Immunology

Introduction

Immunology (from immunis, Latin for "exempt") is the study of the organs, cells, and chemical components of the immune system. The immune system creates both innate and adaptive immune responses. The innate response exists in many lower species, all the way up the evolutionary ladder to humans, and it acts through relatively crude means against large classes of pathogens. The adaptive response is unique to vertebrates, reacting to foreign invaders with specificity and selectivity.

The immune system must maintain a delicate balance, with potent defensive responses capable of destroying large numbers of foreign cells and viruses while refraining from undue destruction of the host's body. When the immune system cannot mount a sufficient defense of the host, there is an immune deficiency; this is seen in HIV infection and SCID (Severe Combined ImmunoDeficiency). If, on the other hand, the immune system acts too vigorously and begins to attack the host, we have autoimmunity. This is a defiance of the integral immune system property of self/nonself recognition. That is, the immune system begins attacking or forming antibodies against the host's own body tissues. Examples of autoimmune diseases include Graves' disease, Hashimoto's thyroiditis, myaesthenia gravis and type I diabetes mellitus.

The History of Immunology

The human immune system recognizes non-self entities and mounts an effector response to neutralize the organism. A faster and stronger memory response may occur upon later exposure. The memory response has been used throughout history to confer immunity upon several populations, even previous to our understanding of the physiological basis for such a response. Thucydides wrote in his History of the Peloponnesian War that persons who had been exposed to plague previously could care for the sick without danger. In the 19th century, variolation was commonplace; this was the removal of smallpox (variola virus) skin pustules which were subsequently put into small cuts in the skin of healthy people. This was itself a crude form of vaccination, with the crusty dry pustules acting as an incubator of attenuated virus. Edward Jenner would later use the cowpox virus to vaccinate (from vacca, Latin for "cow") patients against smallpox, and Louis Pasteur attenuated rabies and injected it into a small boy, naming this substance a vaccine in honor of Jenner's earlier studies in the science of immunology.

As immunology progressed, many people began to question how these vaccines worked. Why should exposure to plague in Thucycides' time confer protection only against plague and not all disease? Why should cowpox, a similar disease to smallpox but clearly a less severe virus, give milk maids sufficient immunity to resist full smallpox infection? In short, what has caused this memory response to be relatively (yet not absolutely) specific as well as selective?

The Basics

First, some vocabulary:

• Serum--liquid, noncellular component of blood after coagulation has occured (and thus devoid of clotting factors).
• Immunoglobulin--a serum fraction (aka gamma globulin) that has antitoxin, precipitin, and agglutin factors (abbreviation: Ig)
• Antigen--"Antibody Generator"; a foreign organism or molecule that generates a humoral immune response, causing the release of antibodies (abbreviation: Ag)
• Epitope--the molecular sidechain of an antigen that each antibody attaches to; there can be many epitopes on a single antigen
• Antibody--refined, Y-shaped proteins that make up the immunoglobulin fraction of serum; antibodies are specific to certain foreign bodies; antibodies can be membrane-bound or free in the serum (abbreviation: Ab)

Note: Often, antibody and immunoglobulin are used interchangably.

There have been several competing theories of the immune response mechanism. The instructional theory of antigen interaction postulated that the antigen itself caused antibodies to fold around the antigen in a certain way; this theory was later disproven. The selection theory states that the body creates many different sidechains on antibodies, and the antigen "selects" the correct antibody; in other words, the body creates every possible permutation of chemical sidechain-specific antibodies, and when an antigen enters the body, it is matched up with antibodies that correspond to its epitopes. The current theory of immune response is known as the clonal selection theory, which states that an individual lymphocyte (specifically, a B cell) expresses receptors specific to the distinct antigen, determined before the antibody ever encounters the antigen. Binding of Ag to a cell activates the cell, causing a proliferation of clone daughter cells.

