrients
(6) They do not remain confined
47. The Humoral Immune Response
is the aspect in the area
of immunity that is mediated by of formation
(c) They divide in an
(a) Antibodies (6) Antigen uncontrolled manner
(c) Dendritic cells (d) They show contact inhibition
(d) Macrophages

ANSWERS
1.(d) 2. (d) 3.(a) 4. (a) 5.(d) 6. (d) 7. (a) 8. (b)
11.(c)
9. (c) 10. (c)
12. (b) 13. (a) 14. (a) 15. (a) 16. (a) 17.(d) 18. (a) 19. (a)
21.(c) 22. (a)
20. (c)
23. (d) 24. (d) 25. (a) 26.(a) 27. (a) 28. (6) 29. (d) 30. (c)
31.(d) 32. (a) 33. (a) 34. (a) 35. (c) 36. (b) 37. (c) 38.(d) 39. (a) 40. (b)
41. (d) 42. (c) 43. (d) 44. (c) 45.(a) 46. (c) 47. (a) 48. (d) 49. (b) 50. (6)
51.(a) 52. (b) 53. (d)
 9.58 IMMUNOLOGY

EXPLANATIONS
1. They are polypeptide growth factors secreted 11. T-cellswhen stimulated by Ag's, liberate
by leucocytes. More than 1°5 interleukins are many soluble substances called aytokines/
isolated. Lymphokines
IL-lis secreted by Monocyte, Jeukocyte 12, The idiotypic determinants are located in the
2. Multiple myeloma (plasma variable parts ofAb. (i.e., Ag reorganization
cytoma)
Ig secreting cells are site).
transformed into
malignant cells, one done is enormously 13. When tuberculin is injected intradermally to a
proliferated. Thus Ig molecules
of the very patient with tuberculosis, an erythematous
same type are produced inducted lesion develops slowly reading it's
in large quantities.
3. Ig's are secreted by plasma cells maximum within 48-72 hours. This is called
belonging to
the B-cells of lymphocytes. delayed type hyper sensitivity. Hyper-sensitivity
4. ELISA-Indirect ELISA method is the over reaction of the immune system,
is used to detect
Antibody (Ab). often resulting in unwanted tissue destruction.
Method: Ag from HN is coated in the wells of
14. In this condition IgM level in blood is increased
a multiwell plate. Patient's serum
is added & considerably with monoclonal peak. This is
incubated. If it contains the Ab; it is fixed and due to malignant proliferation of IgM clones.
the wells are washed. Thisis to remove excess 15. Component C, has 3 subunits; they are Cip
Ab's in serum. Next a second Ab(Ab against C1,, & C,, which are stabilized by Ca*².C,, binds
human Ig) conjugated with HRP. Then colour with Ab.
reagent containing H,0, & diaminobenzidine 16. MHC genes are present on chromosome 6 and
is poured over. If brown colour developed, they are A, C, B, and DR loci. All these loci
D

