SEMESTER III PAPER IX - IMMUNOLOGY & MMUNOTECHNOLOGY

UNIT - II

Antigens - properties, Epitopes, haptens, adjuvant, cross reactivity. Antibodies - properties,

structure (primary & secondary) and isotypes. Diversity and specificity. Anti antibodies.

UNIT - III

Serology - Introduction and classification of antigens and antibody reactions - Agglutination and

precipitation reaction. Strength of antigen and antibody bindings - affinity & avidity.

Complement pathway and complement fixation reaction. Immunofluorscence RIA, RAST,

ELISA and Flowcytometry. Monoclonal antibodies & its applications. (Hybridoma technique)

UNIT - IV

MHC antigens - types and functions. Regulation and response of immune system. Response of

B Cell to antigens. T cell products. Immunity to infectious diseases - Viral, bacterial and

protozoan . Hyper sensitivity reactions .

UNIT V

Transplantation immunology - Tissue transplantation and grafting . Mechanism of graft

acceptance and rejection. HLA typing Tumor immunology. Immunodeficiency diseases and auto

immunity. Vaccines - Types and vaccination methods.

UNIT I

Historical background and scope of immunology, Immunohaematology -ABO and Rh factor.

Cells and organs of immune system. Non immunological defence mechanism - Barriers,

Phagocytosis, inflammation, fever . Types of immunity - HI and CMI.

1. Explain the historical background and scope of immunology.

1718 - Lady Mary Wortley Montagu, the wife of the British ambassador to Constantinople, observed the positive effects of variolation on the native population and had the technique performed on her own children.

1798 - First demonstration of vaccination smallpox vaccination (Edward Jenner)

1837 - First description of the role of microbes in putrefaction and fermentation (Theodore Schwann)

1838 - Confirmation of the role of yeast in fermentation of sugar to alcohol (Charles Cagniard-Latour)

1840 - First "modern" proposal of the germ theory of disease (Jakob Henle)

1850 - Demonstration of the contagious nature of puerperal fever (childbed fever) (Ignaz Semmelweis)

1857-1870 - Confirmation of the role of microbes in fermentation (Louis Pasteur)

1862 - phagocytosis (Ernst Haeckel)

1867 - First aseptic practice in surgery using carbolic acid (Joseph Lister)

1876 - First demonstration that microbes can cause disease-anthrax (Robert Koch)

1877 - Mast cells (Paul Ehrlich)

1878 - Confirmation and popularization of the germ theory of disease (Louis Pasteur)

1880 - 1881 -Theory that bacterial virulence could be attenuated by culture in vitro and used as vaccines. Proposed that live attenuated microbes produced immunity by depleting host of vital trace nutrients. Used to make chicken cholera and anthrax "vaccines" (Louis Pasteur)

1883 - 1905 - Cellular theory of immunity via phagocytosis by macrophages and microphages (polymorhonuclear leukocytes) (Elie Metchnikoff)

1885 - Introduction of concept of a "therapeutic vaccination". First report of a live "attenuated" vaccine for rabies (Louis Pasteur).

1888 - Identification of bacterial toxins (diphtheria bacillus) (Pierre Roux and Alexandre Yersin)

1888 - Bactericidal action of blood (George Nuttall)

1890 - Demonstration of antibody activity against diphtheria and tetanus toxins. Beginning of humoral theory of immunity. (Emil von Behring) and (Shibasaburo Kitasato)

1891 - Demonstration of cutaneous (delayed type) hypersensitivity (Robert Koch)

1893 - Use of live bacteria and bacterial lysates to treat tumors-"Coley's Toxins" (William B. Coley)

1894 - Bacteriolysis (Richard Pfeiffer)

1896 - An antibacterial, heat-labile serum component (complement) is described (Jules Bordet)

1900 - Antibody formation theory (Paul Ehrlich)

1901 - blood groups (Karl Landsteiner)

1902 - Immediate hypersensitivity anaphylaxis (Paul Portier) and (Charles Richet)

1903 - Intermediate hypersensitivity, the "Arthus reaction" (Maurice Arthus)

1903 - Opsonization

1905 - "Serum sickness" allergy (Clemens von Pirquet and (Bela Schick)

1909 - Paul Ehrlich proposes "immune surveillance" hypothesis of tumor recognition and eradication

1911 - 2nd demonstration of filterable agent that caused tumors (Peyton Rous)

1917 - hapten (Karl Landsteiner)

1921 - Cutaneous allergic reactions (Otto Prausnitz and Heinz Kstner)

1924 - Reticuloendothelial system

1938 - Antigen-Antibody binding hypothesis (John Marrack)

1940 - Identification of the Rh antigens (Karl Landsteiner and Alexander Weiner)

1942 - Anaphylaxis (Karl Landsteiner and Merill Chase)

1942 - Adjuvants (Jules Freund and Katherine McDermott)

1944 - hypothesis of allograft rejection

1945 - Coombs Test aka antiglobulin test (AGT)

1946 - identification of mouse MHC (H2) by George Snell and Peter A. Gorer

1948 - antibody production in plasma B cells

1949 - growth of polio virus in tissue culture, neutralization with immune sera, and demonstration of attenuation of neurovirulence with repetitive passage (John Enders) and (Thomas Weller) and (Frederick Robbins)

1949 - immunological tolerance hypothesis

1951 - vaccine against yellow fever

1953 - Graft-versus-host disease

1953 - immunological tolerance hypothesis

1957 - Clonal selection theory (Frank Macfarlane Burnet)

1957 - Discovery of interferon by Alick Isaacs and Jean Lindenmann

1958-1962 - Discovery of human leukocyte antigens (Jean Dausset and others)

1959-1962 - Discovery of antibody structure (independently elucidated by Gerald Edelman and Rodney Porter)

1959 - Discovery of lymphocyte circulation (James Gowans)

1960 - Discovery of lymphocyte "blastogenic transformation" and proliferation in response to mitogenic lectins-phytohemagglutinin (PHA) (Peter Nowell)

1961-1962 Discovery of thymus involvement in cellular immunity (Jacques Miller)

1961- Demonstration that glucocorticoids inhibit PHA-induced lymphocyte proliferation (Peter Nowell)

1963 - Development of the plaque assay for the enumeration of antibody-forming cells in vitro (Niels Jerne) (Albert Nordin)

1964-1968 T and B cell cooperation in immune response

1965 - Discovery of the first lymphocyte mitogenic activity, "blastogenic factor" (Shinpei Kamakura) and (Louis Lowenstein) (J. Gordon) and (L.D. MacLean)

1965 - Discovery of "immune interferon" (gamma interferon) (E.F. Wheelock)

1965 - Secretory immunoglobulins

1967 - Identification of IgE as the reaginic antibody (Kimishige Ishizaka)

1968 - Passenger leukocytes identified as significant immunogens in allograft rejection (William L. Elkins and Ronald D. Guttmann)

1969 - The lymphocyte cytolysis Cr51 release assay (Theodore Brunner) and (Jean-Charles Cerottini)

1971 - Peter Perlmann and Eva Engvall at Stockholm University invented ELISA

1972 - Structure of the antibody molecule

1973 - Dendritic Cells first described by Ralph M. Steinman

1974 - T-cell restriction to major histocompatibility complex (Rolf Zinkernagel and (Peter C. Doherty)

1975 - Generation of the first monoclonal antibodies (Georges Khler) and (Csar Milstein)

1976 - Identification of somatic recombination of immunoglobulin genes (Susumu Tonegawa)

1979 - Generation of the first monoclonal T cells (Kendall A. Smith)

1980-1983 - Discovery and characterization of the first interleukins, 1 and 2 IL-1 IL-2 (Kendall A. Smith)

1981 - Discovery of the IL-2 receptor IL2R (Kendall A. Smith)

1983 - Discovery of the T cell antigen receptor TCR (Ellis Reinherz) (Philippa Marrack) and (John Kappler) (James Allison)

1983 - Discovery of HIV (Luc Montagnier)

1984 - The first single cell analysis of lymphocyte proliferation (Doreen Cantrell) and (Kendall A. Smith)

1985-1987 - Identification of genes for the T cell receptor

1986 - Hepatitis B vaccine produced by genetic engineering

1986 - Th1 vs Th2 model of T helper cell function (Timothy Mosmann)

1988 - Discovery of biochemical initiators of T-cell activation: CD4- and CD8-p56lck complexes (Christopher E. Rudd)

1990 - Gene therapy for SCID

1991 - Role of peptide for MHC Class II structure ([Scheherazade Sadegh-Nasseri] & [Ronald N. Germain])

1992- Discovery of transitional B cells (David Allman & Michael Cancro)

1994 - 'Danger' model of immunological tolerance (Polly Matzinger)

1995 - Regulatory T cells (Shimon Sakaguchi)

1995 - First dendritic cell vaccine trial reported by Mukherji et al.