Innate Immunity

Innate immunity is basic and nonspecific. It includes:

• Phagocytic cells (macrophages, neutrophils; more generally, antigen-presenting cells (APCs))
• Barriers (e.g. skin)
• Antimicrobial compounds
• Inflammation

Phagocytic Cells

Certain cells "eat up" foreign invaders; this is termed phagocytosis. Many of these cells are known as Antigen-Presenting Cells (APCs) because they break apart the ingested pathogen and display certain epitopes of the antigen on their surface. In this way, they localize the presentation of antigen, forming a vital link between the innate response and the adaptive response. Lymphocytes (such as B cells and T-helper cells) will have antigen presented to them, initiating the adaptive response. Monocyte-derived cells are common APCs, and they include tissue macrophages and monocytes within the blood; neutrophils are also APCs. APCs are discussed further below, in the section on cells of the immune system.

Barriers

The skin contains epidermis and dermis. The epidermis contains tightly packed epithelial (cytokeratin positive) cells with keratin waterproofing. The dermis contains connective tissue blood vessels hair follicles, sweat glands, and sebaceous glands. Sebaceous glands secrete sebum; this contains fatty acids and lactic acid, lowering skin pH to 3-5.

Mucous membranes contain normal flora, mucus, and cilia. Mucous membranes are found in the nose, eyes, mouth, urogenital, and anal regions of the body. Flora refers to bacteria that inhabit the human body in a relative steady-state; the gastro-intestinal tract contains a large number of these bacteria, and different areas of the world contain different flora. It is for this reason that people contract Traveler's Diarrhea; in short, flora from one region of the world are more dangerous because the body has not acclimated to their presence. Mucus contains certain mucin proteins, inorganic salts, and water; it is secreted from goblet cells. Cilia acts to sway back and forth during two phases, known as the power stroke and the recovery stroke; this allows mucus to be swept out of the body, either proximodistally from the lungs up the respiratory tract, or down the GI tract through the intestines, culminating in defecation. The enteric nervous system causes contractions of the gut, moving foodstuffs, waste, and bacteria/toxins down the digestive tract. This is just one example of disparate systems of the body working together with the immune system proper. Other examples are skeletal muscles, which squeeze blood along the veins and lymph along the lymph vessels, or the nervous system, which supports a rise in body temperature in response to infection.

Some bacteria can attach to mucous membranes via fimbriae or pili, which attach to special glyoproteins/glycolipids on the epithelial cells of mucous membranes.

Antimicrobial Compounds

Several antimicrobial compounds mediate the innate response.

• Lysozyme--hydrolytic enzyme in tears and mucous; cleaves peptidoglycan in bacterial cell walls
• Interferon--produced by virus-infected cells; binds to nearby cells (it is a paracrine factor), inducing a generalized antiviral state
• Complement--inactive circulating serum proteins that act on pathogen cell membranes
• Collectins--surfactant materials; kills bacteria by disrupting their lipid membranes or agglutinating them
• Toll-Like Receptors---(TLRs) membrane-bound receptors that react via pattern recognition to certain classes of molecules; TLR4, for example, recognizes lipopolysaccharide (LPS) on gram-negative bacteria

Inflammation

Classical biology put several characteristics together as an inflammatory response. These included redness (erythema, rubor), heat (calor), swelling (edema, tumor), pain (dolor), and loss of function (functio laesa). The physiological processes that bring about these symptoms are central to the innate immune response.

Erythema results from constriction of post-capillary venules in tissue beds and vasodilation in pre-capillary arterioles/metarterioles. This results in an increase of hydrostatic pressure in the capillary bed, overcoming the osmotic pressure of the interstitial fluid and causing exudate (high-protein fluid with acute-phase proteins like C-reactive protein and macrophages) to flow into the interstitial tissue. This flowing of exudate also allows the factors of the clotting cascade to enter areas of tissue damage, forming clots and, eventually, scars. Certain inflammatory factors also cause phagocytic cells to enter the damaged tissue. The now-permeable capillaries are traversed by these phagocytes in response to chemotaxis, the release of factors that lure the cells to the site of injury. The cells first approach the side of a capillary (margination), move through the spaces between capillary endothelial cells (diapedesis), and then enter the tissue itself. Histamine helps to mediate this response, and certain factors (such as bradykinin and possibly prostaglandins) stimulate skin pain receptors. Thus, blood flows into the tissue, causing redness, warmer steady-state temperature, swelling, and pain; loss of function is a secondary effect of these four states.