means that the Ab was originally present in together contain more than 150 alleles.
the parent's serum. 17. IFN- a- secreted by leucocytes
6. Ag - Ab reaction leads to activation of Function -Proliferation of Macrophages
complement system, which destroys the foreign
cells. IFN - B - secreted by Fibroblast
IFN-y -T & NK (Natural killer)
The Ab'scan destroy the target cells by the
following mechanisms.
-
Function Antiviral differentiates cells
18. IgG again subdivided into IG, G, G, & G,
) Classical complement pathway
depending on the amino acid sequence of the
() Antigen dependent cell-mediated heavy chain constant region.
cytotoxicity 19. Indirect ELISA is useful to detect the small
(II) Agglutination quantities of Ab's in the blood
(I)Opsinization Ex :-Test for detection of HIVantibody
9. IgE fixes to mast cells & basophils : They are 21. Here electrophoretic separation is followed
cytophilic ab's and mediate allergy, by Ag-Ab reaction. The electrophoresis is
hypersensitivity and anaphylaxis. They have carried out first by applying the patient's
property to fix on mast cells and basophils. When S serum into the wells but out in the agar/
certain Ab's such as penicillin are injected few agarose gel.
times, IgE class Ab's are produced which anchor 26. T- 80% B-20%
on mast cells, when the same chemicals is
27. B cells and T cells recognize different substances
injected next time, the Ag's fix on such Ab's, as antigens and in a different form. The Bcell
causing most celldegranulation and release of uses cell surface-bound immunoglobulin as a
histamine and slow reacting substance.
receptor and the specificity of that receptor is
10. The J chain connects the dimens. The
the same as the immunoglobulin thatit is able
dimens are stabilized against proteolytic to secrete after activation.
enzymes by the secretary piece. This
secretary piece is produced in liver, nelches 28. Antigen processing and presentation are
to the intestinal mucosal cells, where it processes that Occur within a cell.
combines with IgA dimmer to form the 29. Antigen processing and presentation are
secretary IgA which is then released. processes result in fragmentation (proteolysis)
MAUNOLOGY
9.59
,
ofproteins, association of the fragments
with
activation by antigen interaction. B cells are
MHC molecules, and expression
of the an essential component of the adaptive
peptide-MHC molecules at the cell surface immune systen.
by the T cell
where they can be recognized 37. The path leading to the association of protein
receptor on a T cell. fragments with MHCmolecules differs for
an The three main types of antigen
presenting class I and class II MHC, MHC class I
B
and
ells are dendritic cells, macrophages
express class
molecules present degradation products
cells, although other cells, that derived from intracellular (endogenous)
II MHC molecules, (e.g., thymic epithelial proteins in the cytosol. MHC class II
cells) can act as antigen presenting cells in molecules present fragments derived from
some cases. Dendritic cells, which are found extracellular (exogenous) proteins that are
in skin and other tissues, ingest antigens by located in an intracellular compartment.
pinocytosis and transport antigens to the 38. I. Cell-cell interactions of the adaptive
lymph nodes and spleen. immune response are critically important
can
g1, Super antigens are antigens that in protection from pathogens, These
polyclonally activate T cells to produce large interactions are orchestrated by the
quantities of ytokines that can have immunological synapse whose primary
pathological effects. These antigens must be components are the T cell antigen receptor
presented to T cells in association with class (TCR) and the Major histocompatibility
II MHC molecules but the antigen does not complex (MHC) molecule. The major
need to be processed. function of the TCR is to recognize antigen
32. Functional T cells in the-periphery have to in the correct context of MHC and to
recognize foreign antigens associated with transmit an excitatory signal to the
self MHC, because APC or target cells interior of the cell. Since binding of peptide
present foreign antigen associated with self within the MHC is not covalent, there are
MHC. However, an individual does not need many factors while help stabilize the
functional T cells in the periphery that immunological synapse.
recognize antigen (self or foreign) associated II. Class I MHC molecules are composed of
with foreign MHC. An individual especially two polypeptide chains, a long a chain and
does not want functional T cells in the a short B chain called B2-microglobulin.
periphery that can recognize self antigens II. The a chain has four regions.
associated with self MHO because they could 39. The TCR is closely associated withagroup of
lead to damage of healthy, normal tissues. 5 proteins collectively called the CD3complex.
33. Since B cells are not MHC-restricted there is The CD3 complex is composed of one ly, one
no need for positive selection of B cells. 18, two 2e and 2 chains. All of the proteins of
However, negative selection (i.., elimination the CD3 complex are invariant and they do
of self-reactive clones) of B cells is required. not contribute to the specificity in any way.
This occurs during B cell development in the The CD 3 complex is necessary for cell surface
bone marrow. However, negative selection of expression of the TCR during T cell
B cells is not a critical as for T cells since, in development. In addition, the CD3 complex
most instances, cells require T cellhelp in
B