1996-1998 - Identification of Toll-like receptors

2001 - Discovery of FOXP3 - the gene directing regulatory T cell development

2005 - Development of human papillomavirus vaccine (Ian Frazer)

Scope of immunology

Of the four major causes of death injury, infection, degenerative

disease and cancer only the fi rst two regularly kill their victims

before child-bearing age, which means that they are a potential source

of lost genes. Therefore any mechanism that reduces their effects has

tremendous survival value, and we see this in the processes of, respectively,

healing and immunity.

Immunity is concerned with the recognition and disposal of foreign

or non-self material that enters the body (represented by red arrows

in the fi gure), usually in the form of life-threatening infectious microorganisms

but sometimes, unfortunately, in the shape of a life-saving

kidney graft. Resistance to infection may be innate (i.e. inborn and

unchanging) or acquired as the result of an adaptive immune

response (centre).

Immunology is the study of the organs, cells and molecules responsible

for this recognition and disposal (the immune system), of how

they respond and interact, of the consequences desirable (top) or

otherwise (bottom) of their activity, and of the ways in which they

can be advantageously increased or reduced.

By far the most important type of foreign material that needs to be

recognized and disposed of is the microorganisms capable of causing

infectious disease and, strictly speaking, immunity begins at the point

when they enter the body. But it must be remembered that the fi rst

line of defence is to keep them out, and a variety of external defences have evolved for this purpose. Whether these are part of the immune system is a purely semantic question, but an immunologist is certainly expected to know about them.

Non-self A widely used term in immunology, covering everything that

is detectably different from an animals own constituents. Infectious

microorganisms, together with cells, organs or other materials from

another animal, are the most important non-self substances from an

immunological viewpoint, but drugs and even normal foods, which

are, of course, non-self too, can sometimes give rise to immunity.

Detection of non-self material is carried out by a range of receptor

molecules (see Figs 1115).

Infection Parasitic viruses, bacteria, protozoa, worms or fungi that

attempt to gain access to the body or its surfaces are probably the chief raison dtre of the immune system. Higher animals with a damaged or defi cient immune system frequently succumb to infections that normal animals overcome.

External defences The presence of intact skin on the outside and

mucous membranes lining the hollow viscera is in itself a powerful

barrier against entry of potentially infectious organisms. In addition,

there are numerous antimicrobial (mainly antibacterial) secretions in

the skin and mucous surfaces; these include lysozyme (also found in

tears), lactoferrin, defensins and peroxidases. More specialized

defences include the extreme acidity of the stomach (about pH 2), the

mucus and upwardly beating cilia of the bronchial tree, and specialized surfactant proteins that recognize and clump bacteria that reach the lung alveoli. Successful microorganisms usually have cunning ways of breaching or evading these defences.

Innate resistance Organisms that enter the body (shown in the fi gure as dots or rods) are often eliminated within minutes or hours by inborn, ever-present mechanisms, while others (the rods in the fi gure) can avoid this and survive, and may cause disease unless they are dealt with by adaptive immunity (see below). These mechanisms have evolved to dispose of pathogens (e.g. bacteria, viruses) that if

unchecked can cause disease. Harmless microorganisms are usually

ignored by the innate immune system. Innate immunity also plays a

vital role in initiating the adaptive immune response.

Adaptive immune response The development or augmentation of

defence mechanisms in response to a particular (specifi c) stimulus,

e.g. an infectious organism. It can result in elimination of the

microorganism and recovery from disease, and often leaves the host

with specifi c memory, enabling it to respond more effectively on

reinfection with the same microorganism, a condition called acquired

resistance. Since the body has no prior way of knowing which microorganisms are and which are not harmless, all foreign material is usually responded to as if it were harmful, including relatively inoffensive pollens, etc.

Vaccination A method of stimulating the adaptive immune response

and generating memory and acquired resistance without suffering the

full effects of the disease. The name comes from vaccinia, or cowpox,

used by Jenner to protect against smallpox.

Grafting Cells or organs from another individual usually survive

innate resistance mechanisms but are attacked by the adaptive immune response, leading to rejection.

Autoimmunity The bodys own (self) cells and molecules do not

normally stimulate its adaptive immune responses because of a variety of special mechanisms that ensure a state of self-tolerance, but in certain circumstances they do stimulate a response and the bodys own structures are attacked as if they were foreign, a condition called autoimmunity or autoimmune disease.

Hypersensitivity Sometimes the result of specifi c memory is that re-exposure to the same stimulus, as well as or instead of eliminating

the stimulus, has unpleasant or damaging effects on the bodys own

tissues. This is called hypersensitivity; examples are allergies such as hay fever and some forms of kidney disease.

Immunosuppression Autoimmunity, hypersensitivity and, above all,

graft rejection sometimes necessitate the suppression of adaptive

immune responses by drugs or other means.

Immunohaematology -ABO and Rh factor.

Immunohematology, more commonly known as blood banking is a branch of hematology which studies antigen-antibody reactions and analogous phenomena as they relate to the pathogenesis and clinical manifestations of blood disorders. A person employed in this field is referred to as an immunohematologist. Their day to day duties include blood typing, cross-matching and antibody identification

CELLS OF THE IMMUNE RESPONSE

Immune responsive cells can be divided into five groups based on i) the presence of specific surface components and ii) function: B-cells (B lymphocytes), T-cells (T lymphocytes), Accessory cells (Macrophages and other antigen-presenting cells), Killer cells (NK and K cells), and Mast cells. Some of the properties of each group are listed below.

Cell group

Surface components

Function

B-lymphocytes

Surface immunoglobulin (Ag recognition)

Immunoglobulin Fc receptor

Class II Major Histocompatability Complex (MHC) molecule (Ag presentation)

Direct antigen recognition

Differentiation into antibody-producing plasma cells

Antigen presentation within Class II MHC

T-lymphocytes

CD3 molecule

T-cell receptor (TCR, Ag recognition)

Involved in both humoral and cell-mediated responses

Helper T-cells (TH)

CD4 molecule

Recognizes antigen presented within Class II MHC

Promotes differentiation of B-cells and cytotoxic T-cells

Activates macrophages

Suppressor T-cells (TS)

CD8 molecule

Downregulates the activities of other cells

Cytotoxic T-cells (CTL)

CD8 molecule

Recognizes antigen presented within Class I MHC

Kills cells expressing appropriate antigen

Accessory cells

Variable

Phagocytosis and cell killing

Macrophages

Immunoglobulin Fc receptor

Complement component C3b receptor

Class II MHC molecule

Bind Fc portion of immunoglobulin (enhances phagocytosis)

Bind complement component C3b (enhances phagocytosis)

Antigen presentation within Class II MHC

Secrete IL-1 (macrokine) promoting T-cell differentiation and proliferation

Can be "activated" by T-cell lymphokines

Dendritic cells

Class II MHC molecule

Antigen presentation within Class II MHC

Polymorphonuclear cells (PMNs)

Immunoglobulin Fc receptor

Complement component C3b receptor

Bind Fc portion of immunoglobulin (enhances phagocytosis)

Bind complement component C3b (enhances phagocytosis)

Killer cells

Variable

Direct cell killing

NK cells

Unknown

Kills variety of target cells (e.g. tumor cells, virus-infected cells, transplanted cells)

K cells

Immunoglobulin Fc receptor

Bind Fc portion of immunoglobulin

Kills antibody-coated target cells (antibody-dependent cell-mediated cytotoxicity, ADCC)

Mast cells

High affinity IgE Fc receptors

Bind IgE and initiate allergic responses by release of histamine

LYMPHOID TISSUES

Primary

Secondary

(Responsible for maturation of Ag-reactive cells)

(Sites for Ag contact and response)

