Immunology

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Immunology - Study of the defenses of the body against foreign invaders.

Immunity - Exempt; special capacity for resistance; condition of being resistant to an infection.

Ancient Greeks -  Resistance and immunity known about for centuries -

Chinese and Smallpox (11th century) - Conferred immunity to smallpox by inhaling dried pustular material

In Turkey - Turks  injected pustular material into the vein of the person desiring immunity  to smallpox.     

Smallpox (variola) is caused by the Smallpox virus.

Lady Montague (wife of the British ambassador to Turkey) introduced variolation to the western world.  Variolation.

These procedures had great risks.  a) There was no assurance that variolation would result in a mild case of smallpox (people died from smallpox), and b) there was a possibility of transferring other diseases, such as hepatitis.

Edward Jenner (1700's) - Used cowpox material to confer immunity to smallpox.  Vaccination.

Sarah Nelms - a milkmaid from whom Jenner got cowpox material.

James Phipps - The farm boy subject of Jenner used to demonstrate cross-immunity.

Cross-immunity - A rare instance in which permanent immunity to a disease can be conferred by virtue of recovery from another disease.   Examples - smallpox and cowpox; M. tuberculosis and M. bovis.

Louis Pasteur - Father of immunology. Attenuation by aging of microbes, growing microbes at a temperature other than at the temperature normal for pathogenesis, and by drying.  Cholera organism (Pasteuralla multicida), Anthrax bacillus (Bacillus anthracis), and rabies virus.

Attenuation - Lessening of virulence.

Virulence - Measure of the disease-producing power of an organism.

Joseph Meister - Immunized against rabies by Pasteur.

Von Behring and Kitasato (Berlin, 1890) - Demonstrated that protective activity induced by vaccination was present in the blood stream.   Gave the name antibody to the factors in the blood stream with protective activity.

Emil Roux (1894) - Introduced passive immunity.

Clostridium tetani - Gram positive, spore producing rods that produce the toxin tetanospasmin when grown under anaerobic conditions.

Toxoid - A denatured toxin, but which retains the ability to induce antibodies that neutralize the toxin in its native form.

Naturally Acquired Active Immunity - is obtained when a person is exposed to antigens in the course of daily life.  Antigens enter the body naturally; the body produces antibodies and specialized lymphocytes.

Artificially Acquired Active Immunity - results from vaccination.  Antigens are introduced in vaccines; the body produces antibodies and specialized lymphocytes.

Naturally Acquired Passive Immunity - involves the natural transfer of antibodies from a mother to her infant.  Antibodies pass from mother to fetus via the placenta or to the infant in her milk.

Artificially Acquired Passive Immunity - involves the introduction of antibodies (rather than antigen) into the body.  Preformed antibodies in immune serum are introduced into the body by injection.

Time Course of the Immune Response - The primary and secondary immune response to an antigen.

Primary Response - a) Lag phase - Initial phase of the antibody response where antibodies are not detectable.  Length depends on method used for antibody detection, type of antigen inoculated, route of inoculation, amount of antigen inoculated, animal species.   b) Log phase - exponential increase in antibody production.  c) Plateau - production of antibodies is equal to the catabolic breakdown of antibodies. Varies and may be transitory. d) Decline - catabolism exceed synthesis.  Depends on the persistence of the antigen.   The level of antibodies produced in the primary response is small, and therefore the level of protection against an infectious agent  is also low.  The antibodies produced are primarily IgM.

Secondary Response - Also referred to as the anamnestic response, or immunologic memory, or the booster response. e) Injection of a second dose of antigen at a later date results in an initial drop in circulating antibodies resulting from complexing of circulating antibodies with newly injected antigen.  f) Log phase - dramatic increase in circulating antibodies. There is a shorter lag phase than in the primary response with a much higher antibody production.  There is a higher rate and longer persistence of antibodies.  Ab are newly synthesized (not released from a stored compartment).  May be induced at almost any time (even years) after the primary response has taken place.  Repeatable until the physiologic limits of the animal is reached.  The antibodies produced are primarily IgG.

Current Vaccination techniques - Involve repeated booster injections.

See Immunization

Immunogens - Substances capable of inducing a specific immune response in an animal.

    Immunogen ----> animal -------> antibodies, CMI or both

Antigens - Substances that react with products of the specific immune response.

Immunogenecity - The ability of a substance to induce a humoral and/or cell-mediated immune response.

Antigenecity - The ability of a substance to combine with the final products of the humoral or cell-mediated response, i.e. with antibodies and/or cell-surface receptors.

Factors that Govern Immune Responses - a) Foreignness, b) Molecular size, c) Molecular complexity, d) Structural stability, e) Solubility or degradability.

Foreignness - The immunogen must be foreign to the host. Immune responses take place only to substances that are not normally present in the body or normally exposed to the cells of the specific immune system. Under normal conditions, the immune system will discriminate between "self" and "non-self". Generally, the greater the phylogenetic distance between two species, the greater the genetic (and therefore the antigenic) disparity between them.

Molecular size - There is a correlation between the size of a macromolecule and its immunogenecity. The best immunogens have a molecular mass approaching 100,000. Generally substances with a molecular mass less than 5,000 are poor immunogens.

Molecular Complexity - The immunogen must have a certain amount of complexity. Large molecular mass is not enough. Examples: Nylon, Teflon, saran, and homopolymers of amino acids or sugars are not immunogenic because the repeating unit nature of their structure.

Structural stability - Compounds consisting of flexible molecules are weakly immunogenic.

Solubility or degradability - The foreign substance must be processed in order for it to be presented. If insoluble or not attacked by cellular enzyme, the foreign substance will not induce an immune response. Nylon, Teflon, and coal dust are insoluble and not attacked by enzyme. Polymers of D-amino acids are not degraded.

Antigenic Determinant or Epitope - A specific, limited part of an antigen molecule that induces antibody formation.  This portion is also the part of the antigen with which the antibody reacts.

Valence - The number of epitopes that an antigen molecule has, i.e. the number of determinants on an antigen molecule.

Hapten - A small molecule that cannot initiate an immune response unless first bound to an immunogenic carrier molecule. Fully exploited by Karl Landsteiner receiving a Nobel Prize for his work with haptens and for discovery of the blood group antigens.

See Hapten

Cross-Reactivity - Antibodies to one antigen may recognize other antigens. 1) Functional specificity - examples: serum albumin and hemoglobin have different functions, therefore different molecular structures, and are not cross-reactive; bovine serum albumin and sheep serum albumin have a similar function, therefore a similar molecular structure and cross-reactive. 2) Species specificity - Bovine serum albumin and sheep serum albumin are from different species, therefore there are some molecular differences and epitopes that are different in the two molecules. 3) Individual specificity - Molecules, cells or organs of the same type in two individuals may possess determinants that differ, for example the blood group antigen, the transplantation antigen and the Gm factors in immunoglobulin molecules. 4) Organ or cell specificity - Organs or cells within an individual possess determinants that differ, for example the liver has determinants that are not found on the kidney, and macrophages and lymphocytes have antigens that are not common to the two cell types. The CD series of antigens are examples of these differences.

Heterophile Antigens - Antigens with epitopes found on a variety of unrelated molecules.  These antigenic determinants may be found among a variety of phylogenetically unrelated or distant species.  Examples are Group A b hemolytic streptococcal antigen and a heart tissue antigen; an antigen on Proteus vulgaris (strain OX19) and Rickettsii thyphi; Forssman antigen.

Adjuvants - A substance that, when given with an antigen, enhances the immune response. May be immunogenic like gram negative bacteria (Bordetella pertussis) or endotoxin.  May be non-immunogenic like potassium aluminum tartrate (alum), calcium phosphate and mineral oil.

Freund's adjuvant - a) Complete contains Arlecel A, mineral oil and killed Mycobacterium tuberculosis.  b) Incomplete contains only Arlecel A and mineral oil.

TiterMax - An adjuvant that is less toxic than Freund's adjuvant.

See Adjuvant

Resistance - The ability to ward off disease through our defenses. Defenses include specific immunity and nonspecific resistance.

Specific Immunity or Adaptive Immunity - involves a specific defensive response when a host is invaded by foreign organisms or other foreign substances.   The specific immune response elicits a specific immunological memory.   Resistance is improved by repeated exposure to the infectious agent or foreign substance.  Either specific antibodies or specific effector cells (T cells) or both are produced.  These defenses are considered the third line of defense. The immune system also recognizes self and therefore does not mount an immune response.

Nonspecific or Innate Resistance - defenses that protect us against any pathogen, regardless of the species of microbe.  Resistance is not improved by repeated exposure to the pathogen.  These defenses include a first line of defense (skin, mucous membranes and their secretions for example) and a second line of defense (phagocytes, inflammation, fever, and antimicrobial substance).

Mechanical Factors - 

-Skin consists of two portions, 1) the dermis, the skin's inner, thicker portion composed of connective tissue, and 2) the epidermis, the outer thinner portion which is in direct contact with the external environment.  The epidermis contains several layers of tightly packed cells.   The top layer of epidermal cells contains a waterproofing protein called keratin. The epidermis contains Langerhans cells which assist in the generation of the specific immune response. The skin forms a formidable barrier to the entrance of microorganisms. If the skin is broken, a subcutaneous infection often develops. This may result from burns, cuts, stab wounds, etc. If the skin is moist, for example between the toes, fungal infections may develop (athlete's foot).

-Mucous membranes - are composed of 1) an epithelial layer and 2) underlying connective tissue. Mucous membranes line the entire gastrointestinal tract, respiratory tract and the genitourinary tract. The epithelial layer secretes a fluid (mucus) which prevents drying out. Some microbes can thrive on mucus. If the microbes are in sufficient numbers they can penetrate the tissue (Treponema pallidum for example).

-Lacrimal apparatus - The lacrimal glands produce tears which pass under the upper eyelid, and then to the corner of the eye, to two small holes (lacrimal canals) that go into the nose. There is therefore continual washing which keeps microbes from settling on the surface of the eye.

-Saliva - Dilutes the number of microbes on the teeth and mucous membranes of the mouth. This prevents colonization by bacteria.

-Mucus - a slightly viscous substance produced by mucous membranes. Mucus traps microbes.

-Lower respiratory tract - The epithelial cells that line the lower respiratory tract are ciliated. These ciliated cells act as the so-called "ciliary escalator" which keeps the mucus blanket moving up the throat. Sneezing and coughing speed up the escalator.

-Epiglottis - Covers the larynx when swallowing, preventing microbes from entering into the throat.

-Vaginal secretions - Move microbes out of the female body.

Chemical Factors - play an important role in nonspecific defenses.

-Sebaceous glands - Produce oily substance called sebum which prevents hair from drying. Sebum forms a protective covering over the skin. It contains unsaturated fatty acids which prevent certain bacteria and fungi from growing.

-pH of the skin - is between 3 and 5 because of fatty acids and lactic acid. The acid pH prevents microbe from growing. Some bacteria live on sloughed off skin, and the end-product is the cause of body odor. Some microbes may metabolize sebum forming free fatty acids.

-Sweat glands - Perspiration helps maintain body temperature. It also contain lysozyme.

-Lysozyme - Breaks the bond between NAG and NAM of petidoglycan, a b 1-4 glycosidic linkage. Attacks mainly gram positive bacteria. Lysozyme is found on the skin, in tears, saliva, nasal secretions, and tissue fluids.

-Gastric juices - Produced by glands of the stomach. It is mixture of HCl, enzymes and mucus. Its pH is between 1.2 and 3.0, thus preserving the sterility of the stomach.

-Blood - Iron binding proteins called transferrins prevent microbial metabolism because iron is lacking. The blood also contains complement and the acute phase proteins, e.g. C reactive protein and interferon.

-Cells - Phagocytes and natural killer (NK) cells.

Most infectious agents - Enter the body through epithelial surfaces of the nasopharynx, gut, lungs, genitourinary tract.

 Review - 1) Examples of the first line of defense are skin, mucous membranes and secretions. 2) Examples of the second are cells (phagocytes and NK cells, inflammation, fever, antimicobial substances like complement, interferon, and acute phase proteins).

Phagocytosis - Phago = eating; cyto = cell. Eating by the cell.

Elie Metchnikoff - Received the Nobel Prize in 1908 for the discovery of phagocytosis.  Observed that starfish larvae have amoeboid cells that congregate at sites of infection.  Showed that the amoeboid engulf carmine (dye) particles.   Hypothesized that engulfment of foreign particles by cells was a method for removal of microbes.  Injected yeast cells into Daphnia (water flea) that the outcome of the injection (disease or not) was related to the ability of the amoeboid cells in Daphnia to destroy the yeast cells.  He expanded the work to human cells. Made the suggestion that the major defenses against infection were cellular and that antibodies were of minor importance starting a controversy.

Wright - In 1904 showed that antibodies enhance phagocytosis.

Opsonins - Factors that enhance phagocytosis. Substances that function as opsonins include antibodies, complement component breakdown products and fibronectin.

Phagocytic cells - Two complementary system in mammals: 1) the myeloid system which is rapidly phagocytic, extremely efficient, but incapable of a sustained effort; 2) the mononuclear phagocytic system which is slowly acting, but capable of repeated phagocytosis.  This system is also capable of processing antigen and presenting antigenic peptide to cells of the specific immune system. 

Myeloid cells - are also referred to as granulocytes because of the abundance of granules that these cells have in their cytoplasm.  The nucleus of these cells is also lobulated and irregular and are therefore called polymorphonuclear.   There are three types of granulocytes that can be distinguished by their staining properties.  Cells whose granules take up basic dyes (hematoxylin) are called basophils; cells whose granules take up acidic dyes (eosin) are called eosinophils; and cells whose granules (at neutral conditions) take up neither basic or acidic dyes are called neutrophils.

Go to Cells of the Immune System

Ontogeny of Phagocytes -

Neutrophils - Predominant phagocytic cells in the blood, making up 60 to 75% of all WBC.  These cells are produced in the bone marrow from where they enter the blood and circulate for about 12 hours.  They may enter the tissues where they may live for from one to a few days, then die.

-Neutrophil - their cytoplasmic granules stain with neither acidic nor basic dyes at neutral pH.   Their size ranges from 12 to 14 µm in diameter.  They have an abundant cytoplasm.

-Polymorphonuclear - The nucleus has a very irregular shape.  The chromatin is compact and   no longer able to divide. The nucleus is complex, with rounded nuclear lobes joined by thinner   segments.  The segments develop as the neutrophile ages.

-Granulocyte - The cytoplasm has abundant granules.  By EM: 1) Primary granules - electron dense and azurophilic. These granules contain important bactericidal enzymes, myloperoxidases and lysozyme.  Also neutral proteases, e.g. elastase, and acid hydrolases, e.g. b  glucuronidase and cathepsin B.  2) Secondary granules - are electron lucent.  These predominate in aged neutrophils and contain alkaline phosphatase.  They also contain  lysozyme, lactoferrin, collagenase and aminopeptidase.

-Mature neutrophils - have few ribosomes and a sparse endoplasmic reticulum.  Their golgi apparatus is not extensive. They have a small number of mitochondria.  Therefore, most of their proteins are synthesized in the bone marrow during development.  Mature cells probably neither produce nor secrete proteins.

Endocytosis - Ingestion of extracellular macromolecules. Macromolecules within the extracellular fluid are internalized by cells by invagination (inward folding) and pinching off of small regions of the plasma membrane. Endocytosis occurs through 1) pinocytosis or 2) receptor-mediated endocytosis.

Pinocytosis - Non-specific membrane invagination internalizes macromolecules.

Receptor-mediated endocytosis - Macromolecules are selectively internalized after binding to specific membrane receptors.

Phagocytosis - The ingestion of particulate material, including whole pathogenic microorganisms. The process can be divided into arbitrary stages for descriptive purposes.

 -Chemotaxis - directed movement under the influence of external chemical stimuli. Numerous chemotaxins have been described. Those that are chemotactic for neutrophils include 1) IL-8 and NAP-2 (chemokines); 2) complement split products (C3a, C5a, and C5b67); 3) fibrinopeptides; 4) prostaglandins; 5) leukotrienes; 6) soluble bacterial products like formyl methionyl peptides to which neutrophils are strongly attracted. Formyl methionyl amino acids are unique in these peptides released by invading bacteria. Chemotaxins like IL-8, act on neutrophils triggering a G-protein-mediated activating signal that leads to a conformational change in the integrin adhesion molecules, resulting in neutrophil adhesion and subsequent transendothelial migration (part of the inflammatory process). Chemotaxins (chemoattractants) were first described in vitro by use of Boydan Chambers.

-Attachment - Encounter of the neutrophil with the foreign particle. This results in a firm attachment. This is not a spontaneous encounter because of the net negative charge on the phagocyte that must be neutralized. Certain proteins can coat the foreign particle by opsonization to neutralize the charge. Antibodies, C3b, C4b and fibronectin are prime examples of opsonins. It is important that the particles be hydrophobic. Opsonins make particles hydrophobic. For example, Strep. pneumoniae has a hydrophilic capsule. Surface phagocytosis or trapping is an effective mode for attachment.

-Ingestion - Adherence induces membrane protrusions, called pseudopodia to extend around the attached material. Fusion of the pseudopodia encloses the material within a membrane-bound structure called a phagosome. The process requires energy. There are changes in the cytoplasmic microfilaments and microtubules with contraction of myosin and actin. The phagosome enters the endocytic processing pathway in which the phagosome moves toward the cell's interior where it fuses with a lysosome forming a phagolysosome. The primary lysosomes may fuse with secondary lysosomes, each disintegrating and spilling their contents into the phagolysosome.

-Digestion and the destructive forces in phagocytosis - The primary granules contain numerous hydrolytic enzymes. The digestive enzymes can digest the bacterial cell wall. This can kill most ingested microorganisms, but the susceptibility varies. Gram positives are destroyed rapidly, but Gram negatives are more resistant. Listeria can divide within the phagocytes of mice.

-Primary granules - contain 1) myeloperoxidase which potentates bacterial killing by H₂O₂; 2) neutral and acid hydrolases which degrade bacterial macromolecules; 3) cationic proteins which kills Gram positive bacteria; 4) lysoszyme which destroys the cell walls of Gram positive bacteria.

-Secondary granules - contain 1) lysozyme which destroys the cell walls of Gram positive bacteria; 2) lactoferrin which chelates iron preventing bacterial growth; 3) collagenase which degrades bacterial macromolecules.

-Acid pH - Results from intracellular accumulation of lactic acid (glycolysis). The pH is reduced to 4.0 or lower. This low pH is bacteriostatic or may be bacteriocidal.

-The Respiratory Burst - Bacterial killing from the respiratory burst.

 See Phagocytosis

The Fate of Neutrophils - Neutrophils are unable to replenish their energy. Thus phagocytosis in these cells is a single event (1^st line of the phagocytic defense). Neutrophils do not prepare antigen for the specific immune response.

Eosinophils - The cytoplasm is rich in granules which have a high affinity for the acidic dye eosin. Their number ranges from 2% to 5% of the WBC. These cells are produced in the bone marrow, from where they leave in an immature state and enter the spleen where they reach maturity. The cells now enter the blood stream where they circulate for about 30 min minutes, then enter the tissues where they live for about 12 days. Eosinophils are phagocytic cells, but less efficient than neutrophils. Their more specialized functions is to regulate the inflammatory response and to attack and destroy helminth larvae. Helminth larvae are very large in relation to eosinophils, therefore these larvae cannot be phagocytosed. Attachment of eosinophils to larvae activates a respiratory burst, with release of enzymes to the surrounding tissues. The principle enzyme released is peroxidase. Also released is major basic protein (MPB) which is highly toxic to parasitic worms. The released materials from the eosinophil may do damage to surrounding host tissue.