Adaptive Immunity

Adaptive immunity occurs in response to antigen exposure. It is specific, and it shows memory. As long as an antigen is made of the normal chemical elements we experience in biological systems (e.g. Carbon, Nitrogen, Sulfur, Hydrogen, Oxygen), we can form an adaptive response to it. This is how we fight off new diseases, and it has even been shown that we can create antigens in the laboratory that have never before existed on Earth, only to have animals mount competent immune reactions to them. As stated earlier, the adaptive immune system's specificity is tempered with an ability to differentiate between self and non-self antigen; simply put, the body doesn't attack its own cells, unless they have been invaded by virus and ask to be sacrificed for the sake of the host. When the immune system does attack the body, this is a disease state: autoimmunity.

The primary response takes 5-6 days, but the memory (secondary) response will be swifter and deadlier. It includes:

• Lymphocytes
• Antibodies

Lymphocytes

Blood cells are made in the bone marrow of adults. Leukocytes are white blood cells (WBCs). This includes monocytes (which become myriad cell types in the body, most importantly macrophages), granulocytes (neutrophils, basophils, eosinophils), and lymphocytes. Of all the leukocytes, the lymphocyte class are the most preeminent in the adaptive immune response. On a peripheral blood smear, lymphocytes are approximately the size of erythrocytes (red blood cells, RBCs), although they can be larger if activated and have a characteristic "clock-face" nucleus if they are B cells. Lymphocytes are so named because fewer than 1% are present in the circulating blood; the rest lie in the lymph nodes, spleen and other lymphoid organs. T lymphocytes leave the bone marrow, travelling to the thymus gland, where they mature and gain their specificity for the diverse antigens the body might come into contact with; additionally, any T cells that react against the body's own epitopes are selected against (killed) in the thymus, in an effort to stop any possible autoimmunity. A similar process occurs in the bone marrow in the case of B lymphocytes. B cells are conveniently named ("B for bone marrow"), but this is just a coincidence; it turns out that they are named B cells after the Bursa of Fabricius, a small pouch in the cloaca (lower large intestine, cloaca Latin for "sewer") of birds.

B cells leave the bone marrow with their specific membrane bound Ig (antibody) already specified. The Ig itself is made up of two medial heavy chains (both identical) with two lateral light chains (also identical) attached to the "top" of the heavy chains, which form a Y shape. Before they encounter antigen, B cells are known as "naive." Once they encounter antigen, the naive B cells will undergo clonal expansion; an activated B cell will form some daughter memory B cells and some plasma cells. The memory cells will lie in wait for a second encounter with the antigen, while the plasma cells will begin a massive secretion of antibody (Ig). B cells can bind to antigen when it is free and unprocessed in the body, much like APCs can.

Before moving on to T lymphocytes, it should be noted that certain cell surface molecules distinguish each person's unique immune system profile. The Major Histocompatibility Complex (MHC, or HLA) is a type of protein expressed on the surface of host cells that interacts with T-cell receptors (TCRs) of T cells. Virtually all the body's cells, including APCs, express class I MHC (MHC-I) on their surface. Only APCs express class II MHC (MHC-II). Thus, most body cells express MHC-I, while APCs express both MHC-I and MHC-II. When antigen enters a body cell and is broken down, the products of this breakdown are sent to the surface of the cell coupled with MHC-I. This forms the MHC-I/Ag complex, and usually occurs when a virus or bacterium enter a cell and are broken down by intracellular defenses. This can also occur in APCs, but APCs additionally process the antigen that they phagocytose, presenting it as an MHC-II/Ag complex on the surface of their cells.

T cells can be subdivided into two broad types: T helper cells and T cytotoxic cells. T helper cells express a T-Cell Receptor (TCR) that will interact with APC surfaces. Specifically, they interact with the MHC-II/Ag complex on the surface of APCs, and the TCR is stabilized in its binding by a CD4 receptor. CD stands for "cluster of differentiation," and is simply a class of cell receptor which occurs predominantly on T cells. T helper cells contain CD4 receptors and T cytoxic cells contain CD8 receptors. Upon binding, T helper cells release cytokines, which act as chemotactic agents to call for more T helper cells, T cytotoxic cells, APCs, and B cells.