Thymus(T-cell maturation)

Bone marrow

Lymph nodes

Spleen

(T-cell maturation)

(B-cell maturation)

(Expansion of lymphatic system, separate from blood circulation. Deep cortex harbors mostly T-cells, superficial cortex harbors mostly B-cells)

(Similar to lymph nodes but part of blood circulation. Collects blood-borne Ags)

. NON IMMUNOLOGICAL DEFENSE MECHANISM

Body Defenses

Physical barriers: skin & epithelial linings & cilia

Chemical: acids, mucous & lysozymes

Immune defenses internal

Innate, non-specific, immediate response (min/hrs)

Acquired attack a specific pathogen (antigen)

Steps in Immune defense

Detect invader/foreign cells

Communicate alarm & recruit immune cells

Suppress or destroy invader

Microbial killing by phagocytes:

Phagocytosis involves two steps namely attachment and ingestion. Following attachment of the organism,

invagination of the phagocyte results in the formation of a phagosome. Some capsulated bacteria dont attach to the

phagocyte, but they can still be phagocytosed if they are coated with opsonins such as IgG and complement

component (C3b). The engulfed bacteria are held inside a vacuole called phagosome. The formation of phagosome

triggers respiratory bursts and fusion of lysosome with phagosome to form phagolysosome

The phagocytes appear to kill engulfed bacteria by two pathways, oxygen independent pathway and oxygen dependent pathway. The microbicidal mechanisms of the respiratory burst are termed oxygen dependent and phagolysosome formations are termed oxygen independent.Oxygen dependent mechanism involves catalytic conversion of molecular oxygen to oxyhalide free radicals, which are highly reactive oxidizing agents. The phagocyte oxidase present in the plasma membrane and phagolysosome reduce oxygen into reactive oxygen intermediates such as superoxide radicals. Superoxide is converted to H2O2,which is used by enzyme myeloperoxidase to convert unreactive halide ions to reactive hypohalous acids that are toxic to bacteria.Oxygen independent mechanism involves release of lysosomal contents into phagolysosomes. The content of lysosome includes lactoferrin, cathepsin

G, lysozyme and defensins etc.In addition to the phagocyte oxidase system, macrophages have free-radical generating system, namely inducible nitric oxide synthase. This cytosolic enzyme is absent in resting macrophages but can be induced in response to bacterial lipopolysaccharides and IFN-. This enzyme catalyses the conversion of arginine to citrulline, and in the process releases nitric oxide gas. Nitric oxide may then combine with H2O2 or superoxide to form highly reactive peroxynitrite radicals that kill the microbes.

Dendritic cells:

These cells are derived from myeloid progenitor in the bone marrow and are morphologically identified by spiny

membranous projection on their surfaces. Immature dendritic cells are located in epithelia of skin, gastrointestinal

tract and respiratory tract and are called langerhan cells. They express low levels of MHC proteins on their surface

and their main function is to capture and transport protein antigen to the draining lymph node. During their migration

to the lymph node, dendritic cells mature into excellent antigen presenting cells (APC). Mature dendritic cells reside

in the T cell area (paracortex) of the lymph node. Here, they are referred as interdigitating dendritic cells. These

cells are distinct from the dendritic cells that occur in the germinal centers of lymphoid follicles (follicular dendritic

cells) in lymph node, spleen and MALT. The follicular dendritic cells are not derived from the bone marrow and their

role is to present antigen-antibody complex and complement products to B cell.

Lymphoid system:

Lymphoid organs are stationed throughout the body and are concerned with the growth, development and

deployment of lymphocytes. These structurally and functionally diverse lymphoid organs and tissues are

interconnected by the blood vessels and lymphatic vessels through which lymphocytes circulate. The organs

involved in specific as well as non-specific immunity are classified as primary (central) lymphoid organs and

secondary (peripheral) lymphoid organs. The blood and lymphatic vessels that carry lymphocytes to and from the

other structures can also be considered lymphoid organs. Recently, it has become accepted that the liver is also a

hematopoietic organ, giving rise to all leukocyte lineages.

PRIMARY LYMPHOID ORGANS:

Also called central lymphoid organs, these are responsible for synthesis and maturation of immunocompetant cells.

These include the bone marrow and the thymus.

BONE MARROW:

All the cells of the immune system are initially derived from the bone marrow through a process called

hematopoiesis. During foetal development hematopoiesis occurs initially in yolk sac and para-aortic mesenchyme

and later in the liver and spleen. This function is taken over gradually by the bone marrow. During hematopoiesis,

bone marrow-derived stem cells differentiate into either mature cells or into precursors of cells that migrate out of

the bone marrow to continue their maturation in thymus.

The bone marrow produces B cells, natural killer cells, granulocytes and immature thymocytes, in addition to red

blood cells and platelets. It is both a primary and secondary lymphoid organ. The proliferation and maturation of

precursor cells in the bone marrow are stimulated by cytokines, many of which are called colony stimulating factors

(CSFs). The bone marrow also contains antibody secreting plasma cells, which have migrated from the peripheral

lymphoid tissue.

THYMUS:

The thymus is a gland located in the anterior mediastinum just above the heart, which reaches its greatest size just

prior to birth, then atrophies with age. This lymphoepithelial organ develops from ectoderm derived from the third

branchial cleft and endoderm of the third branchial pouch.

Immature lymphocytes begin to accumulate in the thymus of human embryos at about 90-100 days after

fertilization. Initially most of these immature lymphocytes have come from the yolk sac and fetal liver rather than the

bone marrow. Cells from the bone marrow, later migrate to the thymus as precursors and develop into mature

peripheral T cells. Once the immature lymphocytes have passed the blood-thymus barrier they are called

thymocytes. Mature T cells migrate from the thymus to secondary lymphoid organs such as lymph node, Peyer's

patches and spleen.

Ultimately the thymus becomes an encapsulated and consists of many lobes, each divided into an outer cortical

region and an inner medulla. The cortex contains mostly immature thymocytes, some of which mature and migrate

to the medulla, where they learn to discriminate between self and non-self during foetal development and for a short

time after birth. T cells leave the medulla to enter the peripheral blood circulation, through which they are

transported to the secondary lymphoid organs. About 98% of all T cells die in the thymus.

The greatest rate of T cell production occurs before puberty. After puberty, the thymus shrinks and the production of

new T cells in the adult thymus drops away. Children with no development of thymus suffer from DiGeorge

syndrome that is characterized by deficiency in T cell development but normal numbers of B cells.

PERIPHERAL LYMPHOID ORGANS:

While primary lymphoid organs are concerned with production and maturation of lymphoid cells, the secondary or

peripheral lymphoid organs are sites where the lymphocytes localise, recognise foreign antigen and mount

response against it. These include the lymph nodes, spleen, tonsils, adenoids, appendix, and clumps of lymphoid

tissue in the small intestine known as Peyer's patches. They trap and concentrate foreign substances, and they are

the main sites of production of antibodies.

Some lymphoid organs are capsulated such as lymph node and spleen while others are non-capsulated, which

include mostly mucosa-associated lymphoid tissue (MALT).

LYMPH NODE:

Clusters of lymph nodes are strategically placed in the neck, axillae, groin, mediastinum and abdominal cavity,

where they filter antigens from the interstitial tissue fluid and the lymph during its passage from the periphery to the

thoracic duct. The key lymph nodes are the axillary lymph nodes, the inguinal lymph nodes, the mesenteric lymph

nodes and the cervical lymph nodes. Lymph nodes that protect the skin are termed somatic nodes, while deep

lymph nodes protecting the respiratory, digestive and genitourinary tracts are termed visceral nodes.

Each lymph node is surrounded by a fibrous capsule that is pierced by numerous afferent lymphatics that drain

lymph into marginal sinus. The lymph flows through the medullary sinus and leaves through efferent lymphatics.

Each lymph node is divided into an outer cortex, inner medulla and intervening paracortical region. The cortex is

also referred as B cell area, which mainly consists of B cells. The cortex is a high traffic zone where recirculating Tand

B lymphocytes enter from the blood. Aggregates of cells called follicles are present in the cortex, which in turn

may have central areas called germinal centers. Follicles without germinal centers are called primary follicles and

those with germinal centers are called secondary follicles. Primary follicles are rich in mature but resting B cells.