Basophils - The cytoplasm of these cells is rich in granules that stain intensely with basophilic dyes such as hematoxylin. They make up 0.5% of the circulating WBC. The cells contain a multilobed nucleus, few mitochondria, numerous glycogen granules, and electron-dense membrane-bound granules scattered through the cytoplasm. The granules contain vasoactive amines, like histamine and serotonin. They are related to mast cells. Their principle function appears to be to generate acute inflammation. Their activities appear to be regulated by the eosinophil.

Macrophages -

-Development - The stem cell differentiates into monoblasts which gives rise to promonocytes which leaves the bone marrow, enter the blood stream where they undergo further differentiation into mature monocytes. Monocyte circulate in the blood stream for from 1 to 3 days, during which time they enlarge. They then migrate into the tissues and differentiate into specific tissue macrophages. The transition is morphological including biochemical and functional changes. Macrophages replace themselves at a rate of of about 1% per day.

-Alveolar Macrophages - line the alveoli of the lungs.

-Histiocytes - found in connective tissue.

-Kupffer cells - line the sinusoids of the liver.

-Mesangial cells - found in the kidney.

-Microglia cells - present in the brain.

-Free or wandering macrophages - move by amoeboid movement throughout the tissues. Found in the peritoneum, spleen , lymph nodes, and other organs.

The Structure of Macrophages -  They have a wide variety of shapes, but in suspension they are round with a diameter of about 14 to 20 mm. They have an abundant cytoplasm. The nucleus may be rounded, bean-shaped, or indented. The cytoplasm may be divided into perinulear cytoplasm, which contains mitochondria, many lysosomes, rough endoplasmic reticulum, and a Golgi apparatus. The cells can therefore synthesize and secrete proteins. The peripheral cytoplasm is usually devoid of organelles. It is in continuous motion forming projections, pseudopodia, and veil-like ruffles, looking for material to ingest or cells to interact with.

-Glass-adherent - Macrophages adhere to glass surfaces. Cells can be separated into two populations, those that are glass-adherent, and the non-glass adherent population (generally lymphocytes).

-Enzymes - Macrophages are identified as peroxidase and esterase positive. Lymphocytes are peroxidase and esterase negative.

-Radioresistant - Since macrophages do not generally divide, they can carry out some essential functions following x-irradiation. Cells that divide are referred to as radiosensitive. Evidence has shown that some macrophage populations can divide, for example, 70% of the alveolar macrophages come from division in the lungs, whereas 30% come from monocytes.

-Life History - The life span of macrophages is influenced by their experience with foreign particles. If the material is easily digested, the macrophage may live a normal life span; If the material is inert, like a carbon particle, which cannot be digested, the size of the particle plays a determining role in the life span. With a small inert particle, the cell may live a normal life span; if large, several macrophages may surround the particle, and fuse forming a giant cell in an attempt to eliminate the particle. These giant cells may be expectorated or passed into the intestines for elimination. If the material is toxic, the marcrophage may be killed, followed by ingestion by another macrophage which itself will be killed. Enzymes and other factors may be released causing a chronic inflammation with tissue destruction. Asbestosis is a condition caused by toxic asbestos fiber.

The Function of Macrophages -

1. Destroy foreign particles, as well as, dead or dying tissue through phagocytosis. Phagocytosis by macrophages is similar to that described for neutrophils.

-Chemotaxis - from chemoattractants. Examples: AbAgC reaction products like C5a; bacterial products like N-formylmethionyl peptides; factors released by dead or dying neutrophils; cytokines like IL-8; chemokines like NAP-2.

-Attachment - Numerous surface receptors are present on macrophages.

-CD64 is a protein on monocytes and macrophages and to a lesser extent on polymorphonuclear leukocytes. CD64 binds the Fc of human IgG1 and IgG3 with high affinity. It binds both free and Ag bound antibody. Expression of CD64 is enhanced by INFg.

-CD32 and CD16 on human macrophages bind IgG with lower affinity.

-CD35 is the major receptor for C3b of complement

-CD25 is the receptor for IL-2

-CD71 is the receptor for transferin

Receptors and their distribution on different macrophages may be different, for example, Kupffer cells are rich in CD64 and splenic macrophages are rich in CD35.

2. Macrophages as secretory cells - macrophages have a developed rough endoplasmic reticulum, and a Golgi apparatus. Therefore they synthesize and secrete proteins.

 

Example of the importance of the secretory function of macrophages - Fever production - Body temperature is controlled by the hypothalamus. Certain substances set the body's thermostat higher than 37^o C. For example, phagocytes may ingest a gram negative bacterium releasing LPS. The released LPS will caused the phagocyte to release IL-1. IL-1 causes the hypothalamus to release prostaglandins which resets the hypothalamic thermostat to a higher temperature.

3. Macrophages and wound healing

-Macrophages regulate the secretion of collagenase by fibroblasts with released IL-1.

-Macrophages secrete elastase and collagenase, enzymes that break down connective tissue, then remove the damaged tissue.

-Macrophages participates in tissue remodeling by stimulating the fibroblasts to secrete collagen.

-Macrophages secrete angiogenic factors which promote growth of new blood vessels.

4. Antigen Processing and Presentation - antigens are processed differently depending on source.

-Exogenous antigen - Antigens that originate outside the cell and which are endocytosed. Exogenous antigens are processed in the endocytic pathway. These antigens are presented on the membrane by class II MHC molecules.

Endogenous antigen - Antigens that are synthesized within the cell, e.g., virus protein by a virus infected cell. Endogenous antigens are processed in the cytosolic pathway. These antigens are presented on the membrane by class I MHC molecules.

MHC - Major histocompatibility complex or MHC is a region of DNA that codes for proteins associated with the immune response. In man, the MHC is called HLA and is on chromosome 6; in mice, the MHC is called H-2 and is on chromosome 17. There are two classes of proteins coded by the MHC, class I and class II.

Class I MHC molecules - These proteins are associated with cell mediated responses. These proteins are on the surface of virtually all nucleated cells in the body. They are not present on human red blood cells.

Class II MHC molecules - These proteins are associated with antibody responses. These proteins are on the surface of a limited number of cells in the body including macrophages, dendritic cells, Langerhans cells, veiled cells, B cells and other cells.

Dendritic cells - Have long membrane processes resembling dendrites of nerve cells. These cells are difficult to study because the dendritic processes get damaged during the isolation process. One can use gentler dispersion methods and employ enzymes to digest irrelevant material. The differentiation process of these cells is not well understood. Dendritic cells are classified according to the location where they are. Langerhans cells are in the epidermis and mucous membranes; Interstitial dendritic cells populate most organs including the heart, lungs, liver, kidney and gastrointestinal tract; Interdigitating dendritic cells are found in the T cell areas of secondary lymphoid tissue and in the thymic medulla; Circulating dendritic cells are present in the blood (make up 0.1% of the WBC) and in the lymph (veiled cells). All constitutively express high levels of class II MHC molecules and B7.

 Chapter 5. The Combination of Antigen with Antibody

 Background

-von Behring and Kitasato (1890) - demonstrated that the protective activity induced by vaccination was present in the blood stream.

Emil Roux (1894) - introduced passive immunity. Antibodies are present in the serum.

Anticoagulant - Blood obtained in the presence of anticoagulant can be separated into the formed elements (blood cells) and plasma. Plasma contains the blood clotting proteins along with antibodies, complement, albumin and other blood proteins.

No anticoagulant - Blood obtained without anticoagulant will clot. The clot sticks to the walls of a glass test tube. If the clot is separated from the glass (rimmed) with a stick, the clot will retract. The fluid is called serum. Serum contains antibodies, complement, albumin, and other proteins, but not the blood clotting proteins.

Arrhenius (1903) - Introduced the term immunochemistry, stating that the interactions between antibodies and antigens is chemical.

Precipitation Reaction -

Kraus (1897) - Introduced the fluid precipitin test. He took bacterial filtrates, which he injected into rabbits. He later took the serum containing the antibodies (the precipitin) and incubated it with the bacterial filtrates (the antigen) and got a precipitation reaction (AbAg reaction).

Precipitation reactions - can be used to 1) detect antibody in a serum or antigen in solution, 2) determine the relative strength of an antiserum. For example:

1. BSA -----------> rabbit 1 ----> antiserum 1 + BSA ---> precipitation
2. BSA -----------> rabbit 2 ----> antiserum 2 + BSA ---> precipitation

Which of the two antisera has more antibodies to BSA? Do a two-fold dilution of the antigen (BSA) in saline keeping a final volume of 0.5 ml in each tube. Repeat the same procedure in a second series of tubes. Add 0.5 ml of antiserum 1 to each tube in one set of tubes, and 0.5 ml of antiserum 2 to each tube in the second set of tubes. Look for precipitation in the two sets of tubes. Record the reciprocal of the highest dilution in each set showing precipitation. This is the titer. Compare titers.

Quantitative Precipitin Test - BSA - anti-BSA system

Set up 8 tubes and add 0.1 ml of BSA to each tube, but in the following concentrations in mg:

0, 15, 30, 60, 80, 120, 150 in tubes 1 through 8 respectively.

Incubate for a time period, then centrifuge. Remove the supernatant from each tube and for each, add half to a tube containing BSA and the other half to a tube containing anti-BSA. Look for precipitation and record your results.

Antigen: -, -, -, -, -, +, +, + for tubes 1 through 8 respectively.

Antibody +,+, +, +, -, -, -, - for tubes 1 through 8 respectively.

Total protein: 210, 414, 720, 832, 828, 720, 600 in tubes 2 through 8 respectively.

BSA added:  15, 30, 60, 80, 90, 120, 150 in tubes 2 through 8 respectively.

Antibody by difference: 195, 384, 660, 752, 738, 600, 450 in tubes 2 through 8 respectively.

Plot the total protein in each precipitate vs. antigen added to the tubes.

Show antibody excess (pro-zone), equivalence, and antigen excess (post-zone).

Compare this precipitation to precipitation in an inorganic reaction, for example

NaCl + AgNO₃ ----------> AgCl + NaNO₃

There is a discrepancy in antigen excess when comparing with inorganic precipitation. Why does precipitation drop in antigen excess? Why does the curve not level off as in inorganic chemistry?

Ratio Ab/Ag: 14, 12.8, 11, 9.4, 8.2, 5.0, 3.0 for tubes 2 through 8 respectively.

At equivalence 9.4/2.5 = 3.7 or approximately 4 antibodies/antigen

In tube 1, we have 14/2.5 = 5.6 or approximately 6 antibodies/antigen.

Plot the mole ratio Ab/Ag vs Ag added.

We find that the Ab/Ag mole ratio varies linearly. In Ab excess, the mole ration of Ab/Ag is >>> 1. This shows that more than one antibody molecule can combine with one antigen molecule. In extreme antibody excess, one can determine the number of epitopes that a particular antigen has (antigens are multivalent). Here all the epitopes have reacted with antibody molecules. This is referred to as the limiting mole ration of Ab/Ag.

In antigen excess, the antibody antigen combinations are also soluble. To analyze these, free antigen must be separated from the antibody antigen combinations with the ultracentrifuge. Now do a mole ration of Ab/Ag. As antigen excess increases, the Ab/Ag ratio approaches 0.5. This indicates that the combination is 1 Ab for every two antigen molecules. This shows that antibody molecules have two reactive sites, i.e., antibodies are bivalent.

At equivalence, there is maximum precipitation, because here there is the best proportion of antibodies to antigen molecules resulting in lattice formation.

Go to Lattice Formation

1. It gives an exact expression of the amount of precipitating antibodies in an antiserum. One can compare the potency of two or more antisera.

2. It gives proof that antibodies and antigens react in varying proportions.

3. It gives the functional valence of the antigen.

4. It gives an estimation of the relative heterogeneity of the AbAg system.

5. It gives a distinction between precipitating and flocculating antisera.

6. It gives a measure of the contribution of serum complement to the serologic reaction.

7. It gives a quantitative expression of the amount of non-precipitating antibodies present in the system.

Flocculation - A special case of precipitation in which precipitation is inhibited in extreme antibody excess and in extreme antigen excess. Precipitation is observed over a narrow range of Ab/Ag ratios. Flocculation is seen with horse antisera to diphtheria and tetanus toxoids and with antibodies of people who have a disease called Hashimoto's thyroiditis.

Forces effective in the binding of antibodies to antigen - Include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions. The interaction of each of these bonds is weak (compared to a covalent bond), but the overall interaction is strong because of the large number of these noncovalent interactions in the combination of an antigen with an antibody molecule. Additionally, these noncovalent interactions occur over a very small distance forming a strong interaction and a very close fit between antibody and antigen.

Antibody affinity - Affinity is a thermodynamic term by which one can determine the strength of reaction between two reactants. This can be described for a single epitope, or between antibody and a hapten.

If antibody and H (antigen) have a high affinity for each other, then the equilibrium shifts to the right. If of low affinity, then the equilibrium shifts to the left.

Different antibodies have different affinities for particular antigens. Therefore, some antibodies have a high affinity for antigen, others moderate affinity, and other low affinity, and there are antibodies for the antigen in the range between high affinity and low affinity in a given antiserum. It is therefore, difficulty to describe affinity for antibody antigen reactions. Instead, immunologists describe the strength of reaction between antibody and antigen by the term avidity.

Avidity - A non-thermodynamic term used to describe the strength of reaction between multivalent antigen and a complex mixture of antibodies that react with the antigen. Antigens are multivalent (they have more than one epitope). A typical antigen will induce the formation of numerous antibodies of different affinity to each epitope. Thus an "anti-BSA antibody", for example, is a simple reference of a complex number of antibodies of different affinity to BSA. In precipitation (neutralization) there is cooperation among different antibodies reacting with different epitopes.

Homospecificity of Antibodies - Both reactive sites on an antibody molecule are identical, therefore have the same affinity for an epitope.

Monogamous Antibodies - Generally antibodies cross-link two different antigen molecules (polygamy). Occasionally, if the antigen has two identical determinants that are close to each other, the two reactive sites of the antibody may react with the same antigen (monogamy).

Immunodiffusion - Precipitation in gel procedures introduced during the period 1946 to 1948. Oudin introduced the single diffusion technique; Ouchterlony (1948) introduced the double diffusion techniques. The method can detect as little as 10 mg antibody/ml of serum. One can detect the different number of antibody antigen system one may have in a complex reagent. The morphology of the precipitin line can give information on the MW of the two reactants provided the concentrations are equivalent. The most important aspect of double diffusion is in determining whether two or more antigens are identical, not identical, or partially identical.

Radial Immunodiffusion - A procedure for quantifying the amount of antigen or antibody in a solution. 1. Mix agar in buffer with a uniformly dispersed monospecific antiserum. Add the mixture to a glass plate. 2. After the agar solidifies, make uniform wells in the agar. 3. Add increasing known concentrations of antigen to the wells. 4. Add an unknown concentration of antigen to a new well. 5. Incubate the plate overnight at 37^o C. 6. Measure the diameter of the precipitin rings of the known concentrations. 7. Plot, on paper, the diameter of the rings against antigen concentration to obtain a standard curve. 8. Measure the diameter of the unknown and determine its concentration from the standard curve.

Agglutination - The clumping of particulate antigen by antibody.

Hemagglutination - The clumping of red blood cells.

Passive Hemagglutination - A procedure for increasing the sensitivity of antibody - antigen reactions. 1. Treat sheep red blood cells with tannic acid. 2. Incubate the treated red blood cells with an antigen. The antigen sticks to the cell membrane. 3. Incubate the red blood cells with attached antigen with specific antibodies. The red blood cells should agglutinate.

ELISA - Enzyme Linked Immunosorbant Assay is an extremely sensitive technique for detecting antigen. 1. Attach the antigen to bottoms of wells in a microtiter plate. 2. Block sites on the wells that may not have attached antigen by adding unrelated protein to the wells. Powder milk dissolved in buffer generally works well as a blocking agent. 3. Wash excess blocking agent. 4. Add specific antibody (primary antibody) to the wells and incubate for a time period. 5. Wash unreacted antibody. 6. Add antibodies specific (secondary antibodies) to the primary antibodies and incubate for a specified time. The secondary antibodies have been previously conjugated to an enzyme, like horse radish peroxidase. 7. Wash to remove unreacted secondary antibodies. 8. Develop the reaction by adding the substrate for the enzyme to the wells, in this case 4-chloro-1-naphthol and H₂O₂. A color change indicates that the antigen is recognized by the primary antibody.

Electrophoresis - The migration of charged particles in an electrolyte solution, which occurs when an electric current is passed through the solution.

Isoelectric point - The pI of charged particles is the pH at which the negative charges of the particle equal the positive charges of the particle. The pI of a protein, for example, is that pH at which the protein does not migrate in an electric field. Most proteins have a pI that is more acid then pH 8.0.

Electrophoresis of human serum using zone or simple electrophoresis - Different proteins in human serum have different net charges, therefore migrate at different rates and possibly to different poles. 1. Positively charged particles migrate to the cathod (- pole). 2. Negatively charged particles migrate to the anode (+ pole). Because most proteins have a pI that is more acid then pH 8.0, the buffer used is usually at pH 8.0. This gives the proteins a net negative charge, and will therefore migrate to the anode. Electrophoresis can be done on acetate paper and takes between 30 minutes and 2 hours. Following electrophoresis, the paper strip is removed and the proteins stained with a dye to visualize them. One can now determine the concentration of the separated proteins using a desitometer. A typical separation takes place in the order (from the + pole) albumin, a 1, a 2, b 1, b 2, and g -globulins.

In which fraction, in the electrophoresis separation, are antibodies found? One can determine this by comparing antiserum with normal serum. Hyper-immunize a rabbit with polysaccharide of S. pneumoniae. Incubate the antiserum with the polysaccharide in a test tube. After a time period, centrifuge the tube and remove the supernatant. Compare by normal serum to hyper-immune serum and supernatant by electrophoresis. Normal serum has a normal concentration of g -globulins, the hyper-immune serum has an elevated g -globulin fraction, while the supernatant has a highly reduced g -globulin fraction. Antibodies are therefore g -globulins.

Are all g -globulins antibodies? Chemical analysis of isolated antibodies and isolated g -globulins indicate that they have a similar structure.

Do all antibodies arise from previous exposure to antigen? This cannot be answered unequivocally, but recent evidence indicates that exposure to antigen is necessary for antibody production.

How does one explain the titers of antibodies like anti-sheep red blood cells in normal human serum? If one takes normal human serum and incubates it with sheep red blood cells, the red blood cells will agglutinate, even with no known exposure to the sheep red blood cells (how many of you have been immunized with sheep red blood cells?). These antibodies are referred to as "natural antibodies". There are numerous other examples of natural antibodies, which develop without a known exposure to the antigen. Natural antibodies likely develop from exposure to heterophile antigens.

Immunoelectrophoresis - A procedure for detecting multiple antigens with antibodies to them. This procedure was developed by Grabar and Williams (1953). The procedure is based on simple electrophoresis, but done in an agar medium. 1. A rabbit is immunized with a mixture of antigens (human serum for example) to produce anti-human serum. 2. Agar in buffer is pipetted unto a glass slide, and a well made in the agar after the agar has solidified. 3. The antigen (human serum) is pipetted into the well and then electrophoresed. 4. Following electrophoresis, a trough is made along the long axis of the slide to which anti-human serum is added. 5. The slide is incubated overnight. 6. Precipitin lines will form, which correspond to the separated proteins in human serum. There should be a line for albumin, lines for a , b , and g -globulins.