transduces activation signals to the cell

order to become activated. Thus, if a self following antigen interaction with the TCR.
reactive B cell does get to the periphery it 40. Immature B cells are produced in the bone
will not be activated due to lack of T cell help. marrow of most mammals. Rabbits are an
34. A given T cell is restricted to recognize a
exception; their B cells develop in the
peptide antigen only when it is bound to appendix-sacculus rotundus. After reaching
self-MHC molecules. the IgM+ immature stage in the bone marrow,
35. CD4 binding to Class II MCH,which ensures these immature B cells migrate to the spleen,
that Th cells only interact with APCs where they are called transitional B cells, and
36. The principal functions of B cells are to make some of these cells differentiate into mature
antibodies against antigens, perform the Blymphocytes.
role of antigen-presenting cells (APCs) and 41. When the B cell fails in any step of the
eventually develop into memory B cells after maturation process, it will die by a
 9.60
IMMUNOLOGY
of
mechanism called apoptosis, here called 46. The substance of a lymph node consists
clonal deletion. B cells are continuously lymphoid follicles in the outer portion called
produced in the bone marrow. When B cell the "cortex", which contains the lymphoid
receptors on the surface of the cell matches follicles, and an inner portion called "medulla
the detected antigens present in the body, the which is surrounded by the cortex on all sides
B cell proliferates and secretes a free form of except for a portion known as the "hilum»
those receptors (antibodies) with identical The hilum presents as a depression on the
binding sites as the ones on the original cell surface of the lymph node, which makes the
surface. After activation, the cell proliferates otherwise spherical or ovoid lymph node bean.
and B memory cells would form to recognise Shaped. The efferent lymph vessel directly
the same antigen. This information would emerges from the lymph node here. The
then be used asa part ofthe adaptive immune arteries and veins supplying the lymph node
system for a more efficient and more with blood enter and exit through the hilum.
powerful immune response for future 47. The Humoral Immune Response (HIR) is the
encounters with that antigen. aspect of immunity that is mediated by
42. B-l cells express IgM in greater quantities than secreted antibodies (as opposed to cell
IgG receptors show polyspecificity, meaning mediated immunity, which involves
that they have low affinities for many different T- lymphocytes) produced in the cells of the
antigens, but have a preference for other B-lymphocyte lineage (B cell).
immunoglobulins, self antigens and common 48. Leukemia is abnormal increase in number of
bacterial polysaccharides. B-l cells are present leucocytes which are all immature.
in low numbers in the lymph nodes and spleen
49. Osteoporosis is a reduction in bone tissue
and are instead found predominantly in the mass causing weakness of skeletal strength,
peritoneal and pleural cavities.
thus, bones become fragile. It results from
43. I. cells recognize their cognate antigen in
B

excessive resorption of calcium and

its native form. They recognize free phosphorus fromn the bone.
(soluble) antigen in the blood or lymph 50. Glaucoma is not cancer but an abnormality
using their BCR or membrane bound
of eye in which there is an increase in fluid
immunoglobulin. pressure which leads to
(intra-occular)
II In contrast, T cells recognize their cognate permanent damage of optic nerve fibres and
antigen inprocessed form, as a peptide
a
consequently irreversible loss of eye-sight.
fragment presented by an antigen presenting 51. In human beings cancer is caused by extra
cell's MHCmolecule to the T cell receptor,
activation of certain genes called proto
II. criticaldifference between B cells and T
A
oncogenes into cellular oncogenes. Proto
cells is how each lymphocyte recognizes oncogenes typically encode proteins receptors,
its antigen. signal transduction proteins, and transcription
44. Antigens are usually proteins or factors. Mutations in these genes tend to relax
polysaccharides. This includes parts (coats, control mechanisms and accelerate cell
capsules, cell walls, flagella, fimbrae, and division, leading to cell proliferation that is
toxins) of bacteria, viruses, and other characteristic of cancer. Tumor suppressor
microorganisms. Lipids and nucleic acids are gene normally keeps mitosis in check &
antigenic only when combined with proteins prevent cancer from occuring.
and polysaccharides. Non-microbial exogenous 52. Cancer is caused by physical, chemical and
(non-self) antigens can include pollen, egg biological factors. These factors are called
white, and proteins from transplanted tissues carcinogens. Some of the carcinogens and
and organs or on the surface of transfused blood their target tissues are
cells.Vaccines are examples of immunogenic Prostate gland
Cadmium oxide
antigens intentionally administered to induce
Diethylstibestrol (DES) Vagina
acquiredimmunity in the recipient.
Afflatoxin Liver
45. Super antigen - A class of antigens which
cause non-specific activation of T-cells Vinyl chloride (VC) Liver
resulting in polyclonal T cell activation and a
53. Contact Inhibition is property ofnormal cell
massive cytokine release. not of cancer cells.