Germinal centers develop in response to antigenic stimulation and consist of follicular dendritic cells and reactive B

cells. The medulla contains a mixture of B cells, T cells, plasma cells and macrophages. The medulla consists of

medullary cords that lead to the medullary sinus. The cords are populated by plasma cells and macrophages.

Between these two zones, lie the paracotex (T cell area) that contains T lymphocytes, dendritic cells and

mononuclear phagocytes. Most of the T cells (70%) located there are CD4+ helper cells.

SPLEEN:

Situated in the left upper quadrant of the abdomen and weighing about 150 grams, spleen is the largest single

lymphoid organ in the body. It has a dense fibrous capsule with muscular trabeculae extending inward to subdivide

the spleen into lobules. It filters blood and is the major organ in which antibodies are synthesized and released into

circulation. In addition to capturing foreign antigens from the blood that passes through the spleen, migratory

macrophages and dendritic cells also bring antigens to the spleen via the bloodstream. Persons lacking spleen (eg.

splenectomy) are highly susceptible to infections with capsulated bacteria such as pneumococci and meningococci.

Spleen is the major site for phagocytosis of antibody coated bacteria and destruction of aged RBCs. It is supplied by splenic artery, which pierces the capsule at hilum and divides into smaller branches that are surrounded by fibrous trabeculae. The spleen is composed of two types of tissue, the red pulp and the white pulp. The red pulp contains vascular sinusoids, large number

of erythrocytes, resident macrophages, dendritic cells, granulocytes, few plasma cells and lymphocytes. It is the site where aged platelets and erythrocytes are destroyed. The white pulp contains the lymphoid tissue clustered around small arterioles and is known as a periarteriolar

lymphoid sheath (PALS). PALS contain mainly T lymphocytes, about 75% of which are CD4+ helper T cells. Attached to this are lymphoid follicles, some of which contain germinal centers. Follicles and germinal center predominantly contain B cells. The PALS and follicles are surrounded by rim of lymphocytes and macrophages,called marginal zone. Marginal zone is composed of macrophages, B cells, and CD4+ helper T cells. The arterioles end in vascular sinusoids in the red pulp, which in turn end in venules that drain into splenic vein. Antigens and

lymphocytes enter the spleen through vascular sinusoids. Activation of B cells occurs at the juncton between follicle and PALS. Activated B cells then migrate to the germinal centers or into the red pulp.

MUCOSA ASSOCIATED LYMPHOID TISSUE (MALT):

Approximately >50% of lymphoid tissue in the body is found associated with the mucosal system. MALT is

composed of gut-associated lymphoid tissues (GALT) lining the intestinal tract, bronchus-associated lymphoid

tissue (BALT) lining the respiratory tract, and lymphoid tissue lining the genitourinary tract. The respiratory,

alimentary and genitourinary tracts are guarded by subepithelial accumulations of lymphoid tissue that are not

covered by connective tissue capsule. They may occur as diffuse collections of lymphocytes, plasma cells and

phagocytes throughout the lung and lamina propria of intestine or as clearly organised tissue with well-formed

lymphoid follicles. The well-formed follicles include the tonsils (lingual, palatine and pharyngeal), Peyers patches in

the intestine and appendix. The major function of these organs is to provide local immunity by way of sIgA (also

IgE) production. Diffuse accumulations of lymphoid tissue are seen in the lamina propria of the intestinal wall. The

intestinal epithelium overlying the Peyer's patches is specialized to allow the transport of antigens into the lymphoid

tissue. This function is carried out by cuboidal absorptive epithelial cells termed "M" cells, so called because they

have numerous microfolds on their luminal surface. M cells endocytose, transport and present antigens to

subepithelial lymphoid cells.

Majority of intra-epithelial lymphocytes are T cells, and most often CD8+ lymphocytes. The intestinal lamina propria

contains CD4+ lymphocytes, large number of B cells, plasma cells, macrophages, dendritic cells, eosinophils and

mast cells. Peyers patches contain both B cells and CD4+ T cells.

LYMPHOCYTES:

Lymphocytes are stem cells derived cells that mature either in the bone marrow or thymus. Together, the thymus

and marrow bone marrow produce approximately 109 mature lymphocytes each day and the adult human body

contains approximately 1012 lymphocytes. Lymphocytes comprise 20-40% (1000 - 4000 cells/l) of all leukocytes.

The lymphocytes are distributed to blood, lymph and lymphoid organs.

Typically, lymphocyte is small, round, cell with diameter of 5-10m, spherical nucleus, densely compacted nuclear

chromatin and scanty cytoplasm. Though the cytoplasm contains mitochondria and ribosomes, other organelles are

not detectable. Such mature but resting lymphocytes are known as nave cells. They are mitotically inactive but

when stimulated can undergo cell division. Nave lymphocytes have a short life span and die in few days after

leaving bone marrow or thymus unless they are stimulated. Once the lymphocyte is activated (stimulated), they

become large (10-12m), have more cytoplasm and more organelles. Activated lymphocytes may undergo several

successive rounds of cell division over a period of several days. Some of the progeny cells revert to the resting

stage and become memory cells, but can survive for several years in the absence of any antigenic stimulus.

There are three major types of lymphocyte, B lymphocyte, T lymphocyte and NK cells. Different lymphocytes are

identified by certain protein markers on their surface called "cluster of differentiation" or "CD" system. One marker

that all leukocytes have in common is CD45. The presence of the markers can be detected using specific

monoclonal antibodies.

Distribution of lymphocytes

Approximate %

Tissue T-Cells B-Cells NK Cells

Peripheral blood 70-80 10-15 10-15

Bone marrow 5-10 80-90 5-10

Thymus 99 small pre-B cell > immature B cell > mature B cell.

Distribution:

They account for 5-15% of lymphocytes (250 cells/l) in circulation and 80-90% in bone marrow, 20-30% in lymph

node and 50-60% in spleen.

Surface markers:

The most important surface marker on the surface of mature B cell is the surface immunoglobulin. The surface

immunoglobulins are of IgM and IgD type. A B cell will have approximately 109 immunoglubulins of single specificity

on its surface. Markers/Receptors on B cells are Surface Immunoglobulin (IgM and IgD), CD40, B7, ICAM-1, LFA-1,

MHC II, CD32 (Ig Fc receptor), CD35 (Receptor for complement component) and additional markers that distinguish

B cells such as CD19, CD20, CD21 and CD22.

Demonstration of B cells:

EAC (Erythrocyte Amboceptor Complement) Rosettes: When sheep RBCs coated with antibody and treated with

complement and B cells, a rosette is formed due to the presence of complement receptor on B cells. B cells can be

demonstrated by immunofluorescence with fluorescent-labelled monoclonal antibodies against surface markers

such as surface immunoglobulin.

On stimulation by pokeweed mitogen, they undergo blast transformation.

Functions of B-cells:

Direct antigen recognition and Antigen presentation

B cells may differentiate into plasma cells (which secrete large amounts of antibodies) or into memory B cells.

Memory cells can survive 20 years or more.

Plasma cells:

These are the effector cells of the B-cell lineage and are specialised in secreting immunoglobulins. When activated

B cells divide, some of its progeny become memory cells and the reminder become immunoglobulin-secreting

plasma cells. Plasma cells are oval or egg shaped, have eccentrically placed nuclei, have abundant cytoplasm

containing dense rough endoplasmic reticulum (the site of antibody production), perinuclear Golgi body (where

immunoglobulins are converted to final form and packaged). Unlike B cells, immunoglobulins are not present on the

surface of plasma cells. They have a short life span of few days to few weeks.

Sridhar Rao P.N (www.microrao.com)

T LYMPHOCYTE:

Ontogeny:

The name "T-cell" is an abbreviation of "thymus dependent lymphocyte". T lymphocytes arise in the bone marrow

as T-cell precursors, then migrate to and mature in the thymus. After entry into the thymus T-cell precursors are

also referred to as "thymocytes".

In the thymus there are rearrangements at gene segments coding for the variable part of the TCR (T Cell Receptor)

resulting in generation of diversity. T Cell Receptors are then expressed on the surface, which is followed by

expression of either CD8 or CD4 surface molecules. Those cells expressing receptors that can interact with self

MHC molecules are positively selected while those cells that express receptors that recognize peptides derived

from self protein in association with self MHC are negatively selected. Such cells undergo clonal deletion or anergy.