To determine which precipitin lines in human serum have antibody activity - Electrophorse the antiserum by the immunoelectrophoresis procedure. Add antigen to the trough and incubate the slide overnight. The three precipitin lines that develop should correspond to the proteins in the antiserum that have antibody activity, namely IgG, IgM, and IgA, three classes of precipitating antibodies.

Classes and subclasses of antibodies - There are five classes of antibodies in human serum: IgG, IgM, IgA, IgD, and IgE. IgG has four subclasses, IgG1, IgG2, IgG3, IgG4, and IgA has two subclasses, IgA1 and IgA2.

Column chromatography - One can, by column chromatography and various strategies, isolate proteins from mixtures. For example, IgG can be isolated from human serum. There are various column chromatography methods that can used, for example, one can separate proteins of high molecular mass from proteins of low molecular mass using columns made of Sephadex beads. There are several Sephadex bead sizes. Sephadex beads allow molecules of small molecular mass to enter into the beads, but exclude molecules of large molecular mass. Molecules that enter into the beads take longer to pass through a Sephadex column than those that are excluded. One can also separate molecules according to their overall charge using ion exchange column chromatography. The column is made using particles (beads) containing chemically bound charged groups. There are anion exchangers that exchange negative ions and cation exchangers which exchange positive ions. Proteins with a high overall negative charge will adhere very strongly to beads that carry a positive charge, while proteins with a lower overall negative charge will adhere less tenaciously. The adhered proteins can differentially be released from the beads by increasing the molar concentration of an ionizable compound such as NaCl. Ultrcentrifugation can also, in a crude fashion, separate proteins from mixtures.

Porter (1959) - Isolated IgG by ultracentrifugtion obtaining a 7s molecule. IgM has a sedimentation coefficient of 19s. He treated purified IgG with papain for a short time period, then isolated three fragments using carboxymethyl-cellulose. The three fragments were labeled Fragments I, II, and III. The experiments may have been as follows:

1. Ab + Ag -----> ppt.
2. I + Ag -----> no ppt. + Ab -----> no ppt.
3. II + Ag -----> no ppt. + Ab -----> no ppt.
4. III + Ag -----> no ppt. + Ab -----> ppt.

Fragments I and II inhibited precipitation by intact antibody, therefore called fragments that bind antigen. These were referred to as Fab fragments. Fragment III had no antigen binding activity, but was crystallizable, and was therefore called the Fc. This fragment was later shown to have the same carbohydrate content of the original, untreated antibody molecule.

Nisonoff (1960's) - Treated IgG with pepsin and obtained a 5s fragment and small fragments. The 5s fragment was able to precipitate antibody, therefore having both Fab sites. This fragment was referred to as F(ab')₂ since it clearly was bivalent.

Edelman (1960's) - The chain structure of IgG was first suggested by Edelman by his experiments. Porter confirmed Edelman's studies by treated IgG with 2-mercaptoethanol reduction and alkylation, a chemical treatment that irreversibly cleaves disulfide bonds. The sample was chromatographed using a column that separated molecules according to size. Two molecular species were isolated, one of 25,000 and the other of 50,000. Using simple calculations and logic, it was proposed that IgG is composed of 2 L (light) chains of 25,000 kD each, and 2 H (heavy) chains of 50,000 kD each, which equals to 150,000, the molecular mass of intact IgG. Porter and Edelman received the Nobel Prize in 1972 for their contributions to the elucidation of the structure of IgG.

Go to Antibody Structure 

 Hinge - The hinge in immunoglobulins. IgG specific for the hapten, dinitrophenol, was reacted with the divalent form of the hapten, then prepared for observation by electronmicroscopy. The negative stain, phosphotungstic acid was used to make the molecules more dense since this solution penetrates the space between the atoms of the molecules. The proteins were visualized as long objects, triangles, squares, and pentagons. These structures represent complexes of antibodies reacting with divalent hapten, forming dimers, trimers, tetramers. The angle between the arms of the Y-shaped antibody molecules varies in the different complexes, reflecting the flexibility of the hinge region, allowing the IgG molecules to open or close over a 180^o angle. The amino acids proline and cysteine are prominent in the hinge region extending the chain and making them accessible to proteolytic cleavage, for example, by pepsin and papain.

Variations in Immunoglobulin Structure - Electrophoresis of IgG shows a wide range of electrophoretic mobility, from slow g to the a2 globulins. The range of mobilities is due to different net charges on the different IgG molecules. This wide mobility indicates variations in amino acid structure. Even purified IgG against a hapten may show a wide spectrum of electrophoretic mobilities. This represents a variety of IgG molecules to the hapten of varying degrees of fit. Variations result from a polyclonal response to particular antigens.

Myeloma proteins - Multiple myeloma is a cancer of antibody producing cells. These cells divide in an unregulated manner, each resembling its parental cell. They may or may not be capable of secreting antibodies, depending on the particular myeloma. Some synthesize and secrete complete antibodies, others secrete L chains, while others may secrete H chains. Myeloma proteins in the blood stream are referred to as M (monoclonal) proteins. Light chains, because of their size, can be excreted in the urine. These are referred to as Bence- Jones proteins. One can produce antibodies against M proteins or against Bence-Jones proteins in rabbits, then used the rabbit antibodies to study the relationships among different antibodies or L and H chains.

Isolate Bence-Jones proteins from Pt1 and immunize a rabbit to produce anti-Pt1 L chain antibodies. Now use the antibodies to determine whether Bence-Jone proteins from Pt1, Pt2, Pt3, Pt4, Pt5, etc. are recognized. In this scheme, Bence-Jones proteins from some patients are recognized, but not all. Antibodies to Bence-Jones proteins in one of the group that was not recognized by the anti-Pt1 antibodies, recognized a second group of Bence-Jones proteins. By double diffusion testing, it was shown that antibodies to Bence-Jones proteins recognized two different types of L chain. These are called k and l L chains. Antibodies have L chains that are either k or l, but never a mixture of the two.

Using the same strategy, but with H chains from multiple myeloma, the different types of heavy chains were discovered. There are five different types of H chains which are referred to as g, m, a, d, and e. The five classes are IgG (k or l L chains and g H chains), IgM (k or l L chains and m H chains), IgA (k or l L chains and a H chains), IgD (k or l L chains and d H chains), and IgE (k or l L chains and e H chains). There are 4 subclasses of IgG referred to as IgG1, IgG2, IgG3, and IgG4, and two subclasses of IgA referred to as IgA1 and IgA2.

IgG and subclasses - a monomer, having 2 g H chains and 2 k or 2 l L chains. Its molecular weight is 150,000. Its % carbohydrate by weight is 3%. It has two antigen binding sites. It is the most abundant immunoglobulin class in the internal fluids where it fights microbes and toxins. In serum, it makes up about 80% of the total serum immunoglobulins. It has four H chain domains, VH, CH1, CH2, and CH3.

IgG1 - makes up 65% of the total IgG in serum. Its H chain is g1, it has 2 inter H chain S-S bonds, it can activate complement, it can cross the placenta, and has a high affinity to Fc receptors on phagocytic cells. Its half-life is 23 days.

IgG2 - makes up 24% of the total IgG in serum. Its H chain is g2, it has 4 inter H chain S-S bonds, it is relatively inefficient at activating complement, it does not cross the placenta, and has an extremely low affinity to Fc receptors on phagocytic cells. Its half-life is 23 days.

IgG3 - makes up 7% of the total IgG in serum. Its H chain is g3, it has 13 inter H chain S-S bonds, it can activate complement very effectively, it can cross the placenta, and has a high affinity to Fc receptors on phagocytic cells. Its half-life is 8 days.

IgG4 - makes up 4% of the total IgG in serum. Its H chain is g4, it has 4 inter H chain S-S bonds, it cannot activate complement, it can cross the placenta, and has an intermediate affinity to Fc receptors on phagocytic cells. Its half-life is 23 days.

IgM - In serum, IgM is a pentamer, having 10 m H chains and ten k or ten l L chains. Its molecular weight is 900,000. J chain with a MW of 15,000, binds two of the subunits of this Ig by an S-S bond. Its % carbohydrate by weight is 12%. It has ten antigen binding sites, but because of steric hindrence only 5 will bind most antigens. It is a very effective agglutinin; produced early in the response, it is an effective first line of defense against bacteria. In serum, it makes up about 6% of the total serum immunoglobulins with a serum concentration range between 0.5 to 2 mg/ml. Its size prevents it from leaving the blood stream efficiently, but may be found in secretions. It has five H chain domains, VH, CH1, CH2, CH3, and CH4. It activates the classical pathway of complement, it cannot cross the placenta, but does interact with Fc receptors on phagocytic cells. Its half-life is 5 days.

Monomeric IgM, with a MW of 180,000 is expressed as a membrane bound antibody on B cells, acting as the antigen receptor.

IgA - In serum, IgA exists primarily as a monomer, having two a H chains and two k or two l L chains. However, dimers, trimers, and even tetramers are sometimes seen. Its molecular weight ranges from 150,000 to 600,000. It is the predominant immunoglobulin in external secretions such as brest milk, saliva, tears, and mucus of the bronchial, genitourinary, and digestive tracts. In external secretions (secretory IgA) is either a dimer or a trimer possing J chain and secretory piece of MW 70,000. Its % carbohydrate by weight is 7%. It has two, four, or more antigen binding sites, depending its molecular makeup. In serum, it makes up about 6% of the total serum immunoglobulins with a serum concentration range between 1.4 to 4 mg/ml. It has four H chain domains, VH, CH1, CH2, and CH3. It does not activates the classical pathway of complement, but may activate the alternative pathway. It cannot cross the placenta, nor interact with Fc receptors on phagocytic cells. Its half-life is 6 days.

IgE - In serum, IgE is a monomer, having 2 e H chains and 2 k or 2 l L chains. Its molecular weight is 190,000. Its % carbohydrate by weight is 12%. It has two antigen binding sites. It protects external surfaces, is important in generating an inflammatory response, increases in helminth parasitic infection, and is responsible for the symptoms of atopic allergies. In serum, it makes up about 0.002% of the total serum immunoglobulins with a serum concentration range between 17 to 450 ng/ml. It has five H chain domains, VH, CH1, CH2, CH3, and CH4. It does not activate complement, it cannot cross the placenta, but does interact with Fc receptors with high affinity on mast and basophils. Its half-life in serum is 2 days.

IgD - In serum, IgD is a monomer, having 2 d H chains and 2 k or 2 l L chains. Its molecular weight is 150,000. Its % carbohydrate by weight is 12%. It has two antigen binding sites. Because of a highly exposed hinge region, it is highly susceptible to proteolytic cleavage in serum. For example, blood clotting proteases that normally become active when blood is exposed to air, readily hydrolyze IgD. IgD is normally expressed as a membrane bound antibody on virgin B cells, acting as the antigen receptor. In serum, it makes up about 0 to 1% of the total serum immunoglobulins with a serum concentration range between 0 to 0.4 mg/ml. It has four H chain domains, VH, CH1, CH2, and CH3. Its half-life is 3 days.

Immunoglobulins as Antigens - Injection of human antibodies into a rabbit will result in the production by the rabbit of antibodies that react with the human antibodies. There are three principal antigenic determinants on human antibodies, isotypic, allotypic, and idiotypic.

Isotypic determinants - These are determinants that distinguish the immunoglobulin classes and subclasses from each other. These determinants are localized in the Fc region of the heavy chains. For example, antibodies against the m chain recognize IgM, but not any of the other immunoglobulin classes or subclasses, and antibodies against the g chain recognize IgG and not any of the other immunoglobulin classes. All normal humans have five classes of antibodies, four subclasses of IgG, and two subclasses of IgA. Each of the H chains has antigenic determinants that will induce antibody formation. The antibodies against isotypic determinants can be used to distinguish the classes and subclasses.

Allotypic determinants - These are determinants on particular classes of immunoglobulins that can be used to distinguish that class of antibody in one individual from the same class of antibody in another individual. For example, Mary's IgG's can be distinguished from Jim's IgG's based on these determinants. Allotypic markers have been demonstrated for one IgA subclass, the A2m(1) and A2m(2), for all the IgG subclasses, for example G1m(1), and G2m(23), and for the k L chain, the km markers. The allotypes are inherited as co-dominant alleles. For example, some of Mary's IgG2's may have the G2m(23) determinant, whereas the other IgG2's may have the G2m(17) determinant, the G2m(23) coming from the father, and the G2m(17) from the mother.

Idiotypic determinants - These determinants are localized in the antigen binding region of the antibody. Since this part of the immunoglobulin is specific for an antigen, it must differ from antibodies that are specific for another antigen. For example, an anti-BSA antibody should be different from an anti-CEA antibody even if the two antibodies come from the same animal. This difference should be in the antigen combining region, because this region in each antibody has a unique amino acid sequence.

The Generation of Antibody Diversity (Theories of antibody production) - There are two general groups of theories, the selective and the instructive.

Side-chain hypothesis - Paul Ehrlich (1900) formulated the side-chain hypothesis. He believed that cells in the body have side-chain used for interacting with foreign particles. The side-chains are normally used for interacting with food particles, which are ingested and metabolized. Whenever a particular food particle is ingested, a particular side-chain is over expressed to meet the demand. The same thing takes place with toxins. Side-chains that are specific for the toxin interact with the toxin followed by ingestion of the toxin. This results in an over-production of the side-chain, which is then released into the serum. These are the antibodies. This theory is a selective theory.

Karl Landsteiner - Using haptens, Landsteiner showed that antibodies could be synthesized to almost any organic molecule conceivable. It was therefore impossible for a cell to have all the side-chains necessary for the multitude of antigens that the immune system can respond to. There just simply is not enough room on the cell surface to accommodate all the necessary side-chains for this diversity of responses. A theory of antibody production must also account for the variability of antibody molecules.

Early theories- assumed, because the variability of antibodies was so great, that antibodies could not be preformed. They must be synthesized on demand following exposure. Therefore it was suggested that antigen instructs the cell about the specificity of the antibody. Breinl and Haurowitz initially formulated this theory, stating that the antigen serves as a template. Linus Pauling (1940) thought that all antibody molecules are identical before interaction with antigen. Once a molecule interacts with protein, it folds into a specific antibody and subsequently can only react with the specific antigen. His ideas were discarded because experimental evidence did not support them, for example, it could not be shown that antigen was present when antibodies were being synthesized. The template theories were finally discarded when they could not be reconciled with the realities of protein synthesis. In the central dogma, information flows from DNA to RNA to protein and not the reverse.

Germline theories - An alternative group of theories, which state that all the information needed to make all possible antibodies is stored in the animal's genome. Here all that is necessary is that the correct gene be selected following exposure to antigen. The problem is that now one needs literally millions of genes for all possible antibody specificities. There simply is not enough room in the germline DNA for all the possible antibodies that can be synthesized. Now how about all the other genes?

Amino Acid Analysis of L and H chains - Bence-Jones proteins are L chains that can be isolated from the urine of patient with a myeloma. Bence-Jones proteins from different patients were isolated and sequenced. A pattern evolved. The L chains had a region of some 110 amino acids in length starting from the amino terminus that was variable, and starting with amino acid 111 to 214 the amino acid sequence was constant. The H chains from patients with Waldenstrom's macroglobulinemia were also sequenced showing a similar pattern that of a variable region and a constant region. Dryer and Bennett (1965) postulated that both L and H chains are each coded by two genes, one for the variable region and the second for the constant region. A model emerged in which there are many V (variable) genes (DNA), any one of which can associate with a C (constant) gene (DNA) giving a DNA segment that has a contiguous VC stretch of DNA.

Domain structure of IgG - The L chain is about 214 amino acid residues in length. The V region extends from amino acid 1 (NH₂ terminus) to amino acid 110 from where begins the constant region. There is an intrachain disulfide bond between amino acids 22 and 88 forming a domain in the V region. This domain is referred to as the VL domain. A second intrachain disulfide bond between amino acid 134 and amino acid 194 in the constant region forms the CL domain.

The H chain is about 446 amino acid residues in length with a variable region extending from amino acid 1 (NH₂ terminus) to about amino acid 110 where the C region begins. There is an intrachain disulfide bond between amino acid residues 22 and 88 forming the VH domain in the V region. A disulfide bond between residues 144 and 200 form the CH1 domain, the disulfide bond between residues 261 and 321 form the CH2 domain, and the disulfide bond between residues 367 and 425 form the CH3 domain. The CH2 domain has the carbohydrate moiety and is the site for attachment of C1 of the complement system. The CH3 domain serves for bonding to the Fc receptor on phagocytes. Each domain forms a 60 amino acid loop, referred to as the Ig-fold.

Complementarity-determining regions - Amino acid analysis of the VH domain revealed regions of hypervariability, first shown by Kabat and Wu as Kabat-Wu plots. These are referred to as CDR's for Complementarity-determining regions. The H chain has three CDR's, CDR1 between amino acids 30 and 36, CDR2 between amino acids 49 and 65, and CDR3 between amino acids 95 and 103. The L chain also has three CDR's, CDR1 between amino acids 24 and 34, CDR2 between amino acids 50 and 56, and CDR3 between amino acids 89 and 97. The regions between the CDR's which are relatively constant, are referred to as framework regions. The hypervariable regions form the actual site to which the epitope binds. The binding between epitope and antibody involves the cleft that is formed between the L and H chain CDR's. These regions determine the shape of the antigen binding site, and which epitope(s) an antibody will bind.

Genes that code for antibody molecules - From sequencing studies, a number of features about the immunoglobulin molecule were difficult to reconcile with classic genetic models. An explanation was needed for each of the following: 1) the vast diversity of antibody specificities; 2) the presence of a variable region at the amino terminal and a constant region at the carboxyl end of both the H chain and the L chain; and 3) the existence of isotypes with the same antigenic specificity.

Susumu Tonegawa experiment (1976) - He extracted mRNA from a myeloma cell line and labeled the kL chain mRNA with ³²P. He also extracted DNA from the myeloma cells and from embryonic cells, and treated the DNA with restriction enzymes. The restriction fragments were recovered treated to denature to single strands, then electrophoresed and the fragments probed by hybridization with the ³²P labeled mRNA. The gel was developed with photographic film. The myeloma DNA fragments gave one radioactive band, while the embryonic DNA fragments gave two radioactive bands. This implied that there are two segments of DNA that are responsible for coding for the L chain in embryonic cells, and only one in antibody producing cells. This further implies that the DNA gets rearranged during antibody cell development.

The general coding for the L chain is as follows: V (variable) exon codes for amino acids 1 through 95, the J (joining) exon codes for amino acids 96 through 110, and the C (constant) exon codes for amino acids 111 through 214.

• l Light Chain Gene Family - in humans on chromosome 22; in mice on chromosome 16. The mouse l L chain gene family arrangement is 5' L, Vl2, Jl2, Cl2, Jl4, Cl4, L, Vl1, Jl3, Jl1, Cl1 3'. Jl4 and Cl4 are defective. In humans, there are about 100 Vl genes, each preceded by an L segment; The Vl genes are followed by 6 Jl segments, and 1 Cl segment.
• k Light Chain Gene Family - in humans on chromosome 2; in mice on chromosome 6. The mouse k L chain gene family is composed of about 300 Vk segments each preceded by an adjacent L segment; there are 5 Jk one of which is non-functional and 1 Ck. The human k chain family has approximately 100 Vk segments, 5 Jk segments, and 1 Ck segment. There are subclasses for the k chain in either mouse or human.
• The H Chain Gene Family - on chromosome 14 in humans; on chromosome 12 in mice. In mice, there are between 300 and 1000 VH segments followed by a cluster of about 13 DH (D for diversity) segments. Each V segment is preceded by an L segment. The D segments are followed by 4 JH segments, followed by a series of CH gene segments. Each CH segment encodes the constant region of an immunoglobulin heavy chain isotype. The CH gene segments are arranged sequentially Cm, Cd, Cg3, Cg1, Cg2b, Cg2a, Ce, Ca. In humans, there are between 100 and 300 VH segments, 5 DH 9 J segments. The CH gene segments are arranged sequentially Cm, Cd, Cg3, Cg1, Ca1, Cg2, Cg4, Ce, Ca2.