Distribution:

T cell accounts for 70-80% (1500 cells/l) lymphocytes in peripheral blood, 5-10% in bone marrow, 70-80% in

lymph node and 30-40% in spleen.

Surface markers:

The most important surface receptor is TCR. TCR are polypeptides that belong to the immunoglobulin superfamily.

There are two kinds of TCR, one composed of a - heterodimer (TCR2) and the other composed of a -

heterodimer (TCR1). An individual T cell can express either - or - as its receptor but never both. 95% of T cells

express the - heterodimer. The other markers/receptors present on the surface are IL-2R, IL-1R, CD2, CD3,

CD4/CD8, CD28, ICAM-1 and LFA-1. Nearly all the mature T lymphocytes express both CD2 and CD3 on their

surface. CD3, which is always found closely associated with TCR, is necessary for signal transduction following

antigen recognition by the TCR.

Subsets of T Cells:

There are two major types of T cells, Helper (CD4) and Cytotoxic/Suppressor (CD8) T cells.

CD4 cells account for 45% (900/l) of lymphocytes while CD8 cells account for 30% (600/l).

Helper T cells (TH) secrete cytokines that promote the proliferation and differentiation of cytotoxic T cells, B

cells and macrophages and activation of inflammatory leukocytes. TH cells are identified by the presence of

the CD4 marker. They recognize antigen when presented along with Class II MHC molecules. TH cells are

further subdivided into the TH1 and TH2 subsets on the basis of the kinds of cytokines they produce. TH1 cells

produce interleukin-2 (IL-2), interferon-gamma (IFN), and tumour necrosis factor-beta (TNF-) while TH2

cells produce IL-4, IL-5, IL-6, IL-10 and TGF-.

Cytotoxic T cells (TC) lyse cells with foreign antigens, e.g. tumour cells, virus-infected cells, and foreign

tissue grafts. TC cells are identified by the presence of the CD8 marker. They recognize antigen presented

when presented along with Class I MHC molecules. The suppressor T cells have a role in downregulation of

immune response.

Demonstration of T cells:

T cells can be demonstrated by immunofluorescence using fluorescent-labelled monoclonal antibodies

against TCR or other surface markers.

E-Rosette/ SRBC rosette: T cells bind to sheep RBCs at 37oC forming rosettes.

They undergo blast transformation on treatment with mitogens such as phytohemagglutinin (PHA) or

Concanavalin A.

Sridhar Rao P.N (www.microrao.com)

Functions of Helper T-cells (TH):

Promotes differentiation of B-cells and cytotoxic T-cells

Activates macrophages

Functions of Cytotoxic/Suppressor T-cells (CTL):

Kills cells expressing appropriate antigen

Downregulates the activities of other cells

NK CELLS (LARGE GRANULAR LYMPHOCYTES):

Also called Large Granular Lymphocytes (LGLs), these are large lymphocytes containing azurophilic granules in the

cytoplasm. NK cells derive form bone marrow but don't require thymus for development. NK cells are so called

because they kill variety of target cells (such as tumour cells, virus-infected cells, transplanted cells) without the

participation of MHC molecules. They can kill target cell without a need for activation unlike cytotoxic T

lymphocytes. Hence they mediate a form of natural (innate) immunity.

Distribution:

They account for 10-15% of blood lymphocytes. They are rare in lymph nodes and don't circulate through lymph.

Surface markers:

NK cells lack any surface immunoglobulins, TCR or CD4 makers; instead they have CD16 (Immunoglobulin Fc

receptor) and CD56. Approximately 50% of human NK cells express only one form of CD8. Other receptors include

IL-2R, CD2, ICAM-1 and LFA-1.

Functions:

NK cells are activated by recognition of antibody-coated cells, virus infected cell, cell infected with intracellular

bacteria and cells lacking MHC I proteins. Activation of NK cell results in cytolysis of target and cytokine secretion

but no clonal expansion. Interestingly, NK cells are inhibited on contact with MHC I proteins.

NK cells can kill antibody-coated target cells, which is mediated through Fc receptor present on its surface. This is

called antibody-dependent cell cytotoxicity (ADCC).

NK cells also participate

in Graft vs Host reaction in

recipient of bone marrow

transplants. NK cells can

be activated by IL-2 so

that their cytotoxic

capacity is enhanced.

Such cells are called

Lymphokine Activated

Killer cells (LAK) and have

been used clinically to treat tumours. LAK cells have enhanced cytolytic activity and are effective against wide

range of tumour cells. Activated NK cells produce cytokines such as IFN-, TNF, GM-CSF and CSF-1 all of which

are immunomodulators.

LYMPHOCTE RECIRCULATION:

The movement of lymphocytes

via the blood stream and

lymphatics from peripheral tissue

to another is called lymphocyte

recirculation. Lymphocytes are

migratory cells; mature

lymphocytes continually migrate

in and out of all peripheral

lymphoid tissue. At an average

each cell changes location once

or twice each day. At any given

point of time 1-2% of

lymphocytes will be in transit. In

most lymphoid organs, they

enter through blood and exit

through lymphatics, but in spleen they enter and leave directly through blood. As lymphocytes migrate, they can

survey the body for foci of infection or presence of foreign antigens. Such a movement also helps to maintain a

balance in distribution of lymphocytes in the body.

Cell-mediated immunity

The second arm of the immune response is refered to as CellMediated Immunity (CMIR). As the name implies, the functional "effectors" of this response are various immune cells. Cell mediated immunity is produced by the sensetised lymphocytes . It is animmune responsethat does not involveantibodiesorcomplementbut rather involves the activation of macrophages,natural killer cells(NK),antigen-specificcytotoxicT-lymphocytes, and the release of variouscytokinesin response to an antigen and this results in the lysis of the microbial antigens.

Historically, the immune system was separated into two branches:humoral immunity, for which the protective function of immunization could be found in the humor (cell-free bodily fluid orserum) andcellular immunity, for which the protective function of immunization was associated with cells. CD4 cells or helper T cells provide protection against different pathogens.

If an individual is exposed to a particularantigan for the second time, immune response occurs more quickly and more abundantly than during the first exposure. This is known as the secondary response. Both humoral and cell mediated immunity are associated with immunologic memory that is the immune system is able to retain the memory of the first antigenic exposure and there by during the secons similar exposure it produces a quick response.

Cellular immunity protects the body by:

apoptosisin body cells displayingepitopesof foreign antigen on their

surface, such asvirus-infected cells, cells with intracellular bacteria, and

cancer cells displayingtumorantigens;

activating macrophages and natural killer cells, enabling them to destroy

intracellular pathogens

stimulating cells to secrete a variety of cytokines that influence the

function of other cells involved in adaptive immune responses and innate

immune responses.

Cell-mediated immunity is directed primarily at microbes that survivein

phagocytesandmicrobesthat infect non-phagocytic cells. It is most effective

in removing virus-infected cells, but also participates in defending

againstfungi,protozoans,cancers, and intracellular bacteria. It also plays a

major role intransplant rejection.

These functions include:

Phagocytosis and killing of intracellular pathogens

Direct cell killing by NK and K cells

Direct cell killing by cytotoxic T cells

These responses are especially important for destroying intracellular bacteria, eliminating viral infections and destroying tumor cells.

CELLS OF IMMUNE SYSTEM

The immune system operates by producing cells.The important cells as follows

LYMPHOCYTES

Lymphocytes are the mononucleate, nongranular leukocytes of lymphoid tissue, participating in immunity. They are found in blood, lymph and lymphoid tissues such as spleen, lymph nodes, tonsils, peyers patches etc. They are spherical or ovoid in shape. They have a diameter of 7 to 12 microns. They have a large nucleus and cytoplasm.

The lymphocyes are of three types. They are B lymphocytes, Tlymphocytes and null cells.T he null cells constitute about 3%, the B cells about 27% and the T cells about 70% of the total T lymphocytes.

The B lymphocytes mature in bursa of Fabricus in birds or in bone marrow of mammals. The B lymphocytes produce antibodies and hence they are responsible for humoral immunity. The B cells kill bactreria, virus etc

The T lyphocytes mature in the thymus under the influence of thymic hormones. The T lymphocytes bring about the cell mediated immunity. The T cells are responsible for the killing of cancer cells,killing viral infected cells, allograft etc.

The null cells form a third population of lymphocytes. They are intermediate between T and B cells. They have cytotoxic activity.

B LYMPHOCYTES

The lymphocytes that mature in bursa of fabricius or bone marrow and that brings about humoral immunity is called B lymphocytes. The B lymphocytes have a large nucleus. They are found in the blood ad lymph. But they are highly concentrated in lymph nodes and spleen. They contain surface immunoglobulins. The B lymphocytes have the following markers.

Ia(immune associated) protein which binds with the Ia receptor of the T lymphocytes.

Fc protein to bind with the Fc fragment of the immunoglobulin

CRI and CR3 receptors of complement system.

Surface immunoglobulins

The immature B cell changes in to mature B cell which has IgD molecules on the surface in addition to IgM.

PLASMA CELLS

These are the end cells of the B lymphocytes. They secrete immunoglobulins. They are very rarely seen in the plasma of blood but are but are found mainly in the lymph nodes and spleenThe cytoplasm is completely filled with rough endoplasmic reticulam. This is a site of protein synthesis namely synthesis of immunoglobulins.

MACROPHAGE ACTIVATION

Macrophages are the large mononuclear phagocytic cells derived from monocytes. They are components of the reticuloendothelial system. They are distributed through out the body but concentrated in lymph nodes, spleen, bone marrow and liver. They are large lymphoid cells. They have large nucleus. They contain large number of lysosomes.

While the production of antibody through the humoral immune response can

effectively lead to the elimination of a variety of pathogens, bacteria that have

evolved to invade and multiply within phagocytic cells of the immune response

pose a different threat. The following graphics illustrate this dilemma:

Non-encapsulated microorganisms are easily phagocytosed and killed within macrophages.

Encapsulated microorganisms require the production of antibody in order to be effectively phagocytosed. Once engulfed, however, they are easily killed.

Intracellular microorganisms

Intracellular microorganisms elicit the production of antibody, which allows effective phagocytosis. Once engulfed, however, they survive within the phagocyte and eventually kill it.

IFNTNF

Intracellular microorganisms also activate specific T-cells, which then release lymphokines (e.g. IFN, TNF) that cause macrophage activation. Activated ("killer") macrophages are then very effective at destroying the intracellular pathogens.

This process can be further illustrated by considering the following experiment

known as "Koch's phenomenon". Inoculation of an unimmunized guinea pig with

a lethal dose of the intracellular pathogenMycobacterium tuberculosis(MT)

results in death of the animal. Inoculation with a sub-lethal dose induces

immunity.Inoculation of an MT-immunized guinea pig with a lethal dose of MT

causes a local reaction ("delayed hypersensitivity") one to two days later.

Inoculation of an MT-immunized guinea pig with a lethal dose of a different

intracellular pathogen,Listeria monocytogenes(LM) again results in death of

the animal.Inoculation of an MT-immunized guinea pig with a lethal dose of

LMandMT causes a delayed hypersensitivity reaction.These results

demonstrate the specific (T-cell mediated) and non-specific (macrophage

mediated) aspects of this type of cell mediated immunity.

T LYMPHOCYTES

The mononucleated non granular leukocyte that matures in thymus and that

bringa about cell mediated immunity is called T lymphocyte. They vare highly

concentrated in blood and spleen. The T cell markers as follows.

Erythocyte receptor- It recognizes the sheep erythrocytes

T cell antigen receptor It recognizes MHC antigens

The Ia protein receptor etc

1. T helper cells

They are sub population of T lymphocytes that help B cells and T cells in

immune responses They are regulator cells. They help the B and T cells in

many ways . The t helper cells contain glycoprotein molecules called CD4 molecules on the

urface. The HIV infects mainly these cells. The TH cells are activated by very small quantities

of antigen which can not activate other cells. They secrete lymphokines.

2. T suppressor cells

These cells are a sub population of T cells that suppresses the activity of B cells and other T

Cells. They are the regulatory T cells. They inhibit antibody production by B cells. They

suppress the functions of the T killer cells and T helper cells. They are responsible for

immune tolerance by limiting the ability of the immune system to attack a persons own

bodt tissue.

CELL MEDIATED CYTOTOXICITY

The second half of the cell-mediated immune response is involved in rejection of

foreign grafts and the elimination of tumors and virus-infected cells. The

effector cells involved in these processes are cytotoxic T-lymphocytes (CTLs), NK-

cells and K-cells. Each of these effector cells recognizes their target by different

means, described below.

Cytotoxic T-lymphocytes

CTLs, like other T-cells are both antigen and MHC-restricted. That is, CTLs require i) recognition of a specific antigenic determinantandii) recognition of "self" MHC (Click here to review these requirements). Briefly, CTLs recognize antigen via their T-cell receptor. This receptor makes specific contacts with the antigenic determinant and the target cell's class I MHC molecule. CTLs also express CD8, which may assist the antigen recognition process. Once recognition is successful, the CTL "programs" the target cell for self-destruction. This process is thought to occur in one of several possible ways. First, CTLs may release a substance known as perforin in the space between the CTL and its target. In the presence of calcium ions, the perforin polymerizes, forming channels in the target cell's membrane. These channels may cause the target cell to lyse. Second, the CTL may also release various enzymes that pass through the polyperforin channels, causing target cell damage. Third, the CTL may release lymphokines and/or cytokines that interact with specific receptors on the target cell surface, causing internal responses that lead to destruction of the target cell. CTLs principally act to eliminate endogenous antigens.

NULL CELLS

These are lymphocytes with cytotoxic properties. They are neither B cells or T cells. They are intermediate between these. They form less than 3%. Two types namely natural killer cells and killer cells.

NK-CELLS

NK cells are part of a group know as the "large granular lymphocytes". These cells are generally

non-specific, MHC-unrestricted cells involved primarily in the elimination of neoplastic or

tumor cells.The precise mechanism by which they

recognize their target cells is not clear. Probably, there is some type of NK-determinant

expressed by the target cells that is recognized by an NK-receptor on the NK cell surface.

Once the target cell is recognized, killing occurs in a manner similar to that produced by

the CTL.

K-CELLSK-cells are probably not a separate cell type but rather a separate function of the NK group. K-cells contain immunoglobulin Fc receptors on their surface and are involved in a process known as Antibody-dependent Cell-mediated Cytotoxicity (ADCC). ADCC occurs as a consequence of antibody being bound to a target cell surface via specific antigenic determinants expressed by the target cell. Once bound, the Fc portion of the immunoglobulin can be recognized by the K-cell. Killing then ensues by a mechanism similar to that employed by CTLs. This type of CMIR can also result inType II hypersensitivities.

UNIT-II

Antibody

Antibodies (also known as immunoglobulins[1], abbreviated Ig) are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They are typically made of basic structural unitseach with two large heavy chains and two small light chainsto form, for example, monomers with one unit, dimers with two units or pentamers with five units. Antibodies are produced by a kind of white blood cell called a plasma cell. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.[2]

Though the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures, or antigen binding sites, to exist. This region is known as the hypervariable region. Each of these variants can bind to a different target, known as an antigen.[3] This huge diversity of antibodies allows the immune system to recognize an equally wide variety of antigens. The unique part of the antigen recognized by an antibody is called the epitope. These epitopes bind with their antibody in a highly specific interaction, called induced fit, that allows antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules that make up an organism. Recognition of an antigen by an antibody tags it for attack by other parts of the immune system. Antibodies can also neutralize targets directly by, for example, binding to a part of a pathogen that it needs to cause an infection.[4]

The large and diverse population of antibodies is generated by random combinations of a set of gene segments that encode different antigen binding sites (or paratopes), followed by random mutations in this area of the antibody gene, which create further diversity.[2][5] Antibody genes also re-organize in a process called class switching that changes the base of the heavy chain to another, creating a different isotype of the antibody that retains the antigen specific variable region. This allows a single antibody to be used by several different parts of the immune system. Production of antibodies is the main function of the humoral immune system.

Forms

Surface immunoglobulin (Ig) is attached to the membrane of the effector B cells by its transmembrane region, while antibodies are the secreted form of Ig and lack the trans membrane region so that antibodies can be secreted into the bloodstream and body cavities. As a result, surface Ig and antibodies are identical except for the transmembrane regions. Therefore, they are considered two forms of antibodies: soluble form or membrane-bound form (Parham 21-22).