 Variable-Region Gene Rearrangements - Variable gene rearrangements occur in an ordered sequence during B cell maturation in the bone marrow. The variable region genes for the H chain rearrange first, then the variable region genes for the L chain, but in the end process the B cell contains a single V region DNA sequence for its H chain and a single V region for its L chain. This leads to the generation of at least 1.9 x 10⁷ B cells, each with its own membrane bound antibodies with an ability to respond to a particular antigenic epitope.

Generation of Diversity in the Human-

• VJ combinations - k chain: 80 V segments X 5 J segments = 400 possibilities.
• VDJ combinations - H chain: 300 V segments X 5 D segments X 9 J segments = 13,500 possibilities. A = V X D X J
• Number of splice sites - 2 for the H chain and 1 for the L chain.
• Five base variation at each splice site (B=A(X5)ⁿ) - 3 X 10⁵ for the H chain; 2000 for the L chain. n = the number of splice sites.
• N-region insertion at each splice site (C=B(X5461)ⁿ) - 9 X 10¹³ for H chain; 2 X 10³ for L chain. Up to six bases may be inserted at each splice site. This can give 5461 different possible combinations of four bases, that is, 4⁶ + 4⁵ + 4⁴ + 4³ + 4² + 4¹ + 4⁰.
• L and H chain combinations - 1.8 X 10¹⁶. Because sequences (in the splice sites) have to be read in frame, this theoretical number is high. The actual number of antibody possibilities is probably closer to 1.9 X 10⁷.

Overview of B Cell Development - B cells develop in two compartments. The first of the two compartments is the bone marrow. B cell differentiation in the bone marrow is antigen independent. The second compartment is the periphery (lymph nodes, spleen), where B cell differentiation  is antigen dependent. In the bone marrow, the lymphoid stem cells differentiate into Pro-B cells, which express B220 (a lymphoid cell marker), but not Ig on their surface. The H chain genes rearrange in Pro-B cells giving rise to Pre-B cells, which express m chains in their cytoplasm. The m chain associates with a surrogate L chain.  The L chain genes rearrange in Pre-B cells giving rise to immature B cells. Immature B cells express mIgM. A change in RNA processing in immature B cells results in the synthesis of d chains and the formation of mature B cells. Mature B cells express both mIgM and mIgD. These cells leave the bone marrow, enter the periphery (lymph nodes, spleen) by circulating through the blood stream. Mature B cells are activated by interacting with antigen. Activated B cell differentiate into plasma cells (which secrete antibodies) and into memory cells (which are long lived). Activated B cells may also switch Ig classes.

See B Cell Development

Free Antibody vs Antigen-bound Antibody - Antigen-bound antibody acquires new activites because of conformational changes that occur when bound to antigen.  For example, antigen-bound antibody acquires the ability to bind to phagocytic cells by the Fc receptor and act as an opsonin.  New antigenic determinants on antigen-bound antibodies may provoke the immune system to produce autoantibodies like rheumatoid factor.  New sites, like on the CH2 domain on the Fc region, are also exposed, and with which C1 of complement can interact. 

Complement Discovery - Pfieffer (1894) immunized one set of guinea pigs by injecting them i.p. with V. cholerae.  A second set of guinea pigs was injected i.p. with saline and used as a control. Later, both sets were injected i.p. with V. cholerae, then a short time later the peritoneal exudate was examined for the microorganism.  In those that had been immunized, the microorganisms had disintegrated, whereas in the control group, the microorganisms were found intact.   Incubation of serum from an immunized animal with the microorganism also resulted in disintegration. 

        Studies by others gave the following results:  1) Bacteria ---> animal ---> serum + bacteria ---> disintegration.   2) SRBC ----> animal ---> serum + SRBC ---> lysis.  The material in serum that lysed bacteria and red blood cells was given the name "alexin" which in Greek means "to ward off".  The material was thought to be antibody.

Bordet - Conclusively demonstrated that the serum component was not antibody by using three reagents and the following set of experiments.  1) Goat anti-BRBC + BRBC ---> lysis.  2) Normal goat serum + BRBC ----> no lysis.   3) Goat anti-BRBC left at room temperature for several days + BRBC ----> no lysis.  4)  Goat anti-BRBC, 56^o C for 30 min + BRBC ---> no lysis.  5) Goat anti-BRBC, 56^o C for 30 min + fresh normal goat serum + BRBC ---> lysis.   Alexin was renamed and now called complement.  Bordet received a Nobel Prize for this work.  Complement is now well known.  There are two pathways, the classical pathway and the alternate pathway. 

Reaction Sequence in Immune Cytolysis -

1.  E + A ----> EA    where E = erythrocyte; A = antibody.  Activation of complement requires one IgM or two IgG molecules.  The two IgG molecules must be closely spaced for complement activation.  There is no complement activation with anti-Rh antibodies.  The interaction of the antibody with antigen causes a conformational change of the antibody molecule exposing sites on the CH2 site of IgG which can now react with C1q of complement.

2.  EA + C1 + Ca²⁺ -----> EAC1    C1 = C1q, C1r, C1s which are connected loosely (non covalent bond) by Ca²⁺.   C1q is the recognition unit.  It interacts with the CH2 domain of IgG.   C1q is composed of 6 triple stranded subunits.  These are 6A + 6B + 6C = 18 peptides.  The molecule looks like 6 tulips which extend from a vase.  C1r and C1s together form a complex of two molecules each.  These are intersparsed within the peptides of C1q which extend from vase portion of C1q.  They look like a figure 8 between C1q strands. 

    C1q binding to the Fc results in a conformational change of C1q that is transmitted to C1r.  C1r becomes an active enzyme whose substrate is C1s.  A small peptide is cleaved from C1s, the remainder now becoming an active enzyme whose substrate is C4.  C1 may also be activated directly by E. coli, Klebsiella pneumoniae, C reactive protein, and myelin.

3.  EAC1s + C4 ------> C4b + C4a    C4b binds to the Fab of the antibody or to the membrane directly, or may bind to the membrane of nearby cells.

4.  EAC1s + C4bC2 ----> C4bC2b + C2a      C2 can bind to C4b attached to the Fab.  This interaction requires Mg²⁺.  C2a has potent vasoactive activity.

5.  C4bC2b + C3 ---> C3b + C3a    C4bC2b complex is an enzyme referred to as the classical pathway C3 convertase.  Its substrate is C3.  This is a critical reaction because the reaction is amplified (C3 is the complement in highest concentration in serum). Up to 200 C3 molecules can be cleaved by one C4bC2b complex.  C3b attaches to the membrane at different sites and to the membrane of nearby cells.  C3b can opsonize particles like bacteria or cells.   Opsonized particles and cells are readily phagocytosed because phagocytes have receptors on their membrane for C3b.  C3a is an anaphylotoxin capable of degranulating mast cells and basophils.  C5 attaches to C3b.

6.  C4bC2b + C3bC5 ---->   C5b + C5a      If the C5 which is attached to C3b is close to the C4bC2b complex it will be cleaved by the C4bC2b into C5b and C5a.    C5a is an anaphylotoxin, and is also a chemotaxin.  Cleavage of C5 begins the terminal portion of complement and the membrane attack complex, the MAC.

10. C5C6C7C8 initiates the polymerization of C9. 

11. C9 is polymerized on the cell membrane  From 10 to 18 molecules of C9 polymerize completing the MAC.  Polymerized C9 forms a tube of pore size ranging from 70 to 100 A.  During polymerization, the C9 molecules undergo a hydrophilic-amphiphilic transition, therby inserting into the membrane.  Pore formation results in osmotic lysis of the cell.

Alternate Pathway - (May be referred to as the alternative pathway). Under normal circumstances, low levels of C3 breakdown into C3a and C3b.  C3b is either inactivated or it initiates the alternate pathway.  This depends on the surface that C3b attaches to and its affinity for Factor H.  If C3b attaches to Factor H, C3b is cleaved by Factor I into C3bi, an inactive form of C3b.  Sialic acid enhances attachment of C3b to Factor H.  If sialic acid is deficient, for example as in bacterial cells, C3b binding to Factor H is depressed and C3b becomes activated.   C3b activation can result from attachment to virus-infected cells, some tumors, cell walls of bacteria (peptidoglycan, LPS), and other cells and molecules.

1. Activated C3b + Factor B ---> C3bB   C3b binds Factor B forming the C3bB complex.

2. C3bB ------> C3bBb + Ba    Factor D, an enzyme cleaves B of the C3bB complex into Bb and Ba which is inactive. C3bBb is known as the alternate pathway C3 convertase.

3. C3 ----> C3b + C3a    C3 is cleaved by C3bBb.   C3bBb may interact with Factor P (properdin) and may become stabilized. Many C3's can now be cleaved.  C3b can attach to cell membranes.  If C5  interacts with C3b, it can be attacked by C3bBb to form C5b and C5a leading to formation of the MAC.

Regulation - The consequences of complement activation are significant and may be potentially dangerous.  Therefore complement needs regulation.

Control Proteins -

• C1 inhibitor (C1-INH) - binds to and blocks activaties of active C1r and C1s.  A congenital deficiency of C1-INH results in a disease called hereditary angioedema.  In this condition the C2a fragment is produced in high quantities.   C2a has vasoactive activity.
• C4 binding protein - binds C4b, preventing formation of C4b2b.
• Factor S - binds soluble C5b67 and prevents its insertion into the cell membrane.
• Other - include Factors H, I, and P.

Complement Receptors -

• CR1 (CD35) - Present on RBC, neutrophils, eosinophils, monocytes, macrophages, B cells, some T cells.
• CR2 (CD21) - Present on B cells.  Binds C3d.
• CR3 (CD11b/CD18) - Binds C3bi.
• CR4 (CD11b/CD18) - Binds C3bi.

Consequences of Complement Activation

• Destroys invading microbes directly or via inflammation.
• Complement-mediated opsonization - CD35 binds C3b, C4b and C3bi.
• Complement-mediated secretion - non-primate platelets have CD35.  Interaction with C3b causes the release of pharmacological agents from platelets.
• Removal of immune complexes - It is important to remove antibody-antigen complexes from circulation.  Ab-Ag complexes interact with complement producing C3b.  This results in removal of the complexes by phagocytes.  In homozygous deficiencies of C1q, C1r, C1s, C4 or C2 results in an increase of immune complex disease, for example systemic lupus erythematosus, glomerulonephritis, and vasculitis. There is a need to generate C3b for solubilization and clearance of immune complexes.
• Complement -mediated cytolysis - generation of the MAC.
• Complement-mediated chemotaxis - generation of C5a, C5b67, C3e, Bb
• Complement-mediated inflammation - generation of C3a and C5a.
• Complement-mediated immune regulation.

Early Experiments - 1) Antibody production by splenectomized guinea pigs and normal guinea pigs was compared.  Normal guinea pigs produced greater amounts of antibody than splenectomized guinea pigs. The conclusion was that antibodies were synthesized in the spleen and at other sites. 2) Removal of other organs like the pancreas, and parts of the small intestine had no specific action on the immune response. 3) Aschoff (1924) delineated the reticuloendothelial system (RES).  The RES is a collection of cells of different origin and morphology that are united by their common property of phagocytosis.   Shortly thereafter, others proposed that phagocytic cells constituted the major, if not the sole source of antibody.  This conclusion came from experiments like a) injection of India ink into an animal results in macrophages becoming engorged with India ink because of phagocytosis.  When antigen was injected into these animals they produced less antibody than animals not injected with India ink prior to injection of antigen. b) Animals subjected to x-rays, than injected with antigen, produce less antibodies than animals not subjected to x-rays, because the x-ray kill macrophages (x-rays also kill other cells). c) The histologic studies by Sabin (1939) detected antigen in the RES after intradermal and subcutaneous injection of antigen.  Antigen was detected in alveolar macrophages and Kupffer cells when antigen was injected by intravenous route. Shortly after antigen detection in the phagocytes, antibodies were produced.  From these observation, it was falsely concluded that macrophages synthesize antibody.

Experiments by Burnet, Erhlich, Harris - Antigen injected into the footpads of rabbits resulted in hyperplasia of the draining lymph node.  The afferent and efferent lymph channels were cannulated to obtain lymph which was tested for the presence of specific antibodies.  The efferent lymph contained specific antibodies in much greater quantity than the afferent lymph. This showed that antibodies are also produce in lymph nodes.

See Lymph Node

Wagner and Chase and Adoptive Transfer Experiments - Antigen was injected into rats.  Later lymph node cells were removed and injected into other rats of the same strain which then produced the specific antibodies. This showed that cells in the lymph nodes produce antibodies.

Bjornebe and Gormsen (1943) - Hyperimmunized rabbits by injecting them with eight different strains of S. pneumoniae.  This resulted in hyperglobulinemia (lots of antibodies). There was also a pronounced production of plasma cells in lymph nodes.  The number of plasma cells was proportional to the concentration of antibody protein found in the serum.

Coons, et al. - developed the fluorescent antibody sandwich technique.   The idea was to detect the antibody producing cells.  Antigen was injected into an animal, then the antibodies were purified and labeled with fluorescein isothiocyanate (FITC).  Lymph nodes were removed from an animal immunized with the same antigen.  The tissue was sectioned, covered with the antigen, washed, then covered with the labeled antibody. Cells with specific antibody on their membrane captured the antigen.  The captured antigen now reacted with the FITC-labeled antibody forming a sandwich (cell-antibody-antigen-antibody-FITC).  The tissue was observed with a fluorescence microscope. Cells with specific antibody were found in colonies called germinal centers in the cortex of the lymph node.  These cells multiply forming large cells which migrate to the medulla where they transform into plasma cells.

See Lymphoid System

Nossal - introduced the microdroplet technique.  Nossal took lymph nodes from a rat immunized with flagella from Salmonella, teased the lymph node tissue forming a cell suspension.  By diluting the suspension, he transferred individual cells into small chambers (like a 96-well plate) containing nutrient medium.   He then added motile Salmonella into each chamber.  The idea here was to look for immobilization of the Salmonella as evidence that the cells had produced antibodies that reacted with the flagella. He was also able to stain the cells and identify them.  He observed 601 cells, of which 93 produced antibodies that immobilized Salmonella.  Of the antibody producing cells, 91 were clearly identified as plasmablasts.  This experiment confirmed that the antibody producing cells are of the plasmacyte series.

Thymus -

J.F.A.P. Miller (1962) - The disease, lymphocytic leukemia in certain strains of  mice, have the thymus as the target organ.  Miller was studying the effect of thymectomy on the production of the disease.  He thymectomized some mice at birth and others at various intervals after birth.  He observed that in mice that had be thymectomized as neonates had a marked depletion of lymphocytes as they matured.   The depletion of lymphocytes gave rise to a serious impairment of immune function.  

• Neonatal thymectomy - 1) These mice occasionally became runted. 2) There was a drop in circulating lymphocytes, 3) a marked depression in their ability to mount an immune response, i.e. they become very susceptible to infectious agents.  4) Their capacity to reject graft became depressed. 5) Antibody-mediated immunity also became depressed, but less obvious.
• Adult thymectomy - These mice had no immediate obvious effect, but later there was a progressive decline in the number of circulating lymphocytes and cell-mediated immune responses.

DiGeorge (1965) - Described a congenital disease in human infants characterized by 1) an absence of a thymus at birth.  As result, there was a serious impairment of immune function. 2) The serum contained less than normal amounts of immunoglobulin. 3) These patients suffered from severe viral, fungal, and bacterial infections which became chronic. 4) If the individuals were vaccinated, for example with Vaccinia virus (attenuated virus), the patients succumbed to the virus. 5) There was no delayed type hypersensitivity in these patients. 6) The lymph nodes and the spleen had a decreased number of small to medium sized lymphocytes.  The plasma cell number was also less than normal.  One patient was reconstituted immunologically with a thymus graft from a fetus that had been aborted at 13 weeks.  This patient displayed normal T cell activity within 48 hours.  He was vaccinated against smallpox one month later.

Thymus Size - 1) Newborn - has the greatest relative size. 2) Puberty - greatest absolute size.  3) Post-puberty - the thymic parenchyma atrophies.   Thymic remnants may persist until old age.

Bruton (1952) - Described a congenital agammaglobulinemia in man.   These patients 1) have an absence or a marked deficiency of immunoglobulins in the serum. 2) They have no plasma cells in the lymphatic tissue. 3) They have a prevalence of severe bacterial infections.

Bursa of Fabricius - A lymphoepithelial organ in birds, but not in mammals.  The organ lies dorsal to the cloaca.

Glick (1954) - at Ohio State University, was a graduate student in poultry science, working with chickens.  He wanted to know whether removal of the bursa of Fabricius shortly after birth influenced the size of the chicken or the quality of chicken meat much like castration produces capons.  His experiments were fruitless.   Timothy Chang used the bursectomized chickens to demonstrate antibody production to the immunology class.  Of nine bursectomized chickens, six died immediately following injection with Salmonella typhimurium O antigen, and the three that survived produced no antibodies against Salmonella. The experiments with bursectomized chickens were repeated.  A manuscript was prepared and submitted to Science which rejected the paper because the results were not of wide scientific interest.  The paper was later published in Poultry Science, and overlooked until 1960.  Bursectomy was shown to deprive birds of the humoral immune response, i.e. a a much depressed antibody production. A search, thus began for the bursal equivalent in mammals.  Candidates included the tonsils, the appendix, and the gut associated lymphoid tissue (GALT).

Two Populations of Lymphocytes - 1) T lymphocytes are processed by or in some way are dependent on the thymus.  2) B lymphocytes are bursa-dependent and function as antibody synthesizing cells.  In mammals, the B lymphocytes are derived from the bone marrow.

Ontogeny - Development of immunocompetent cells.  1.) In chickens, the pluripotent stem cells from the yolk sac enter the primary lymphoid organs, the thymus and the bursa.  The stem cells that enter the thymus undergo differentiation becoming T cells; stem cells that enter the bursa undergo differentiation becoming B cells.  T and B cells, by way of the circulation, populate the secondary lymphoid organs.  The secondary lymphoid organs in birds include the spleen and the lymph masses. 2.) In mammals, the stem cells come from the yolk sac.  After progenitor T cells formed during hematopoiesis enter the thymus, they undergo maturation, leave the thymus and populate the secondary lymphoid organs.  The B cells are formed in the fetal liver in early fetal development.  Later the fetal spleen becomes the principle site where B cells are formed.  Late in fetal development the bone marrow begins to take over this function and becomes the sole organ for B cell development after birth.  The cells through the circulation populate the secondary lymphoid organs, the spleen and the lymph nodes.  The primary lymphoid organs regulate the production and differentiation of lymphocytes.

The Thymus -  is a flat bilobed organ surrounded by a capsule.   A cross-section of a lobe reveals that the organ is divided into lobules.  The lobules are section by trabeculae (connective tissue) and each lobule having a cortex, the outermost part of the lobule, and a medulla, the centermost portion of a lobule. The cortex is densely populated with lymphocytes called thymocytes, while the medulla is less densely populated by thymocytes.  Both the cortex and the medulla are criss-crossed by a cellular network (stroma) composed of epithelial cells, interdigitating dendritic cells, and macrophages.  These cells contribute to the maturation of T cells.   Certain epithelial cells in the outer cortex, called nurse cells, appear like the letter C, and have been observed to surround as many as 50 thymocytes.  Other epithelial cells have long extensions which interact with with numerous thymocytes.   Interdigitating dendritic cells also have long extensions that interact with developing thymocytes.  Structures called Hassall's corpuscles are prominent in the medulla. Its function is not clear.

Thymic Hormones - At least four different hormones have been isolated from the thymus purported to be synthesized by thymic epithelial cells. Thymic hormones that have been characterized are a???????1-thymosin, b???????4-thymosin, thymopoietin, and thymulin.   Precursor T cells in the presence of a thymic hormone will acquire T cell characteristics.  Thymic stromal cells also secrete IL-7 which stimulates thymocyte growth.

T Cell Diversity - T cells have receptors specific for antigen, each T cell developed in the thymus having its own specific receptor.  The receptor is called the T cell receptor (TCR).  The TCR is generated in the thymus by random gene rearrangements analogous to immunoglobulin H and L chain gene rearrangements.  It is estimated that 1 X 10⁶ precursor T cells enter the thymus daily, each developing its specific TCR.  Among these thymocytes, some will develop TCR's that are autoreactive and potentially dangerous to the host. As developing thymocytes begin to express antigen-binding receptors, they become subject to a selection process.  Only T cells that can recognize antigenic peptide in association with MHC molecules can leave the thymus.  Thymic stromal cells which express high levels of class I and class II MHC molecules play a role in this selection process.  T cells unable to recognize self-MHC molecules or which have a high affinity for self-MHC molecules are eliminated by apoptosis (programmed cell death).  It is estimated that between 95% and 99% of all thymocyte progeny undergo apoptosis. The external signal (if present) that triggers apoptosis is not known, but may be a glucocorticoid, since cortical thymocytes are extremely sensitive to glucocorticoids.  Apoptosis is gene activated.

Programmed Cell Death - Cells die by apoptosis in order to maintain a steady-state level of hematopoietic cells.  Each of the hematopoietic cells, whether myeloid, mononuclear, or lymphoid have a limited life span.  For example neutrophils in the circulation have a life span of about 1 day, then die by programmed cell death.   1.) Cells undergoing apoptosis experience a mild convolution, chromatin compaction and segregation, and condensation of the cytoplasm.  The nucleus fragments and the cell forms blebs releasing them to the surrounding medium (apoptotic bodies).  The apoptotic bodies are removed by phagocytosis. 2.) Cells undergoing a necrotic death differ in characteristics.  There is chromatin clumping, swollen organelles, and flocculent mitochondria in these cells.  The cells disintegrate with release of intracellular contents.  The end result is inflammation.

Bursa - A lymphoepithelial organ in birds.  Reaches maximum size on to two weeks after the chick has hatched, then undergoes a gradual involution.   Bursectomy at hatch produces a drop in circulating lymphocytes.  The bird produces very small quantities of antibodies and suffers the loss of antibody producing plasma cells.  The bursa may produce hormones needed for B cell differentiation.   The hormone bursin, isolated from the bursa, activators B cells, but not T cells.

Chapter 9.  Lymphocytes and B Cell Responses

Morphology - The size ranges from small to large, ranging in size from 7 mm to 15 mm in diameter.  The cells are spherical, with a nucleus that is large, round, and stains intensely and evenly with dyes.  The cytoplasm is thin with respect to the nucleus, containing some mitochondria, free ribosomes, and a small Golgi apparatus.  By scanning electron microscopy, the surface is smooth in some, but other lymphocytes may have hairy projections or villous processes.  Lymphocytes cannot be differentiated on the basis of morphology.  They are defined on the basis of operational terms, including ontogeny, surface receptors, enzyme content, and characteristics of cell surface antigens.

Identification of T and B cells - From a morphological stand point, there are little differences between T and B cells using conventional stains and either light or electron microscopy.

T lymphocytes - 1) Mouse - In the 1960's, mouse brain cells were injected into a rabbit to produce anti-brain cells antibodies.  These antibodies recognized T cells but not B cells.  The antigen on mouse T cells recognized by these antibodies came to be known as the Thy-1 antigen.  Treatment of mouse spleen cells with anti-Thy-1 + complement lysed T cells, but not B cells.  2) Human - Incubation of blood lymphocytes with sheep red blood cells produced spontaneous rosettes.  The T cells were surrounded by the SRBC's, but not the B cells.  The receptor on T cells for SRBC's is the membrane protein called CD2.

B lymphocytes - In the 1960's, immunoglobulins were injected into a rabbit to produce anti-Ig  antibodies.  These antibodies recognized B cells but not T cells. B cells therefore have immunoglobulins (antibodies) on their membrane. 

EA Rosettes - Incubation of SRBC with anti-SRBC antibodies produces EA's.  Incubation of EA's with lymphocytes results in B cells, but not T cells, surrounded with the EA's.  Therefore, B cells have a receptor for Fc of IgG, since EA's are red blood cells covered by antibodies reacting with the Fab's and having the Fc's project away from the cells.

EAC Rosettes -  Incubation of SRBC with anti-SRBC antibodies produces EA's.  If the EA's are further incubated with less than lytic amounts of complement, the EA's will be coated with C3b, and are now referred to as EAC's.  If the EAC's are incubated with lymphocytes, the B cells, but not the T cells, will be surrounded by the EAC's.  Therefore, B cells have a receptor for C3b of complement.

Fc Receptors for g chain (IgG) -

• FcgRI - CD64; 75 KD; on monocytes and macrophages; high affinity receptor; functions in phagocytosis.
• FcgRII - CD32; 39-48 KD; on B cells, macrophages, and eosinophils; medium affinity receptor; functions in phagocytosis for macrophages, but inhibits B cells activity.
• FcgRIII - CD16; 50-65KD; on NK cells, granulocytes, and macrophages; low affinity receptor; functions in phagocytosis for granulocytes, has killer activity for NK cells (ADCC).

Isolation of Lymphocytes From Blood - Overlay whole blood (blood collected with an anti-coagulant) on a Ficoll-Hypaque cushion in a test tube.  The Ficoll-Hypaque has a density higher than the density of lymphocytes, but lower than the density of red blood cells or the other white blood cells.  Following centrifugation, the lymphocytes can be recovered from the interface between the plasma and the Ficoll-Hypaque.  The red blood cells and the other white blood cells will centrifuge to the bottom of the test tube.

Characteristics of Lymphocytes in Human Blood -

• B Cells - 16 to 28% of human blood lymphocytes have Ig on their membrane.  Of these, 4 to 12.7% have IgG on their membrane, 1 to 4.3 have IgA, 6.7 to 13% have IgM, and 5.2 to 8.2 have IgD.
• T Cells - 65 to 75% of human blood lymphocytes form spontaneous (E) rosettes and have CD2 on their membrane.
• NK Cells (or Null Cells) - 9% of human blood lymphocytes do not have Ig on their surface nor form E rosettes.

Lymphocyte Mitogens - Substances that induce cells to undergo mitosis.   Phytohemagglutinin (PHA), a lectin isolated from the red kidney bean, is a mitogen for T cells.  Concanavalin A ( Con A), a lectin isolated from the Jack bean, is also a T cell mitogen.  Pokeweed mitogen (PWM), isolated from the pokeweed plant, induces both T and B cells to divide.  LPS is a mitogen for mouse B cells.  One can demonstrate mitogen activity by incubating lymphocytes in culture with the mitogen for 48 hours, then adding [3H]thymidine and further incubating the culture for 5 hours.  The cells are now harvested and the incorporated radioactivity determined with a scintillation counter.  Control cells (no mitogen) should have cpm of less than 300, whereas those with mitogen will have counts of over 10,000 cpm. Thymidine gets incorporated into the DNA of dividing cells. Mitogens interact with surface molecules on lymphocytes, sending a signal to the nucleus inducing the cells to divide.  A parallel can be observed with specific lymphocytes and antigen, resulting in cell division.

Synthesis of Antibodies -

Jerne Plaque Assay - Mice are injected with SRBC's.  Five days later, the spleen (or lymph nodes) is removed, the cells teased apart, and placed in a petri dish with agarose and a uniform distribution of SRBC's.  The plate is incubated overnight, then complement is added.  Lymphocytes producing anti-SRBC antibodies will have a clear lytic zone surrounding them.  The antibodies bind to the SRBC's which complement lyses.  This direct method of enumerating antibody producing cells measures IgM producing cells since it only takes one IgM to activate complement.  In the indirect method, anti IgG is added to the culture, which will interact with IgG on the RBC's.  Now there are at least two closely spaced IgG antibodies on the RBC's and complement can be activated.  This indirect method measures both IgG and IgM. Subtraction of IgM from the total will give the IgG producing cells. Using specific markers for B and T cells, it can shown that B cells produce antibodies and not T cells.

Cooperation Between T and B cells in Antibody Formation - 1) If one thymectomizes mice as neonates, they lose their T cells.  They also lose their ability to synthesize normal amounts of antibodies.  2) Mice that are exposed to lethal doses of X-irradiation die, unless infused with white blood cells.   (Irradiation kills dividing cells, such as skin cells, cells in the intestine, and the white cells).  Irradiated mice thus became living test tubes, adaptable to studying  the interaction of T and B cells in the immune response.  Thymus and bone marrow cells were obtained from compatible mice.  Three groups of irradiated compatible mice were then prepared for study.  Thymus cells were given to group I, bone marrow cells to group II, and a combination of thymus and bone marrow cells to group III.  The three groups of mice were then immunized with SRBC's, and later examined for the production of anti-SRBC antibodies.  Group I produced no anti-SRBC antibodies, group II produced low amounts of anti-SRBC antibodies, but group III produced normal amounts of anti-SRBC antibodies.  This demonstrated that both T and B cells were necessary for production of normal amounts of antibody, the B cells producing antibodies and the T cells acting as helper cells.

Two Classes of Antigens - 1) T-dependent antigens - require T cell help, for example SRBC's. The vast majority of antigens are T-dependent.  2) T-independent antigens - do not require T cell help for antibody production.   Polymerized flagellin (repeating subunits) is an example.

In Vitro Immunization - developed by Mishell and Dutton in the late 1960's.  Spleen cells in culture medium are incubated with SRBC's for 3 to 5 days, then complement is added to the culture. The SRBC that surround antibody producing cells are lysed.  In vitro immunization provided a means for studying the generation of antibody producing cells in vitro.  For example, cells could be separated according to their glass adherent properties.  Following separation, neither glass adherent cells nor non-adherent cells in culture alone when incubated with SRBC's generated anti-SRBC antibodies.  However, if adherent and non-adherent cells were incubated together and with SRBC's the cells produced normal amounts of anti-SRBC antibodies.   Thus adherent and non-adherent cells were seen to cooperate in the generation of antibody-forming cells.  Glass adherent cells are macrophages; non-adherent cells are lymphocytes.

The Three Cell Concept - Antigen is phagocytosed by macrophages and processed.  The antigenic determinant is presented on the cell surface to specific T and B cells which have receptors for the determinant.  The macrophages secrete IL-1 which induces both T and B cells to divide and undergo differentiation.  The activated T cells now secrete IL-2 which further influences B cell differention into plasma cells which secrete specific antibodies, and into memory cells.

B Cell Surface Molecules - B cells bind antigen through their antigen receptor.  The antigen receptors are antibodies on the surface of B cells.   There are between 20,000 and 200,000 antibody molecules per cell each on a given cell having the same specificity.

Response of B Cells to Antigen - The first experience of an animal with an antigen results in a primary response. Immature B cells have mIgM and mIgD on their surface. Both surface antibodies have the same antigen recognition ability, but different constant regions of their respective H chains.  Immature B cells that do not interact with antigen are short lived (they live for a few days than die by apoptosis).  B cells that interact with antigen divide into plasma cells, which produce IgM (primary response), and into memory cells.  Memory cells are long lived (they may live 10 to 12 years).  During maturation, the memory cells undergo a class switch from IgM to some other class of antibody.  For example, an immature B cell's mIgM H chain may have V23D2J3Cm gene segments, whereas the memory cell's mIgG H chain will have V23D2J3Cg3 if the mIgG is IgG3.  The V region remains the same, but the C region changes.  In a secondary response, the memory cell will be activated and the generated plasma cells will secrete IgG3 antibodies.

Go to LC Produce AB

The B Cell Receptor (BCR) - In order for the B cell to become activated, its activation genes must be activated from a cell surface signal.  A surface signal comes from interaction of the antibody on the membrane with antigen.   However, because the cytoplasmic tails of antibodies are too short, they are unable to transmit a signal to the nucleus.  mIgM and mIgD have cytoplasmic tails that are only three amino acid residues in length; mIgA's cytoplasmic tail is 14 amino acids in length; mIgG and mIgE have cytoplasmic tails of 28 amino acid residues.  These cytoplasmic tails are too short, and cannot therefore associate with intracellular signaling molecules, for example with tyrosine kinase, and G protein.  The membrane bound immunoglobulins are associated with two peptide, Iga which has a 61 amino acid cytoplasmic tail, and Igb which has a 48 amino acid cytoplasmic tail.  The B cell receptor is therfore composed of antibody in association with Iga and Igb. Interaction of membrane bound antibody with antigen transmits a signal to the Iga and Igb peptides which can now become phosphorylated. The biochemical details of B cell activation are fairly well known.

Polyclonal Response - Antigens are polyvalent, having more than one epitope.  Injection of a polyvalent antigen into an animal will produce a polyclonal response. It is estimated that for each epitope from 50 to 300 antibodies of different affinity will be produced. The affinity of these antibodies will range from low affinity to high affinity.  There is one B cell for each of the 50 to 300 antibodies, each having its own unique antigen receptors on its membrane.  Each of the 50 to 300 B cells will be activated by the epitope, each dividing producing B cell clones.  If the antigen has four epitopes, then between 200 and 1200 clones of B cells will be produced.

Monoclonal Response - Plasma cell tumors may produce complete antibodies.  Because these tumors are derived from a single parental cell, the cells are of a single clone and the antibodies that they produce are monoclonal.  These antibodies have all have the same specificity and affinity.  It is generally not known what antigen the antibodies will react with.

Hybridomas - Kholer and Milstein first developed hybridomas that produce monoclonal antibodies in 1975.  They received a Nobel Prize for their work in 1982.  With this technology, one can produce monoclonal antibodies to almost any antigen.  Hybridoma formation is based on cell fusions by incubating two different cells in the presence of a surface active agent.  Characteristics of the two cells - 1) NS-1 cell - This is an established myeloma cell line (immortal) derived from the BALB/c mouse. It is a non-secretor meaning that it does not secrete antibodies.  It also lacks the enzyme hypoxanthin-guanine-phosphoribosyl transferase (HGPRTase).  This enzyme is needed in one of the salvage pathways of nucleotide biosynthesis.  These cells in culture must continuously synthesize nucleotides de novo using the folic acid pathway.  If one adds aminopterin, a folic acid analogue, to the culture medium, de novo synthesis will cease.  Because these cells lack HGPRTase, they cannot synthesize nucleotides by the salvage pathway, even though one may provide the nucleosides hypoxanthin and thimidine.  2) The antibody synthesizing cell - These cells are not established, and will therefore die after 10 to 12 days in culture. They can, however, synthesize nucleotides de novo via the folic acid pathway and can also recover nucleosides from the culture medium by the salvage pathway because they have HGPRTase.

Monoclonal Antibodies - An antigen is injected into BALB/c mice.   The spleen is removed after the mouse produces antibodies to the antigen, and teased to obtain spleen cells.  The spleen cells are fused to NS-1 cells using polyethylene glycol (PEG) to produce hybridomas.  The differnet hybridomas produced will be B:NS-1, T:NS-1, B:B, T:T, NS-1:NS-1, and others.  The fused cells are cultured in hypoxanthin-aminopterin-thmidine (HAT) medium.  NS-1:NS-1 hybridomas cannot survive because de novo synthesis is blocked in these cells (aminopterin) and they lack the HGPRTase enzyme to recover the nucleosides hypoxanthin and thymidine. B:B, T:T, B:T die after 10 to 12 days in culture because they are not established (they lack the immortality genes).  The only cells that survive are hybridomas formed between spleen cells and NS-1 cells, i.e. B:NS-1, T:NS-1.  The cells are cultured in 96 well plates.  Later, the supernatants are tested for specific antibody.  The cells in antibody producing cultures are then isolated by limiting dilution so that a well in a microtiter plate will contain one cell.  For example, if you had 100 cells in 10 ml, and the distribution of the cells in the medium was uniform, each 100 ml would contain 1 cell.  Therefore, transferring 100 ml to a well would transfer one cell.  The cells divide in the presence of IL-2, and essentially the cell has been cloned and the antibodies that the cells produce are monoclonal.  Hybridomas can be frozen, thawed and cultured, sent to anywhere in the world, to collegues who can culture the cells and produce the same monoclonal antibody.

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Lyt Antigen - Antigens associated with functional characteristics of T cells in mice.  These antigens were used to demonstrate the existence of subpopulations of thymus-derived lymphocytes.  There are three well defined Lyt antigens: 1) Lyt 1 is a glycoprotein of 67,000 daltons and is encoded by a gene on chromosome 19 in the mouse.  2) Lyt 2 and Lyt 3 are glycoproteins of 35,000 daltons and encoded by genes on chromosome 6 in the mouse.  If one treats lymph node T cells with anti-Lyt 1 and complement, the Lyt 1 cells lyse.  Treating lymph node T cells with anti-Lyt 2,3 and complement  results in lysis of Lyt 2, 3 cells.

Test for Helper Activity in Antibody Production Using the Mischell-Dutton Method - Spleen cells are taken from a mouse and four variations of an experiments set up. 1) Spleen cells are treated with normal mouse serum (NMS)and complement (C).  2) Spleen cells are treated with anti-Thy-1 and C. 3) Spleen cells are treated with anti-Lyt 1 and C.  4) Spleen cells are treated with anti-Lyt 2,3 and C.  In each, the surviving cells are cultured with SRBC's for five day, then C added to determine whether spleen cells producing anti-SRBC's have been generated.  Spleen cells treated with NMS and C produce normal amounts of antibody producing cells (this is the control).  Spleen cells treated with anti-Thy-1 and C generated very few antibody producing cells (no helper T cells).  Spleen cells treated with anti-Lyt 1 and C generated very few antibody producing cells, but cells treated with anti-Lyt 2,3 and C generated normal amounts of antibody producing cells.  This indicates that the helper T cells are cells having the Lyt 1 antigen since treatment of these cells with anti-Lyt 1 and C destroyed the helper activity of T cells.  Destruction of the Lyt 2, 3 + cells had no effect on antibody cell generation indicating that Lyt 2,3 T cells are not helper T cells in the generation of antibody producing cells.

Helper T Cell Phenotype - Thy 1⁺, Lyt 1⁺, Lyt 2,3^- .  Helper T cells are now referred to as CD4 cells because they have CD4 on their membrane.

The Response of T cells to Antigen - T cells have different functions, which collectively are referred to as cell mediated immunity. T cells are essential for protection against 1) intracellular bacteria, 2) viruses and virus-infected cells, 3)   and some tumors.  4) They are also primarily responsible for rejection of foreign grafts, 5) they mediate the inflammatory response called delayed type hypersensitivity (DTH), 6) and they regulate the immune response in which T helper cells up-regulate the response and T suppressors cells down-regulate the response.  7) T cells also differentiate into T memory cells, and 8) produce lymphokines.