The membrane-bound form of an antibody may be called a surface immunoglobulin (sIg) or a membrane immunoglobulin (mIg). It is part of the B cell receptor (BCR), which allows a B cell to detect when a specific antigen is present in the body and triggers B cell activation.[7] The BCR is composed of surface-bound IgD or IgM antibodies and associated Ig- and Ig- heterodimers, which are capable of signal transduction.[8] A typical human B cell will have 50,000 to 100,000 antibodies bound to its surface.[8] Upon antigen binding, they cluster in large patches, which can exceed 1 micrometer in diameter, on lipid rafts that isolate the BCRs from most other cell signaling receptors.[8] These patches may improve the efficiency of the cellular immune response.[9] In humans, the cell surface is bare around the B cell receptors for several thousand ngstroms,[8] which further isolates the BCRs from competing influences.

Immunoglobulin A

Isotypes

Name

Types

Antibody Complexes

IgA

2

IgD

1

IgE

1

IgG

4

IgM

1

Antibodies can come in different varieties known as isotypes or classes. In placental mammals there are five antibody isotypes known as IgA, IgD, IgE, IgG and IgM. They are each named with an "Ig" prefix that stands for immunoglobulin, another name for antibody, and differ in their biological properties, functional locations and ability to deal with different antigens, as depicted in the table.[13]

The antibody isotype of a B cell changes during cell development and activation. Immature B cells, which have never been exposed to an antigen, are known as nave B cells and express only the IgM isotype in a cell surface bound form. B cells begin to express both IgM and IgD when they reach maturitythe co-expression of both these immunoglobulin isotypes renders the B cell 'mature' and ready to respond to antigen.[14] B cell activation follows engagement of the cell bound antibody molecule with an antigen, causing the cell to divide and differentiate into an antibody producing cell called a plasma cell. In this activated form, the B cell starts to produce antibody in a secreted form rather than a membrane-bound form. Some daughter cells of the activated B cells undergo isotype switching, a mechanism that causes the production of antibodies to change from IgM or IgD to the other antibody isotypes, IgE, IgA or IgG, that have defined roles in the immune system.

Structure

Antibodies are heavy (~150kDa) globular plasma proteins. They have sugar chains added to some of their amino acid residues.[15] In other words, antibodies are glycoproteins. The basic functional unit of each antibody is an immunoglobulin (Ig) monomer (containing only one Ig unit); secreted antibodies can also be dimeric with two Ig units as with IgA, tetrameric with four Ig units like teleost fish IgM, or pentameric with five Ig units, like mammalian IgM.[16]

Several immunoglobulin domains make up the two heavy chains (red and blue) and the two light chains (green and yellow) of an antibody. The immunoglobulin domains are composed of between 7 (for constant domains) and 9 (for variable domains) -strands. See also:

The variable parts of an antibody are its V regions, and the constant part is its C region.

Immunoglobulin domains

The Ig monomer is a "Y"-shaped molecule that consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds.[13] Each chain is composed of structural domains called immunoglobulin domains. These domains contain about 70-110 amino acids and are classified into different categories (for example, variable or IgV, and constant or IgC) according to their size and function.[17] They have a characteristic immunoglobulin fold in which two beta sheets create a sandwich shape, held together by interactions between conserved cysteines and other charged amino acids.

Heavy chain

There are five types of mammalian Ig heavy chain denoted by the Greek letters: , , , , and .[3] The type of heavy chain present defines the class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively.[4] Distinct heavy chains differ in size and composition; and contain approximately 450 amino acids, while and have approximately 550 amino acids.[3]

1. Fab region2. Fc region3. Heavy chain (blue) with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3) domains.4. Light chain (green) with one variable (VL) and one constant (CL) domain5. Antigen binding site (paratope)6. Hinge regions.

In birds, the major serum antibody, also found in yolk, is called IgY. It is quite different from mammalian IgG. However, in some older literature and even on some commercial life sciences product websites it is still called "IgG", which is incorrect and can be confusing.

Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains , and have a constant region composed of three tandem (in a line) Ig domains, and a hinge region for added flexibility;[13] heavy chains and have a constant region composed of four immunoglobulin domains.[3] The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain.

Light chain

.

In mammals there are two types of immunoglobulin light chain, which are called lambda () and kappa ().[3] A light chain has two successive domains: one constant domain and one variable domain. The approximate length of a light chain is 211 to 217 amino acids.[3] Each antibody contains two light chains that are always identical; only one type of light chain, or , is present per antibody in mammals. Other types of light chains, such as the iota () chain, are found in lower vertebrates like Chondrichthyes and Teleostei.

CDRs, Fv, Fab and Fc Regions

Some parts of an antibody have unique functions. The arms of the Y, for example, contain the sites that can bind two antigens (in general identical) and, therefore, recognize specific foreign objects. This region of the antibody is called the Fab (fragment, antigen binding) region. It is composed of one constant and one variable domain from each heavy and light chain of the antibody.[18] The paratope is shaped at the amino terminal end of the antibody monomer by the variable domains from the heavy and light chains. The variable domain is also referred to as the FV region and is the most important region for binding to antigens. More specifically, variable loops of -strands, three each on the light (VL) and heavy (VH) chains are responsible for binding to the antigen. These loops are referred to as the complementarity determining regions (CDRs). In the framework of the immune network theory, CDRs are also called idiotypes. According to immune network theory, the adaptive immune system is regulated by interactions between idiotypes.

The base of the Y plays a role in modulating immune cell activity. This region is called the Fc (Fragment, crystallizable) region, and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody.[3] Thus, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen, by binding to a specific class of Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils.[13][19]

Function

Activated B cells differentiate into either antibody-producing cells called plasma cells that secrete soluble antibody or memory cells that survive in the body for years afterward in order to allow the immune system to remember an antigen and respond faster upon future exposures.[20]

At the prenatal and neonatal stages of life, the presence of antibodies is provided by passive immunization from the mother. Early endogenous antibody production varies for different kinds of antibodies, and usually appear within the first years of life. Since antibodies exist freely in the bloodstream, they are said to be part of the humoral immune system. Circulating antibodies are produced by clonal B cells that specifically respond to only one antigen (an example is a virus capsid protein fragment). Antibodies contribute to immunity in three ways: they prevent pathogens from entering or damaging cells by binding to them; they stimulate removal of pathogens by macrophages and other cells by coating the pathogen; and they trigger destruction of pathogens by stimulating other immune responses such as the complement pathway.[21]

The secreted mammalian IgM has five Ig units. Each Ig unit (labeled 1) has two epitope binding Fab regions, so IgM is capable of binding up to 10 epitopes.

Activation of complement

Antibodies that bind to surface antigens on, for example, a bacterium attract the first component of the complement cascade with their Fc region and initiate activation of the "classical" complement system.[21] This results in the killing of bacteria in two ways.[6] First, the binding of the antibody and complement molecules marks the microbe for ingestion by phagocytes in a process called opsonization; these phagocytes are attracted by certain complement molecules generated in the complement cascade. Secondly, some complement system components form a membrane attack complex to assist antibodies to kill the bacterium directly.[22]

Activation of effector cells

To combat pathogens that replicate outside cells, antibodies bind to pathogens to link them together, causing them to agglutinate. Since an antibody has at least two paratopes it can bind more than one antigen by binding identical epitopes carried on the surfaces of these antigens. By coating the pathogen, antibodies stimulate effector functions against the pathogen in cells that recognize their Fc region.[6]

Those cells which recognize coated pathogens have Fc receptors which, as the name suggests, interacts with the Fc region of IgA, IgG, and IgE antibodies. The engagement of a particular antibody with the Fc receptor on a particular cell triggers an effector function of that cell; phagocytes will phagocytose, mast cells and neutrophils will degranulate, natural killer cells will release cytokines and cytotoxic molecules; that will ultimately result in destruction of the invading microbe. The Fc receptors are isotype-specific, which gives greater flexibility to the immune system, invoking only the appropriate immune mechanisms for distinct pathogens.[3]