Demonstration of T cell Activity -

Delayed Type Hypersensitivity (also referred to as Type IV Hypersensitivity) - Koch (1890) in an experiment injected viable M. tuberculosis subcutaneously into a group of non-sensitive guinea pigs and observed a mild reaction at the site of injection.  He also injected viable M. tuberculosis subcutaneously into sensitive (M. tuberculosis immunized) guinea pigs. These guinea pigs experienced a slowly evolving, but intense reaction at the site of injection.  Koch's observations were repeated by others using other microorganisms.  The reaction was commonly referred to as Koch's phenomenon.

Cutaneous Reaction - Delayed type hypersensitivity reactions are used in the United States as diagnostic tests to demonstrate either a clinical or a sub-clinical experience of a person with a particular microorganism.  DTH, for example, can be observed following injection of  0.1 mg of PPD (purified protein derivative, an extract from M. tuberculosis) intradermally.  A sensitized person will see erythema (redness) and swelling (induration, hard to the touch) at the site of injection, which appears gradually 10 hours after injection.  Maximum intensity and size will occur between 24 and 72 hours (48 hours average) following injection.  The reaction will gradually subside and disappear over several days. Skins tests have been developed to TB, mumps, histoplasmosis, Candida, and to numerous other microorganisms.

Transfer of DTH - 1) Injection of PPD into guinea pigs can sensitize them.  If PPD is injected intracutaneously into sensitized guinea pigs, a DTH reaction will occur (they are said to be skin test positive).  Injection of serum from a PPD sensitized guinea pig into a non-sensitized guinea pig (which is skin test negative) will not transfer DTH reactivity. That is, the non-sensitized guinea pig will not become skin test positive, showing that serum does not transfer reactivity and that antibodies are not responsible for DTH.  2) Lymphocytes from a PPD sensitized guinea pig injected into a non-sensitized guinea pig (skin test negative) will transfer DTH reactivity. That is, the non-sensitized guinea pig will become skin test positive.   This shows that lymphocytes are capable of transferring DTH reactivity from a sensitized guinea pig to a non-sensitized guinea pig, and that lymphocytes are responsible for DTH.  If the lymphocytes are treated with anti-T cell antibodies and complement, the surviving cells cannot transfer DTH.  This shows that T cells are responsible for DTH.  The phenotype of T cells involved in DTH appears to be heterogeneous.   These cells are referred to as TDH cells.

Cytotoxic T Cells (Cytotoxic Lympholysis)- 1) Mixed Lymphocyte Reaction - Lymphocytes from two different individuals (or different mouse strains) are mixed (two-way test) and incubated in culture for 5 days, after which ³H-Thy is added to the culture.  Five hours later the cells are harvested, and if they recognize foreign antigens, they will incorporated ³H-Thy into their DNA because they have divided (mounted an immune response). Cells treated with mitomycin C are unable to divide when exposed to foreign antigens, because mitomycin C is an inhibitor of cell division. Therefore, by treating one of the two cell sources with mitomycin C, one can show cell division in the other cell source. This is referred to as a one-way test. 2) Cytotoxic Lympholysis (CTL) - Lymphocytes from two different individuals (or different mouse strains), one of which has been treated with mitomycin C, are mixed and incubated for 5 days, after which ⁵¹Cr-labeled target cells (cells with the same surface antigens as the cells that were treated with mitomycin C) are added to the culture.   Five hours later, the supernatant of the culture is recovered and will be radioactive if the target cells have been attacked and lysed (the ⁵¹Cr has been released) by the cells that were not treated with mitomycin C.  This response is a cell-mediated immune response in which cytotoxic (killer) cells have been generated.   The cells responsible for cytotoxic lympholysis (the effector cells) are T cells and their phenotype in mice is Thy-1^+, Lyt 1^-, Lyt 2,3⁺.   These cells are now referred to as CD8⁺ T cells. CD8⁺ T cells require CD4⁺ (Thy-1^+, Lyt 1⁺, Lyt 2,3^-) T cells (helper cells) to mediate the response.  In the mixed lymphocyte reaction outlined above, the cells that divide are both CD4⁺ T cells and CD8⁺ T cells.  Therefore to mount a cytotoxic response, both CD4⁺ T cells and CD8⁺ T cells recognize the antigen on the foreign cells and divide (like in the mixed lymphocyte reaction), the CD4⁺ T cells being required for activating the CD8⁺ T cells and making them killer cells.  The CD4⁺ T cells produce IL-2 which is self-activating (autocrine) inducing CD4⁺ T cells to divide, and also activates (paracrine) the CD8⁺ T cells inducing them to divide, differentiate and become effector (killer) cells and memory cells.  CD4⁺ T cells also become memory T cells.

Suppressor T Cells - These cells were demonstrated by the following experiment:  A high dose of keyhole limpet hymocyanin (KLH) was injected into mice.   Later spleen cells or thymus cells were injected into genetically identical mice, then injected with KLH conjugated to a hapten.  The response to the hapten was low.   Repeat experiments, but with donor cells first treated with anti-T cell antibodies, resulted in normal responses to the hapten.  These experiments were interpreted as T cells suppressing the anti-H response. Suppressor T cells are actually CD8⁺ T cells.

Major Surface Molecules That Characterize Human Cells Involved in the Immune Response -

• All T Cells - CD2⁺ (molecules involved in cell to cell adhesion interactions, and coincidentally also bind to a surface molecule on SRBC's), CD3⁺, CD11⁺
• Helper T Cells - CD4⁺
• Suppressor and Cytotoxic T Cells - CD8⁺
• All B Cells - sIg⁺; Epstein-Barr virus receptors (It is not known why B cells have a receptor for this virus), Fc receptors.
• Macrophages, Neutrophils, Eosinophils, Basophils, and Mast Cells - Complement component receptors and Fc receptors.

The T Cell Receptor (TCR) - The TCR is a heterodimer composed of either a and b or g and d chains.  The TCR is associated with a signal transducing molecular complex called CD3.  About 95% of T cells in humans express the ab heterodimer, whereas 2% to 5% of T cells in humans express the gd heterodimers. Both a and b chains have two external domains containing intrachain disulfide bonds that bind spans of about 60 to 75 amino acids. These domains highly resemble the Ig domains in size and molecular conformation. Molecules with this conformation (the Ig fold) are members of the Ig superfamily.  There is a marked sequence variation in the amino terminal (first) domains of each of the two TCR chains, whereas the second domain is conserved.  Thus the TCR domains, one V and one C, are structurally homologous to the V and C domains of antibodies.  The two chains are interconnected by an interchain disulfide bond which is located close to the membrane.   A transmembrane span, consisting of 21 to 22 amino acids anchors the TCR to the membrane. The transmembrane span of each chain is unusual in that they contain positively charged amino acids, which enables the TCR heterodimer to interact with CD3.  Each chain has a short cytoplasmic domain of 5 to 12 amino acids at the carboxyl terminal end. These cytoplasmic domains are too short for signal transduction. Therefore the TCR is associated with CD3 which serves for signal transduction.  The TCR associated with CD3 is referred as the TCR-CD3 complex.  TCR diversity is generated in an analogous fashion to the diversity generated for the Ig molecules.  Approximately 10¹⁵ ab TCRs and 10¹⁸ gd TCR's can be generated, more than enough to cover any foreign antigen that the T cells may encounter.

CD3 - This is a complex of five invariant chains that associate to form three dimers.  One heterodimer is composed of ge chains, a second of de and a homodimer of zz (zeta zeta) chains or zh (zeta eta) chains.  The cytoplasmic tails of the zz chains (and zh chains) have three amino acid segments called immunoreceptor tyrosine-based activation motifs (ITAMs), whereas the ge chains and de chains each has one ITAM, motifs that are similar to those found in the Ig-a and Ig-b heterodimers of the B cell receptor.  ITAMs interact with tyrosine kinases playing an important role in signal transduction to the genes in the nucleus.

Ig Superfamily Members - Immunoglobulin, T-cell Receptor, CD3, Major Histocompatibility Complex molecules, CD2, CD4, CD8, Fc receptor, and other molecules.

Response to Foreign Tissue Grafts - Why graft rejection since transplantation is entirely artificial?  There is no counterpart in nature.  In the twentieth century, surgical grafting and organ transplatation procedures have been perfected.  With the use of immunosuppressive drugs, grafts can be accepted for prolonged periods. Graft rejection is an immune response to foreign tissue possessing foreign antigens, by a mechanism much like the mixed lymphocyte reaction and cytotoxic lympholysis. However, with the use of immunosuppressive drugs, there is also an increase in risk to the development of tumors.  It is possible that foreign grafts are rejected because the body needs a system that can recognize abnormal cells and reject them.  Foreign or abnormal cells include 1) foreign grafts, 2) cells infected with viruses, 3) chemically altered cells, and 4) cancer cells.   It is probable that this form of immune response is largely directed against tumors. 

Types of Grafts - 1) Allograft - these are grafts from a donor and a recipient who differ genetically.  These grafts are regularly rejected.  2) Autograft - the grafting of tissue from one part of the body to another part, for example skin from the buttocks to the face (cheek to cheek). Autografts are  accepted. 3) Isograft - the donor and the recipient are in this type of grafting are genetically identical, for example a graft identical twins, or from a BALB/c mouse to a BALB/c. Inbred strains of mice are genetically identical.  4) Xenograft - the graft is between genetically different individuals who are members of different species.  Examples are grafts between a mouse and a cat, of a chimpanzee and a human.

The Rejection Process - Rejection is mediated by lymphocytes.   Rejection of a skin allograft takes about 12 to 14 days. At first the grafted skin appears healthy.  The blood vessels anastomose connecting the graft to underlying tissue.  The skin graft takes on a healthy color, becoming functional (hair may grow, the sweat glands function; if the graft had been an organ like a kidney, it would start producing urine).  By day seven, the graft begins to pale, and to degenerate. The tissues surround the newly formed vessels become increasingly infiltrated with white blood cells, especially lymphocytes.  The graft finally dies, and if not removed, it is shed.  Destruction of the graft occurs because the blood supply is cut off.   There is degeneration of the connecting blood vessels which is brought about by lymphocytes.  This slow rejection process is known as a first set rejection.    Now if a graft is again done using the same donor and the same recipient, the rejection rate becomes more rapid with complete rejection in 5 to 7 days.  In this rejection process, the underlying tissues may also become inflamed.  This rejection is referred to as a second-set rejection.  Comparing the intesity and rate of rejection, the first set rejection process is slower and less intense than the second set rejection.  This shows that immunological memory is involved in graft rejection.

Comparison of an Antibody Response With Graft Rejection - 1) Antibody response - In the primary response, antibody production is low.  In the secondary response, antibody production is high and requires less time for antibody to be produced following antigen stimulation. 2) Graft rejection - In the first set rejection, rejection is delayed (12 to 14 days).  In the second set rejection, rejection is accelerated (5 to 7 days) and the rejection is more intense.  Both antibody and graft rejection involve memory and both are specific responses.  That is, for a secondary antibody response or second set rejection, the antigen or graft must be the same as in the primary antibody response or first set rejection process.

Examples of Grafts - 1) A graft from a BALB/c mouse to a C3H mouse will result in a first set rejection.  A graft from a second BALB/c mouse to the same C3H recipient will result in a second set rejection. 2) A skin graft from a BALB/c mouse to a C3H mouse will result in a first set rejection.  Transplantation of a kidney from BALB/c moust to the same C3H recipient will result in an accelerated rejection.   3) Grafting of BALB/c tumor cells into a BALB/c recipient will result in tumor growth.  Grafting of BALB/c tumor cells into a C3H recipient will result in tumor cell death (rejection).

Acceptance of Allografts - 1) Privileged sites - allografts may flourish in privileged for prolonged periods without inducing immunity. Privileged sites include a) the meninges of the brain, b) anterior chamber of the eye, c) cheek pouch of the hamster (good for growing tumors).  2) Pregnancy - A child inherits antigens, called histocompatibility antigens, from both parents.  Those that the child inherits from the father should be foreign to the mother and theoretically should mount an immune response and the child aborted.  This intrauterine foreign allograft (fetus) however, is not rejected.  The reason is not completely clear, but may be a result of mucinous secretion masking the antigens on the trophoblast.

Graft vs Host Reaction (GVHR) - Infusion of adult spleen cells into a neonate (especially if the neonate has undergone a thymectomy) results in a graft vs host reaction.  The neonate undergoes what is described as the runting syndrome.  It fails to gain weight normally, it develops skin lesions and has diarrhea.  It will probably die within a few weeks. The spleen becomes greatly enlarged (splenomegaly).   The grafted spleen cells (which are immunocompetent) recognize the host (neonate) cells as foreign and mount an immune response.  Transplantation in adults generally involves immunosuppression of the host to avoid organ, tissue or cell rejection.   Implantation of bone marrow (stem) cells into an immunosuppressed host may result in GVHR if the bone marrow cell preparation contains mature T cells.

Histocompatibility Antigens - Allografts are rejected because antigens on donor cells are different from the antigens on recipient cells.  The foreign antigens are recognized and an immune response is mounted against them resulting in graft rejection.  The recognized antigens on the donor cells are referred to as histocompatibility antigens.  Some antigens are referred as minor provoking a small response, and others are referred to as major histocompatibility antigens, which provoke the major response in graft rejection.

The Major Histocompatibility Complex (MHC) - Every individual possesses its own characteristic set of major histocompatibility antigens.  The set is acquired from its parents, one-half coming from one parent, and the other half of the set coming from the second parent.  These genes are therefore co-dominant.  The mating of two homozygous, but dissimilar animals, results in offspring that have one set of histocompatibility antigens that are compatible with one parent and a second set which are compatible with the second parent.  Therefore, the offspring can accept grafts from either parent, but neither parent can accept grafts from the offspring.  The MHC is a segment of DNA with genes that code for proteins important in the immune response.   In man, the MHC is referred to as the HLA (human leukocyte antigen), and in mice the MHC is referred to as the H-2 complex. 

H-2 Complex - The H-2 of mice is on chromosome 17.  The gene sequence is K, I , complement genes, TNF-a, TNF-b, D, L, R, Qa, and Tla.  K codes for H-2K, I codes for IA and IE, complement genes code for certain complement components, D codes for H-2D, L codes for H-2L, and R codes for H-2R.  H-2K, H-2D, H-2L, and H-2R are class I molecules;  IA and IE are class II proteins, and complement proteins have been referred to as class III. The Qa gene codes for Qa, and the Tla gene codes for Tla.   Qa and Tla are strong antigens that appear at specific times during differentiation. 

The HLA - The HLA is on chromosome 6.  The gene sequence is DP, DQ, DR, complement genes, TNF-a, TNF-b, B, C, and A.  DP codes for HLA-DP, DQ codes for HLA-DQ, and DR codes for HLA-DR proteins.  These molecules are MHC class II molecules.  The complement genes code for certain complement components.  B codes for HLA-B, C codes for HLA-C, and A codes for HLA-A.  These molecules are MHC class I molecules.

Comparison of MHC Class I and Class II Molecules -

• Class I - The molecules include HLA-A, -B, and -C in man, and H-2K, -D, -L, and -R in the mouse.  They can be detected by serology or by PCR.  Their distribution is on most nucleated cells.   Their function is to present antigen to CD8⁺ T cells.  The result of presentation is the generation of cytotoxic T cells.
• Class II - The molecules include HLA-DP, -DQ, and -DR in man, and H-2IA, and -IE in the mouse.  They can be detected by serology, MLR or by PCR.  Their distribution is on antigen presenting cells (APC's) like macrophages, dendritic cells, and B cells. Their function is to present antigen to CD4⁺ T cells.  The result of presentation is the generation of  T cell-mediated help.

HLA Molecules - Membrane-bound glycoproteins, concerned with cell to cell interactions in the immune response. 

Molecular Description of Class I Molecules - A class I molecule is a dimer composed of an a chain associated by non-covalent, hydrophobic interactions with a b chain.  Only the a chain is coded by the MHC.  The a chain has three external domains, a1, a2 and a3, a transmembrane segment, and a short cytoplasmic tail.   The a3 domain is formed by an interchain disulfide bond which bridges a 90 to 100 amino acid span, analogous to the Ig domains.  Therefore the a chain belongs to the Ig superfamily.  The a1 and a2 domains associate with each other forming a groove in which antigenic peptides of 12 to 20 amino acids can interact.   There is specificity, but which is considerably less than that seen with antibodies and the TCR.  The b chain is referred to as b???????2 microglobulin.  It also has the Ig fold hence belonging to the Ig superfamily.  This protein is a peripheral protein which serves to stabilize the a chain.

Polymorphism of Class I Molecules - In the human population, there are greater than 23 different HLA-A a chain alleles, 49 different HLA-B a chain alleles, and 8 different HLA-C a chain alleles.  The polymorphism results from the variations in amino acids in the a1 and a2 domains.  The a3 domain, transmembrane segment and the cytoplasmic tail is highly conserved.  The polymorphism arises from a high rate of mutation.  Being that most persons are heterozygous (two chromosomes), the nucleated cells of the offspring will generally each have six different class I molecules. Each is associated with a b??????????????2 microglobulin molecule.  Graft rejection is the result of recognition of an MHC class I molecules by the recipient.

Function of MHC Class I Molecules - Experimentally in vitro, lymphocytes from the spleen of a virus immunized strain of mouse, like  BALB/c, will efficiently kill BALB/c target cells infected by the virus, but will not kill virus infected cells from an allogeneic (non-compatible) mouse like the C3H.  For efficiently killing, the immune system must recognize self and also foreign antigen.   In this case, self is MHC class I molecule and foreign is the virus (Peter Doherty and Rolf Zinkernagel shared the Nobel Prize in 1996 for their major contribution to understanding how cytotoxic cells develop). The T cells that are responsible for the killing are CD8⁺ T cells.  These cells recognize the virus with their specific TCR, and self MHC with the CD8 molecule. Class I molecules interact with endogenous antigens, for example, viral proteins synthesized within the infected cell. Endogenous antigens are processed in the cytosolic pathway. The association of class I with antigenic (virus) peptide occurs in the lumen of the endoplasmic reticulum, transported by vesicle to the golgi, then to the cell surface by vesicle where antigenic peptide is presented to CD8⁺ T cells.  Activated T cells release cytokines that inhibit replication of the virus and cytokines that kill the infected cells.   The host cell dies; the cycle of viral replication stops.

MHC Class II Molecules - In humans, MHC class II molecules are coded by genes DP, DQ, and DR.   The gene products are DPa and DPb, DQa and DQb, and DRa and DRb.

• MHC class II molecules are glycoproteins expressed primarily on antigen-presenting cells (APC's) which include B cells,  macrophages, and dendritic cells (including Langerhans cells).
• The MHC class II molecules are composed of two glycoproteins, a and b, that are non-covalently linked.  The a chain has a molecular mass  between 31,000 and 34,000, and the b chain has a molecular mass between 25,000 and 29,000.   The a chain has two external domains, a1 and a2, a transmembrane segment and a cytoplasmic tail.  The b chain also has two external domains, b1 and b2, a transmembrane domain, and a cytoplasmic tail.  The external domains of the two chains are formed by intrachain disulfide bonds approximating the size of the Ig domains, including the two class II peptides in the Ig superfamily.  In the cytoplasm, the a and b chains are associated with a 32,000 dalton invariant g chain.
• The a1 and b1 domains of the two chains associate forming a groove that can accommodate 12 to 24 amino acid peptides.   There is considerable polymorphism in class II molecules because of amino acid variations in the a1 and b1 domains of the two chains.