Natural antibodies

Humans and higher primates also produce natural antibodies which are present in serum before viral infection. Natural antibodies have been defined as antibodies that are produced without any previous infection, vaccination, other foreign antigen exposure or passive immunization. These antibodies can activate the classical complement pathway leading to lysis of enveloped virus particles long before the adaptive immune response is activated. Many natural antibodies are directed against the disaccharide galactose (1,3)-galactose (-Gal), which is found as a terminal sugar on glycosylated cell surface proteins, and generated in response to production of this sugar by bacteria contained in the human gut.[23] Rejection of xenotransplantated organs is thought to be, in part, the result of natural antibodies circulating in the serum of the recipient binding to -Gal antigens expressed on the donor tissue.[24]

Immunoglobulin diversity

Virtually all microbes can trigger an antibody response. Successful recognition and eradication of many different types of microbes requires diversity among antibodies; their amino acid composition varies allowing them to interact with many different antigens.[25] It has been estimated that humans generate about 10billion different antibodies, each capable of binding a distinct epitope of an antigen.[26] Although a huge repertoire of different antibodies is generated in a single individual, the number of genes available to make these proteins is limited by the size of the human genome. Several complex genetic mechanisms have evolved that allow vertebrate B cells to generate a diverse pool of antibodies from a relatively small number of antibody genes.[27]

Domain variability

The hypervariable regions of the heavy chain are shown in red, PDB 1IGT

The region (locus) of a chromosome that encodes an antibody is large and contains several distinct genes for each domain of the antibodythe locus containing heavy chain genes (IGH@) is found on chromosome 14, and the loci containing lambda and kappa light chain genes (IGL@ and IGK@) are found on chromosomes 22 and 2 in humans. One of these domains is called the variable domain, which is present in each heavy and light chain of every antibody, but can differ in different antibodies generated from distinct B cells. Differences, between the variable domains, are located on three loops known as hypervariable regions (HV-1, HV-2 and HV-3) or complementarity determining regions (CDR1, CDR2 and CDR3). CDRs are supported within the variable domains by conserved framework regions. The heavy chain locus contains about 65 different variable domain genes that all differ in their CDRs. Combining these genes with an array of genes for other domains of the antibody generates a large cavalry of antibodies with a high degree of variability. This combination is called V(D)J recombination discussed below.[28]

V(D)J recombination

Simplistic overview of V(D)J recombination of immunoglobulin heavy chains

Somatic recombination of immunoglobulins, also known as V(D)J recombination, involves the generation of a unique immunoglobulin variable region. The variable region of each immunoglobulin heavy or light chain is encoded in several piecesknown as gene segments. These segments are called variable (V), diversity (D) and joining (J) segments.[27] V, D and J segments are found in Ig heavy chains, but only V and J segments are found in Ig light chains. Multiple copies of the V, D and J gene segments exist, and are tandemly arranged in the genomes of mammals. In the bone marrow, each developing B cell will assemble an immunoglobulin variable region by randomly selecting and combining one V, one D and one J gene segment (or one V and one J segment in the light chain). As there are multiple copies of each type of gene segment, and different combinations of gene segments can be used to generate each immunoglobulin variable region, this process generates a huge number of antibodies, each with different paratopes, and thus different antigen specificities.[2]

After a B cell produces a functional immunoglobulin gene during V(D)J recombination, it cannot express any other variable region (a process known as allelic exclusion) thus each B cell can produce antibodies containing only one kind of variable chain.[3][29]

Somatic hypermutation and affinity maturation

Following activation with antigen, B cells begin to proliferate rapidly. In these rapidly dividing cells, the genes encoding the variable domains of the heavy and light chains undergo a high rate of point mutation, by a process called somatic hypermutation (SHM). SHM results in approximately one nucleotide change per variable gene, per cell division.[5] As a consequence, any daughter B cells will acquire slight amino acid differences in the variable domains of their antibody chains.

This serves to increase the diversity of the antibody pool and impacts the antibodys antigen-binding affinity.[30] Some point mutations will result in the production of antibodies that have a weaker interaction (low affinity) with their antigen than the original antibody, and some mutations will generate antibodies with a stronger interaction (high affinity).[31] B cells that express high affinity antibodies on their surface will receive a strong survival signal during interactions with other cells, whereas those with low affinity antibodies will not, and will die by apoptosis.[31] Thus, B cells expressing antibodies with a higher affinity for the antigen will outcompete those with weaker affinities for function and survival. The process of generating antibodies with increased binding affinities is called affinity maturation. Affinity maturation occurs in mature B cells after V(D)J recombination, and is dependent on help from helper T cells.[32]

Mechanism of class switch recombination that allows isotype switching in activated B cells

Class switching

Isotype or class switching is a biological process occurring after activation of the B cell, which allows the cell to produce different classes of antibody (IgA, IgE, or IgG). The different classes of antibody, and thus effector functions, are defined by the constant (C) regions of the immunoglobulin heavy chain. Initially, nave B cells express only cell-surface IgM and IgD with identical antigen binding regions. Each isotype is adapted for a distinct function, therefore, after activation, an antibody with a IgG, IgA, or IgE effector function might be required to effectively eliminate an antigen. Class switching allows different daughter cells from the same activated B cell to produce antibodies of different isotypes. Only the constant region of the antibody heavy chain changes during class switching; the variable regions, and therefore antigen specificity, remain unchanged. Thus the progeny of a single B cell can produce antibodies, all specific for the same antigen, but with the ability to produce the effector function appropriate for each antigenic challenge. Class switching is triggered by cytokines; the isotype generated depends on which cytokines are present in the B cell environment.[33]

Class switching occurs in the heavy chain gene locus by a mechanism called class switch recombination (CSR). This mechanism relies on conserved nucleotide motifs, called switch (S) regions, found in DNA upstream of each constant region gene (except in the -chain). The DNA strand is broken by the activity of a series of enzymes at two selected S-regions.[34][35] The variable domain exon is rejoined through a process called non-homologous end joining (NHEJ) to the desired constant region (, or ). This process results in an immunoglobulin gene that encodes an antibody of a different isotype.[36]

INTRODUCTION:

Antigen (Ant Installer Generator) is a tool to take an Ant build script, combine it with a GUI and wrap it up as an executable jar file. Its primary purpose is to create powerful graphical installers from Ant scripts.

ANTIGENS PROPERTIES:

Antigens- may possess:

Immunogenicity,

Antigenicity,

Allerogenicity, or

Tolerogenicity

Immunogenicity

Property that allows a substance to induce a detectable immune response (humoral or cellular) when introduced into an animal. Such substances are termed Immunogens.

Antigenicity

Property that allows a substance to combine specifically with antibodies or TcR,whether or not they are immunogenic. Therefore, all immunogens are antigens but not all antigens are immunogens.

Allerogenicity-

Property that allows a substnace to induce an allergic response. Such substances are termed allergens.

Tolerogenicity-

Property that allows a substance to induce specific immunologic non-responsiveness in either the humoral or cell-mediated branch. Such substances are termed tolerogens.

Haptens

Low-molecular weight compounds including many drugs and antibiotics, are non-immunogenic but when coupled to immunogenic proteins, the resulting conjugates stimulate the production of antibodies which can bind to the low-molecular weight component. Such molecules are termed haptens.

Epitope

The part of an antigen that combines with a specific antibody or T cell receptor. Previous term used was antigenic determinant.

Immunogens

For the induction of humoral immunity (antibody response), the most potent immunogens are macromolecular proteins or glycoproteins, but polysaccharides, synthetic peptides, and other synthetic polymers such as polyvinylpyrrolidone are immunogenic under appropriate conditions. Pure nucleic acids or lipids are not immunogenic but antibodies which react with them can be induced by immunization with nucleoproteins or lipoproteins.

In general, proteins serve as immunogens for T cell-mediated immunity. (Recall that these proteins must be processed into peptides and the peptides must be presented by an APC in association with MHC proteins.

Requirements for Immunogenicity

The requirements are somewhat dependent upon the experimental conditions (mode of immunization, organism being immunized, sensitivity of detection methods, etc.). However, certain conditions must be met in order for a molecule to be immunogenic:

A. Foreignness: [Rabbit albumin not immunogenic in another rabbit but would be immunogenic in a mouse.]

B. Molecular Size: [Certain minimum size is required for immunogenicity. The most potent immunogens are macromolecular proteins with

molecular weights greater than 100,000. Substances