Function of Class II Molecules - Newly synthesized MHC class II a and b chains associate with g chain in the lumen of the rough endoplasmic reticulum.  The association of the g chain with a and b chains prevents the MHC class II molecules from interacting with peptides derived from endogenous antigens or other intracellular proteins.  The a and b chains with associate g chain enter the Golgi complex to the endocytic pathway.  The g chain is degraded by proteolytic enzymes inside an endocytic vesicle, leaving a small peptide, called the CLIP, bound to the peptide-binding cleft of the class II molecule. The vesicle may now associated with a phagosome containing antigenic peptide, and the CLIP is  replaced by antigenic peptide.  The class II MHC molecules containing the antigenic peptide now moves to the plasma membrane where the antigenic peptide will be presented to a CD4+ T cells.

General Rule 1- B cells recognize antigen with Ig on their membrane.  B cell also secrete antibodies. T cells recognize antigenic peptide with their TCR, but only when antigenic peptide is associated with MHC molecule. T cells do not secrete their receptor, like B cells do.

General Rule 2 - If the antigen is endogenous, the antigenic peptide will be presented to T cells by class I molecules; endogenous peptides are generally presented to CD8⁺ T cells.  If the antigen is exogenous, the antigenic peptide will be presented to T cells by class II molecules; exogenous peptides are generally presented to CD4⁺ T cells.  CD8⁺ T cells become cytotoxic T cells, whereas CD4⁺ T cells become helper T cells in either the generation of cytotoxic T cells or in the generation of enhanced antibody production.

Cytokines - Low Molecular weight proteins important in cell-to-cell communication.  These proteins are secreted by white blood cells and a variety of other cells in the body in response to certain stimuli. 

• Autocrine - A secreted cytokine binds to a receptor on the same cell that secreted the cytokine stimulating a response.
• Paracrine - A secreted cytokine binds to a receptor on a neighboring cell stimulating a response.
• Endocrine - A secreted cytokine binds to a receptor on a cell that is in a distant part of the body stimulating a response there.

Interleukins - Cytokines secreted by white blood cells that act on other white blood cells.  Interleukins 1 through 18 have presently been identified.

Chemokines - A group of low molecular weight cytokines that play an important role in the inflammatory response.

Examples of Interleukins -

• IL-1 - This interleukin is produced by many different cells, for example, dendritic cells, neutrophils, T cells, B cells, fibroblasts, but especially by activated macrophages.  There are two types of IL-1: IL-1a and IL-1b.   IL-1 activates T cells and promotes T cells to release other lymphokines.   IL-1 and IL-2 induce B cells to proliferate and undergo differentiation.  IL-1 also is important in regulating the maturation of B and T cell precursors.  IL-1 interacts at the cell membrane with IL-1R (IL-1 receptor).
• IL-2 - Activated T cells and NK cells secrete IL-2.  IL-2 induces B cells to proliferate and differentiate, thus enhancing Ig production.  IL-2 interacts with activated T cells inducing these T cells to proliferate and produce other lymphokines, and enhancing cytotoxic activity.  IL-2 also activates NK cells enhancing their activity, and activates macrophages, increasing their cytotoxic activity and enhancing their IL-1 secretion.  IL-2 interacts at the cell membrane with IL-2R (IL-2 receptor).

TH1 and TH2 cells - Two different T helper cell populations have recently been discovered in mice based on their ability to secrete different cytokines.  The cytokines that a cell secretes reflects the different biological functions of that cell.

• TH1 - Secrete IL-2, IFN-g, TNF-b, GM-CSF, and IL-3.
• TH2 - Secrete GM-CSF, IL-3, IL-4, IL-5, IL-10, and IL-13.

Functions of TH1 and TH2 cells - TH1 cells are responsible for classical cell-mediated functions, for example, for Delayed Type Hypersensitivity and activation of Tc cells; The TH2 cells function primarily as helpers for B cell activation.

• Help for total antibody production - TH2 cells are better than TH1 cells.
• Help for IgE production - TH2 cells provide help for IgE production; TH1 cells do not.
• Help for IgG2a production - TH1 cells are better than TH2 cells.
• Eosinophil and mast cell production - TH2 cell function; TH1 cells do not.
• Macrophage activation - TH1 cells but not TH2 cells.
• Delayed Type Hypersensitivity - TH1 cells but not TH2 cells.
• Tc cell activation - TH1 cells but not TH2 cells.

Inflammation - The response of irritated or damaged tissues to injury. There are five cardinal signs of inflammation in its classic form.  1) Redness - due to capillary dilation;  2) Swelling - the edema is due to an increase in vascular permeability with outflow of fluid and cells;  3) Heat - a result of the increased blood flow at the inflammatory site; 4) Loss of function - a result of the damage or irritation at the inflammatory site.   Inflammation is of critical importance as a vital protective mechanism.  Inflammation provides a means by which protective factors, such as antibodies, complement, and phagocytic cells, which are normally confined to the blood stream, can penetrate tissues and gain access to the site of foreign invasion.

See Inflammation

Vasoactive factors - These are inflammatory mediators.  They include molecules like histamine, serotonin, leukotrines, prostaglandin E2, anaphylotoxin, lymphokines, and kinins (bradykinin).

Hypersensitivity - Are all immune responses beneficial?

Portier and Richet (1902) - They were studying the toxicity of extracts of sea anemone in an attempt to develop a serum for treatment.  An extract was injected into a dog.  Later the dog was injected with the extract with unexpected results.  The dog became acutely ill and died within a few minutes following the injection.   Richet called the response anaphylaxis (ana = against; phylaxis = protection). 

Von Pirquet - Introduced the term allergy (Greek for altered response) to cover any altered response induced by previous exposure to an antigen.   Hypersensitivity and allergy today, are for the most part, synonymous.

Coombs and Gell - Defined four types of hypersensitivity reactions, which they referred to as Type I, Type II, Type III, and Type IV.

Type I Hypersensitivity (Anaphylactic Hypersensitivity) - Three steps are required to demonstrate systemic anaphylaxis: 1) Injection of the sensitizing antigen; 2) Incubation or latent period, which leads to the sensitized state; 3) Introduction of the eliciting or shocking dose.  Systemic anaphylaxis can be demonstrated rather easily in guinea pigs by injecting them with 1 mg of egg albumin, wait 2 to 3 weeks, then injecting them with egg albumin to elicit shock.  Within minutes after administering the shocking dose, a guinea pig becomes restless, its hair bristles, it coughs, kicks, and rubs its nose with its forepaws.  Its respiration slows and becomes labored.   Convulsions and death may follow.  A postmortem reveals that the heart is still beating.  There is active intestinal peristalsis, and there may be visceral congestion.  The lungs are fully inflated.  The animal dies of asphyxiation.   The active organs all have smooth muscles, which have contracted.  Other symptoms (which are also common to other animals undergoing shock) include 1) decreased blood pressure, 2) decreased body temperature, 3) decreased number of circulating leukocytes, and 4) there is often decreased blood coagulability.  These symptoms result because the leukocytes and the platelets attach to the endothelium of small blood vessels.  The mechanism is the result of the release of pharmacologically active substances, which produce contraction of smooth muscles, and capillary dilation which results in edema.  The target organs of guinea pigs and humans are the bronchi of the lungs.  In dogs the target organ is the liver, which may accumulate up to 60% of the animal's blood.  The target organs of the rabbit are the heart and lungs.

Pathway to Anaphylactic Shock - Are antibodies involved?   Injection of an allergen (antigen) into a sensitized animal results in shock.   Shock may occur even though antibodies may not be detected in the serum.   Deductions that one can make from this observation is that 1) anaphylaxis is a more sensitive detector of circulating antibodies than are standard in vitro serological tests, or 2) one is dealing with a different form of antibodies which may not be free in the circulation.  An argument against antibody involvement is that animals with a high titer of precipitating antibodies are sometimes refractory to shock.

• Passive sensitization - Demonstrates that a serum factor is responsible for the reaction.  Sensitivity to an allergen may be transferred form a sensitized animal to a non-sensitized animal by injecting the non-sensitized animal with serum from a sensitized animal.  1) If one waits a few minutes following transfer of serum, then injects the allergen, there is no shock.  2) If one waits several hours to one day following transfer of serum, then injects the allergen, shock will be elicited.   Therefore a latent period is required following injection of serum for shock to be elicited.
• Smooth Muscle Contraction - Is smooth muscle contraction a result of antibodies attaching to the muscles?  There is no evidence of this.
• Anaphylotoxins - In 1909, the thought was that antibody reacted with antigen giving rise to the release of anaphylotoxins which were then responsible for shock.  Now it is known that antibody-antigen complexes can activate complement with production of C3a and C5a.  C3a and C5a are now known as anaphylotoxins because their formation can lead to shock.
• Transfer of Serum From an Animal Undergoing Shock - Inject this serum into the skin of a normal animal and one will see an immediate "wheal and flare" reaction at the site.  The wheal and flare reaction is an acute, transitory, circumscribed area of erythema and edema.  One can elicit a similar wheal and flare reaction by injecting histamine into the skin of an animal.  Histamine is present in mast cells and in circulating basophils.

Mast Cells - These were first described by Paul Ehrlich.  These cells are widely distributed in the body.  They are found especially in connective tissue, blood vessels, spleen, lungs, liver, heart, uterus and kidneys.  Mast cells contain between 200 and 500 granules, each with a limiting membrane.  The endoplasmic reticulum of mast cells is poorly developed.  Mast cells have few mitochondria. The granules contain heparin, histamine, Zn ions, and other agents.

Mechanism of Anaphylaxis - An antigen in introduced into an animal which produces antibodies specific for the antigen.  These antibodies attach by the Fc to mast cells and basophils which bear an Fc receptor.  At a second experience of the sensitized animal with the antigen, the cell bound antibodies are cross-linked by the antigen.  This cross-linking results in degranulation of the cells with release of pharmacological agents.  The pharmacological agents cause smooth muscle contraction and dilation of microcirculatory vessels.

Antibodies That Bind to Mast Cells and Basophils - In humans, IgE binds to mast cells and to basophils.  Some reports have indicated that IgG4 also binds to these two cell types. 

Action of Pharmacological Agents Released From Mast Cells and Basophils - Pharmacological agents that may be released varies according to animal species.

• Histamine - This agent alters smooth muscle cells in the vascular system, causing edema and erythema by dilating and increasing the permeability of small blood vessels.  Histamine also causes contraction of smooth muscles, for example of the bronchi which may lead to asphyxia.  Histamine is also a potent stimulator of the exocrine system.
• Heparin - impairs blood coagulability.
• Serotonin (5-hydroxytryptamine) - This agent contracts certain smooth muscles rapidly.  It also increases capillary permeability.
• Leukotrienes - LTA4, LTC4, and LTD4 are examples.  Minute amounts promote slow but prolonged contraction of certain smooth muscles.  These agents are not preformed, but are produced as a consequence of antibody - antigen complex formation at the cell surface of mast cells and basophils. Leukotrienes are formed as a result of arachidonic acid metabolism.
• Bradykinin - A nine amino acid peptide.  Certain smooth muscles react slowly to bradykinin, but the result is a powerful contraction.  Bradykinin has greater vasodilator action than any other substance known.

Controls of Mast Cell Degranulation - Crosslinking of IgE on mast cells causes the release of histamine, leukotrienes, platelet activating factor, and eosinophil chemotactic factor, for example.  The eosinophil chemotactic factor draws eosinophils to the site where mast cells are degranulating where they release histaminase which breaks down histamine and its breakdown products.  Eosinophils also release aryl sulphatase which breaks down the leukotrienes, and phospholipase D which acts on platelet activating factor.  Control agents, such as these, keep degranulation of mast cells regulated, allowing inflammation to take place, but generally not permiting anaphylaxis.

IgE - Protects external body surfaces, and recruits antimicrobial agents through the generation of inflammation.  IgE also increases during parasitic infections.

Other Mechanisms for Release of Histamine - C3a and C5a are called anaphylotoxins.  They can cause the release of histamine from mast cells and basophils.

Cutaneous Anaphylaxis - A local anaphylactic reaction in a sensitive person, characterized by transient redness and swelling.  Mast cells in the skin, for example, have IgE bound to them.  Injection of allergen will cause a cross-linking of the antibodies resulting in degranulation and release of pharmacological agents like histamine.  Histamine causes dilation of the vascular bed with resultant erythema and edema at the site.   There is complete return to normal in about 30 min.   Physicians can determine whether an individual is sensitive to particular allergens by skin testing (cutaneous anaphylaxis).

Passive Transfer of Cutaneous Reaction (Prausnitz - Kustner or PK reaction) - Kutsner (1921) was extremely sensitive to certain fish.  His serum incubated with fish extracts gave no detectable reaction in vitro.  An in vivo test was done by Prausnitz in which he took Kutsner's serum and injected a small amount intracutaneously in a volunteer.  Twenty-four hours later, fish extract was injected intracutaneously into the volunteer at the same site.  An immediate wheal and flare reaction occurred at the site because Kutsner's serum contained IgE antibodies that bound to mast cells, and the fish extracts 24 hours later, cross-linked the antibodies causing the mast cells to degranulate.  The resulting histamine release caused the wheal and flare reaction.   The P-K test is very rarely used today because of the possibility of transferring disease from one person to another (hepatitis, for example), and because of in vitro tests that are adequate. The P-K test may be used to determine whether a young child has a particular allergy and the direct skin test is contra- indicated, or in adults with a disseminated skin disease where it may be difficult to administer the test.

Atopy - Out of place; foreign.  A group of diseases characterized by a local type of anaphylaxis.  Atopic diseases are better known as allergies.   The local anaphylaxis affects primarily the skin, the respiratory tract, and the G.I. tract.  About 10% of the US population is especially prone to this type of hypersensitive response.  Atopic diseases are heritable, i.e., they run in families.    These family members are not born with allergies, but have a predisposition toward hypersensitivity.  These individuals become allergic to a variety of environmental antigens.  They become allergic to pollens of ragweed, grasses and trees, as well as to fungi, animal danders, different foods, and house dust.  House dust is generally dirty dust, i.e., containing organic material like fungal components and cockroach components.   An atopic person comes in contact with an allergen, and he/she produces IgE antibodies.  Later, another experience with the allergen results in mast cell degranulation with release of histamine.  This results in expression of symptoms associated with hayfever, asthma, or urticaria (hives).  Non-atopic individuals produce IgG when they come in contact with potential allergens.

Desensitization - An allergic person is given small, but increasing doses of allergen at time intervals, of, for example, weekly.  The hope is that IgG or IgA (blocking antibodies) will be produced, but not IgE.  The strategy is for the IgG or IgA antibodies to interact with allergen before the allergen can interact with IgE antibodies on the mast cells.  Setbacks are that therapeutic benefits are not consistently evident.  Also, a rise in the titer of blocking antibodies does not mean that the individual has been cured or aided.

Comparison of IgE and IgG -

• IgE - Active in the P-K test; is persistent in human skin for up to 6 weeks; is labile (inactivated if heated at 56^o C for 4 hours); does not cross the placenta.
• IgG - Is not active in the P-K test, but may inhibit; persists in the skin for up to two days; is stable (not inactivated if heated at 56^o C for 4 hours); crosses the placenta.

Type II Hypersensitivity (Antibody-Dependent Cytotoxic Hypersensitivity) - 1) Red blood cells + anti-RBC antibodies results in red blood cell lysis or death from a) phagocytosis (phagocytes have an Fc receptor as well as a receptor for C3b should complement get activated), and b) from activation of complement leading to formation of the MAC (membrane attack complex).  2) Cells with antibodies directed to their surface may also be killed by ADCC's (antibody-dependent cell-mediated cytotoxicity).   Cells with Fc receptors (phagocytes, neutrophils, B cells) may kill by this method.   The significance of ADCC is that these cells can kill target cells, especially if the cells are too large to ingest, for example, large parasites, or solid tumors.  3) Isoimmune reactions are another example of Type II hypersensitivity.

Isoimmune Reactions -

The Blood Groups - Landsteiner (1902) found that human blood could be divided into different types.  He made multiple crosses of human red blood cells with serum from himself and from his associates.    Serum + RBC ------> agglutination or no agglutination.    He designated the blood groups as A, B, and O depending on whether they were agglutinated or not with particular sera.   Later, the AB group was found.   Differences in blood types result from differences in alloantigens on the red blood cells.  

Alloantigens are antigens found in certain individuals of a species, but not in other members of the same species.   Group O blood cells have a ceramide on their surface with a terminal Gal - Fuc.  Group A blood cells have a gene, called gene A, that codes for a glycosyl transferase that attaches N-acetylgalactosamine to the Gal of the Gal-Fuc ceramide on the surface of the cells.   Group B blood cells have a gene, called gene B, that codes for a glycosyl transferase that attaches to Gal of the Gal-Fuc ceramide on the surface of the cells.

• Group A - 42% of U.S. citizens have antigen A on their RBC; they also have anti-B antibodies in their serum.
• Group B - 10% of U.S. citizens have antigen B on their RBC; they also have anti-A antibodies in their serum.
• Group AB - 3% of U.S. citizens have antigen A and antigen B on their RBC; they do not have anti-A or anti-B antibodies in their serum.
• Group O - 45% of U.S. citizens have neither antigen A or antigen B on their RBC; they have anti-A and anti-B antibodies in their serum.

Landstiener's Rule - Antigen and specific antibodies do not coexist in the body.

Immunologic Mystery - How do antibodies against the blood groups develop?  The following facts are listed.

• The antigens to the ABO system are present before birth.  These antigens are present on most cells tissue except the brain and they are not present in the spinal fluid.  78% of all persons also have the blood group antigens in their secretions like in the saliva, urine, and gastric juices.  These individuals are referred to as secretors. 
• Antibodies, in general, are barely detectable at birth.  After the first few months, the titer develops and increases.
• Incubation of E. coli O86 with anti-B results in agglutination of the E. coli cells.   Incubation of Streptococcus pneumoniae type 15 with anti-A results in agglutination of these cells.  This indicates that the bacteria have antigens that are the same as those specified for the blood groups.  These antigens are therefore heterophile antigens (identical or very similar antigenic determinants found in either related or unrelated animals or plants).
• Enteric bacteria begin to colonize newborns shortly after birth. Food is also ingested. It is believed that the immune system produces antibodies against bacteria.  Thus, cross-reactive antibodies against the blood group antigens also develop.  This is supported by germ-free studies.  The antibody titer of animals in the natural environment increases significantly in the first six months.  Antibodies against the blood antigens also develop. The antibody titer of animals raised in a germ-free environment produce significantly less amounts of antibodies than normal.  Antibodies against the blood group antigens also develop poorly. 
• Natural antibodies - Antibodies in normal serum which arise without apparent antigenic stimulation.  However, current thought is that these antibodies do indeed arise from antigenic stimulation, and some may cross-react with antigens in related or unrelated life forms.
• Individuals with blood type A do not develop anti-A antibodies because the immune system recognizes this antigen as part of self;  similarly, individuals with blood type B do not develop anti-B antibodies because the immune system recognizes this antigen as part of self.

Rh Factor - Landstiener (1940) described the Rh factor.  He took rhesus monkey red blood cells and injected them into rabbits.  The rabbits produced antibodies against the rhesus monkey red blood cells.  Later, the anti-rhesus red blood cell antibodies (anti-Rh) were incubated with human red blood cells from different individuals.  The RBC's of 85% of the humans tested, were agglutinated by the anti-Rh antibodies.  These individuals were called Rh⁺.  Now there are at least 30 different Rh antigens that have been described.  There are different nomenclature systems.  The Fisher-Race system is based on CDE/cde genes which code for the Rh antigens.  Of the three pairs of genes, which show dominance and recessiveness, only five (C,D,E,c,e) have been demonstrated to produce product (Ag).   The recessive d may simply represent the absence of D.  D is the original antigen described by Landstiener.  It is now known that 85% of Americans have the D gene and they are Rh⁺; 15% do not have D and are therefore Rh^-.

Rh Phenotypes and Genotypes -

• Rh^-  -  cde/cde; 14.6%
• Rh⁺  - cDE/cde; 12.8%
• Rh⁺  - CDe/cde; 31.0%
• Rh⁺  - CDe/cDE; 13.5%
• Rh⁺  -CDe/CDe; 16.3%
• Other with D; 12.0%

The Presence of D = Rh⁺ ; Test for Rh:  incubate human RBC + anti-D and look for agglutination. 

Alloantibodies - There are no natural alloantibodies produced against the Rh factor.  For humans to acquire antibodies to the Rh antigen, they must be Rh^- and be exposed to Rh⁺ cells, therefore, only from alloimmunization.  Human red blood cells (Rh⁺) ----->  Rh^- individual ----> produces anti-Rh antibodies.

Erythroblastosis fetalis - A severe form of hemolytic disease of the newborn.  An Rh^- woman conceives an Rh⁺ child because the father is Rh⁺.  At birth, there is trauma as the child passes through the birth canal, and the Rh⁺ blood cells of the baby enter the Rh^- mother's circulation.  She now produces antibodies against the Rh factor.   Normally, nothing untoward happens to the first child.  If the Rh^- mother conceives a second child who is Rh⁺, this child is at risk because the mother's anti-Rh antibodies can cross the placenta.  The result is that the fetus' red blood cells are lysed.  The fetus may be aborted, the fetus is stillborn, or born alive with evidence of hemolytic disease.  Examination of the blood cells of the newborn will reveal an outpouring of immature red blood cells, which are nucleated, into the blood.  The nucleated red blood cells are called erythroblasts, hence the name for the disease.

Mechnism of Red Blood Cell Lysis - Anti-Rh antibodies do not normally activate complement, and therefore complement is not involved in RBC lysis.  The red blood cells are lysed by ADCC.

Determination of whether the Rh^- Mother has Anti-Rh Antibodies - The mother's serum is incubated with Rh⁺ cells.  Look for agglutination of the red blood cells.  IgM can agglutinate the red blood cells, but not IgG.   Therefore with some sera there is a problem in detecting anti-Rh antibodies.   IgM cannot cross the placenta, whereas IgG can, which are the antibodies that can cause the damage.  The mother may have anti-Rh antibodies and not cause agglutination of the red blood cells.  Early immunologists thought that the anti-Rh antibodies were structurally incomplete, thinking that the antiboies may only have a single Fab segment.   There is no evidence for this to be the case; anti-Rh antibodies are structurally the same as "complete" antibodies.  To test for anti-Rh IgG antibodies use Coomb's reagent, which is an anti-Hu IgG antibody.

Direct Coomb's Test - Obtain blood from a newborn who has developing signs of hemolytic disease (jaundice).  Incubate the red blood cells with anti-Hu IgG.  Agglutination means that the red blood cells have anti-Rh on their surface.   The anti-Hu IgG will cross-link the anti-Rh antibodies on the RBC surface.  A transfusion may be necessary to save the child's life.

Indirect Coomb's Test - The maternal serum is incubated with Rh⁺ cells.  Anti-Hu IgG is then added to the red blood cells.  Agglutination indicates that the mother has anti-Rh antibodies evidenced by cross-linking of the anti-Rh antibodies by the anti-Hu IgG antibodies.  Anti-Rh antibodies detected early in pregnancy constitutes a serious medical problem.

Prevention of Anti-Rh Response - Prevention is simpler than treatment.   Simultaneous injection of antigen and specific antibodies blocks the formation of antibodies to the antigen.  This is referred to as immunoglobulin induced immunosuppression.  One can thus prevent the formation of anti-Rh antibodies by administering anti-Rho (anti-D) to the Rh^- mother carrying her first Rh⁺ child.  This treatment blocks the formation of antibodies to the Rh factor.   Administration of anti-Rh can be done at 28 weeks of pregnancy, since "silent" antipartum feto-maternal bleeds may occur.

Other Human Blood Groups - The MN system was discovered by Landstiener (1927).  The MN system consists of over two dozen antigens.  The Kell system designates other blood antigens.

Blood Transfusions - The recipient must receive his own type of blood, or at least as closely matched as possible.  Type A should not receive type B (type A has anti-B and these antibodies will destroy the infused type B cells).  Conversely, type B should not receive type A.  Both type A and type B can receive type O blood (type O has anti-A and anti-B, but these antibodies become diluted in the recipient.   Thus there is insignificant damage).   Type O is therefore called the universal donor.  Type AB can receive from all donors and is called the universal recipient.

Cross Matching -

• The Major Cross Match - The serum of the recipient is incubated with blood cells of the donor.  Agglutination means that antibodies in the recipient will destroy the cells of the donor.
• The Minor Cross Match - The blood cells of the recipient are incubated with serum from the donor.  Agglutination means that the donor's serum has antibodies that can attack the recipient's cells. Only in true emergencies can blood be give if there is no agglutination in either of the two cases.  In blood banking, other antigens must be taken into account, for example, the Rh factor and the MN series of antigens.

Type III - Complex-Mediated Hypersensitivity - Antibodies and antigen combine in vivo leading to antibody-antigen complexes.  This may lead to a disease state, especially if the complexes are large.

Serum Sickness - An example of type III hypersensitivity.   Passive immunizations involve the injection of persons with preformed antibodies that are produced in another individual or animal.  Anti-tetanus toxin, for example, is produced in horses.  These antibodies are given to individuals who are at risk of Clostridium tetani infection, such as from a deep puncture wound.  Eight to twelve days following treatment with the horse anti-tetanus toxin antibodies, the individual experiences generalized swelling of the lymph nodes, with itching, urticaria or erythmatous eruptions.  Often, there is edema of the eyelids, face and ankles.   The underlying mechanism is as follows:  Following treatment with the antibodies, the patient's immune system recognizes the horse antibodies as foreign, producing antibodies to them.  The newly synthesized antibodies interact with the antigen (horse antibodies) still in circulation forming antibody-antigen complexes.   These complexes become deposited in the vascular walls where complement may become activated producing the anaphylotoxins, C3a and C5a.  The anaphylotoxins degranulate mast cells releasing histamine.  The histamine causes vessels to dilate allowing fluid to leave the vessels (edema).  Complement activation results in the generation of C3b which may attach to innocent bystander cells which may lyse due to formation of the MAC.  The cells of the basement membrane of the glomeruli in the kidneys may be attacked.  Complement activation will also lead to formation of C5a, a chemotaxin.   The chemotaxin attracts neutrophils which engulf the Ab-Ag complexes.  This may result in release of proteolytic enzymes and polycationic proteins from lysosomes to the surrounding area.  These molecules may cause severe arthritis and in the kidney may affect the glomeruli and the basement membrane leading to serious permanent kidney damage.  C3b may also cause platelet aggregation, as will the Ab-Ag complexes.   The platelets may release vasoactive amines contributing to edema.  Platelet aggregation may also become microthrombi which may occlude small vessels.  Following the episode, the complexes are removed in about 2 days and the reaction subsides.

Arthus Reaction - Another example of type III hypersensitivity. 

• A rabbit is injected with subcutaneous doses of foreign protein at intervals of a few days.   The first few injection cause no untoward reaction.  The late injections, however, produce local infiltrates.  These develop into abscesses which may become necrotic.
• Better yet, strongly immunize a rabbit with a series of I.V. injections.  Challenge by intracutaneous route rather than subcutaneous route.  This will demonstrate the Arthus Reaction better.
• The mechanism - Antigen is introduce into the vascular bed where antigen combines with antibodies resulting in AB-Ag precipitates.  Complement may be activated with generation of the anaphylotoxins and C5a.  C5a attracts neutrophils which release enzymes causing local damage.  If the precipitates are large, they may physically block small vessels, preventing exchange of gases, and elimination of tissue waste.   The food supply to the tissues may also be blocked.  This also contributes to the local tissue damage.
• The Arthus response peaks between 3 and 8 hours after antigen injection.

Serum Allergy - Serum from another species (horse, for example) is given to a person for treatment or prophylaxis.  Much later, the person is given horse serum for another reason.  The person may now suffer from an immediate anaphylactic shock reaction which may be fatal.  Serum allergy is the result of IgE to serum proteins binding to mast cells.  The later serum injection results in cross-linking of the IgE on the mast cells by the serum proteins resulting in degranulation and release of histamine.  Serum allergy is a Type I hypersensitivity reaction.

See Immunopathology

Type IV (Delayed Type) Hypersensitivity - This is a cell-mediated immune response involving T cells as discussed above.  Peak development ranges between 24 and 72 hours, with a common peak development of 48 hours.

Tolerance - A state in which an animal becomes specifically unresponsive to a particular antigen.  The ability of an individual's immune system to distinguish between his own and foreign antigen.

Paul Ehrlich's Doctrine of "Horror Autotoxicus" (1900) - One can form antibodies to almost anything, except components of one's tissues.  This is now called self-tolerance.  Serious medical consequences follow if self-tolerance breaks down resulting in autoimmune disease. 

Experimental Conditions - Foreign antigen may be treated in the same way as self-antigen, in which the antigen not only fails to act like an immunogen, but becomes a tolerogen establishing a specific unresponsive state.   Ag (tolerogen) ----> animal ---> no antibodies or CMI. 

Pseudotolerance - Antigens, at moderately high levels are injected into an animal.  The animal produces antibodies, but which are not detected because the newly formed antibodies combine with injected antigen.  Thus antibodies get eliminated before they can be detected.  This is not true tolerance because the animal produced antibodies.  A truly tolerant animal does not have specific antibodies in circulation and also lacks the cells that synthesize the antibodies (at least, even if present, the cells are non-functional).

Conditions Promoting Tolerance -

• Dosage - Demonstrated by Felton (1940).  0.01 to 1.0 mg of Streptococcus pneumoniae polysaccharide type II injected into mice resulted in protection against S. pneumoniae type II.   Antibodies against type II polysaccharide were also found in the blood stream of the mice.   Injection of the mice with 1000 mg of S. pneumoniae polysaccharide type II resulted in no protection of the mice to S. pneumoniae type II.  Antibodies to type II polysaccharide were also not found in circulation.  The condition was called immune paralysis which was characterized by 1) persistence (long lasting), and 2) specific.  The tolerance did not affect the production of antibodies to another type of S. pneumoniae polysaccharide, i.e., mice reacted normally to immunogenic doses of other antigens including capsular polysaccharides.  Not only is there specific unresponsiveness at high antigen dose levels, but also by doses just below the threshold for initiation of antibody synthesis.   Thus have high zone tolerance as well as low zone tolerance.
• Route of Administration - Antigens are immunogenic when given with adjuvants and injected into the tissues.  Antigens are tolerogenic when given alone and by I.V. route (high circulation of antigen).
• Physical State - Proteins in an aggregated state tend to be immunogenic; in the monomeric form, proteins are tolerogenic.
• Particulate Antigen - It is difficult to establish tolerance to particulate antigens like red blood cells.  Particulate antigens have numerous antigens on their surface.  One may, in effect, establish tolerance to an antigen on the surface of a particulate antigen, but other surface antigens may be immunogenic.   Therefore, it is difficult to detect or demonstrate tolerance to particulate antigens.

Newborn vs. Adult - Burnet and Fenner (1949) made observations from experiments of others.  They hypothesized that there are recognition centers on self-antigens that cause them to be ignored as antigens by one's antibody making machinery.  They suggested that this self-identification occurred in embryonic life when the antigens were first formed and at a time before the immune system was functioning.

Cattle -      Bull X Dam -------> calves from different pregnancies have different blood groups.

                   Bull X Dam -------> identical twin calves have identical blood groups.

In one publication, it was reported that a Dam had been impregnated by two different bulls giving rise to dizygotic twins.  The dizygotic twins had 1) obvious phenotypic differences, because they were genetically different.  2)  It appeared that the two had the same blood types?  Subsequently, it was learned that all the red blood cells did not have the same blood antigens, but in fact there were two populations of red blood cells in each of the two animals.  These were referred to as blood chimeras.   Blood chimeras arise from anastomoses of the blood vessels which leads to exchange of hematopoietic tissue.  The "foreign" red blood cells were not destroyed in these calves, but persisted for long period.  The "foreign" red blood cells were recognized as self.  Adults would have destroyed transplanted red blood cells possessing foreign antigens. (Burnet received a Nobel Prize for this work and for his studies on clonal selection).

Medawar (1953) - did the following experiment:  Skin grafts from CBA mice (which is brown) unto A strain (albino) mice are rejected in 12 to 14 days.   Medawar injected tissues from CBA mice by I.V. route into fetuses of the A strain between the 16th and 17th day of fetal life.  The A strain mice were born and allow to grow to adults when they were skin grafted with CBA mouse skin.  These skin grafts were not rejected because the CBA mouse tissue was introduced into the A strain mice before the immune system developed.  Medawar received a Nobel Prize for his work on tolerance.

T Cell Tolerance –

Selection of TCR - There are two types of TCR’s (T cell receptors), gd which are expressed by 2% to 5% of T cells in humans, and ab which is expressed by more than 95% of T cells in humans. Pro-T cells, derived from stem cells, are TCR^-, enter the thymus and become double positive (CD4⁺ and CD8⁺) T cells. These cells have their TCR genes rearranged and if successfully express the TCR, remain viable, otherwise die by apoptosis. The cells that successfully express the TCR are now either positively selected or negatively selected.

• Positive Selection – Experimental evidence has shown that binding of thymocytes to class I or class II MHC molecules within the thymus leads to positive selection of MHC restricted T cells. These T cells carry appropriate TCR’s useful to the host. MHC molecules are carried by thymic epithelial cells.
• Negative Selection – T cells bearing a TCR that is inappropriate, i.e. to self-antigen, is potentially harmful to the host. These T cells are negatively selected in the thymus because they either have a high affinity for self-MHC molecules alone, or react with self-antigen expressed by self-MHC molecules. These T cells die by apoptosis.

Question – Are all self-reactive cells eliminated in the thymus? If this were the case, then the thymus must express all possible self-antigens. If the antigen is not encountered in the thymus, then the T cells become available for self-reaction.

Haplotype – The particular alleles at loci within a gene complex, for example in the H-2 of mice. Haplotype H-2^a indicates that genes aaaa are at loci K I S and D, and haplotype H-2^k indicates that genes kkkk are at loci K I S and D.

Transgenic Mice – Mice that carry foreign genes through genetic manipulation. For example, one can splice a class I (H-2^k) gene next to the insulin promoter, then inject the construct into a fertilized H-2^b ovum. The ovum is implanted into a false pregnant female who is H-2^b. All the cells of the H-2^b mouse will have H-2^b molecules on their surface including the b cells of the pancreas. The b cells of the pancreatic islets, however, will also express H-2^k class I molecules. This mouse has become tolerant to the H-2^k even though H-2^k is not present in the thymus.

 Go to Transgenic mice

Clonal Anergy – Some T cells with autoreactivity leave the thymus. Therefore there is a need for self-tolerance for animal survival. These T cells are subject to powerful suppression, i.e., they become functionally inactivated. The turning-off of a clone of T cells is called clonal anergy. At least two signals are needed for T cell stimulation shown below.

• CD4⁺ interact with antigenic peptide with the TCR. The antigenic peptide is presented by MHC class II molecules on the surface of an APC (antigen presenting cell). This interaction is the 1^st signal. The APC secretes IL-1, the 2^nd signal, activating a CD4⁺ T cell, which now secretes IL-2. If either signal is missing, there is no production of Il-2, which results in clonal anergy.
• Aggregated BSA is engulfed by an APC, processed and presented with the MHC class II molecule to a CD4⁺ T cell. The APC secretes IL-1, which activates the CD4⁺ T cell that has a specific TCR for the antigenic peptide of BSA. This cell secretes IL-2 resulting in an immune response. With monomeric (low dose) BSA, the BSA molecule interacts directly with the CD4⁺ T cell. The APC is not involved, therefore the 2^nd signal is missing. This results in clonal anergy.

B Cell Tolerance – For B cell stimulation, APC, CD4⁺ T cell, and IL-2 are necessary. If any one of the three is not present, antibodies will not be produced.

Immature B Cell – Low doses of antigen results in clonal abortion.

Mature B Cell –

• Exhaustive antigen challenge – all B cells become plasma cells with no generation of memory cells. This is called clonal exhaustion.
• Functional Deletion – Results from the following: 1) TH are absent; 2) there are excessive TS cells; 3)there is excessive T-independent antigen (this may also result in receptor blockade.

Regulation – 1) Excessive antigen results in an increase in the following conditions: increase in allergies, autoimmune disorders, amyloidosis, and lymphoid tumors. 2) Antigens at immunogenic doses results in a normal response and elimination of foreign agents. 3) Insufficient antigen results in increased susceptibility to infections and an increase in the number of tumors.

The Immune Response is antigen driven. Antigen is needed to start the immune response. Eliminate antigen and the response subsides.

Antibody-Regulated Immune Responses – Antibody and immune complexes exert a negative influence on immune responses. 1) For example, immunize a rabbit with antigen, but remove the antibodies by plasmapheresis as antibodies are being formed. This rabbit will continue to produce more and more antibodies many times greater than normal if one continues to remove the antibodies as they are formed. 2) One can also demonstrate the influence of antibodies on the immune response by adoptive transfer using syngeneic (genetically identical at H-2) mice. Immunize a mouse, then test the spleen cells, before there is a high titer of antibodies in circulation, for antibody production (Jerne plaque assay). They will show 10⁴ pfu’s. Transfer spleen cells to a compatible mouse and later test the spleen cells of the second mouse for antibody production before there is a high titer of antibodies in circulation. There will now be10⁶ pfu’s. Spleen cells from the second mouse are transferred to a third compatible mouse and tested in the same manner. There are now 10⁸ pfu’s, etc. No antibodies in circulation increases the number of antibody specific cells produced.

In General – IgG depresses production of IgM and IgG; IgM tends to depress further synthesis of IgM.

Prevention of Rh factor Disease – Rh⁺ red blood cells introduced into an Rh^- mother results in the mother producing anti-Rh antibodies. If the mother is given anti-Rh antibodies (RhoGam) at the time of her exposure to fetal red blood cells, she will be completely blocked from producing anti-Rh antibodies.

Idiotypic Networks – Review isotypic, allotypic and idiotypic determinants. Idiotypic determinants are located in the antigen binding region of antibodies. Some of these determinants may interfere with antigen binding, but some may not and are external to the antigen binding groove. However, because these determinants are new (antibodies are newly synthesized) and never previously been seen by the immune system, they very likely will induce antibody formation. These antibodies are referred to as anti-id antibodies, and may interact with circulating idiotypes. They may also interact or with the idiotypic determinants on the original B cell clone bearing the idiotype, and in this way regulate antibody formation. The anti-idiotypic antibodies have idiotypic determinats of their own which can induce new antibody formation. These antibodies are now anti-anti-idiotypic antibodies, and since these new antibodies have idiotypic determinants, they can in turn stimulate formation of new antibodies, etc.