T cytotoxic cells encounter body cells that have been invaded and are presenting MHC-I/Ag on their surface. Tc cells extend their TCR to the MHC-I/Ag complex and stabilize this interaction with their CD8 receptor arms. Upon binding, the CD8+ cells differentiate, much like naive B cells, into memory T cells and cytotoxic T lymphocytes (CTLs), effector cells that cause the MHC-I/Ag-presenting cell (the "altered self cell") to die (apoptose). CTLs trigger apoptosis by secreting a perforin that allows the entry of a serine protease (Granzyme B) which activates intracellular executioner caspases. It should be noted that cancer cells can also become altered self cells, and CTLs are very important in the destruction of cancerous cells. T cells can interact with antigen only after it has been processed, either by a normal body cell (MHC-I) or by an APC (MHC-II).

Difference between the two types of T cells:

• T helper cells react to exogenous antigen, phagocytosed by APCs, presented on MHC-II, via binding with TCR and CD4.
• T cytotoxic cells react to endogenous antigen (such as viral or cancer proteins), broken down by lysosomes in many types of body cells, presented on MHC-I, via binding with TCR and CD8.

Note that it is both the antigen and the MHC that is presented to T cells; each person has a unique MHC. This is why we must type for bone marrow transplants--we don't want people producing tons of new immune cells in bone marrow that think every MHC in the body is actually just antigen; when this does occur, it is called graft-vs.-host disease. Not only would the new bone marrow make cells that attacked every body cell's MHC, but when the body did present Ag on MHC, the lymphocytes from the transplant would not be able to recognize this MHC-I/Ag complex (although the MHC-II complex cells would be made in the new bone marrow, and thus APCs could, in some cases, still present). Unfortunately, these APCs would be very busy presenting the host's own body cells; clearly MHC typing (also known as HLA typing) for bone marrow transplants is a necessary result of the elegant self-nonself recognition of the human immune system. It is also worth noting that we are only as good as our presenting molecules. Some people have MHC genes that are not as good at presenting antigen, and thus some people have more vigorous immune respones than others.

Antibodies

The cellular and humoral responses of the adaptive immune system are linked via the T helper cell-B cell interaction. The T helper cell secretes cytokine factors to encourage chemotaxis of B cells to the site of infection as well as B cell differentiation and growth. B cells themselves, in their effector plasma B cell form, release antibodies into the blood. Antigens, or foreign substances that enter the body,are very harmful to the Immune System. But when specific antibodies are produced to bind with an antigen, the foreign substance becomes harmless and is delivered to the lymph. Antibodies are also very important in the complement system; this is an example of the vertebral adaptive immune response making good use of the relatively primitive innate immune response. Antibodies to the body's own cells are a very real danger. Type I diabetes may be caused by an autoimmune response, where the body makes antibodies to its own Beta cells in the pancreatic Islets of Langerhans. Graves' disease is a condition where the body produces antibodies to receptors in the follicular cells of the thyroid; these Ab keep the cells constantly activated, giving the patient hyperthyroidism and increasing their metabolism, to adverse effect. The interesting counterpoint to this is Hashimoto's thyroiditis; here the body makes antibodies to thyroid follicular cells' receptors, but in this case the cells are shut down by the Ab, and the patient endures hypothyroidism.

Autoimmunity due to antibody overreaction (hypersensitivity) is a huge problem, and it is a much greater problem in women than in men. Women tend to have a higher titer of immunoglobulin (antibody), and thus they exert a stronger (and sometimes overwhelming) immune response. For example, for every one man who gets Graves' disease, 8 women will contract it.

Antibodies are often given to produce a short-lived humoral immunity in emergency situations. Many antivenoms are actually horse immunoglobulin, produced in order to bind the venom until it can be cleared from the body.

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Theory Meets Function

It should be noted that all of the above-mentioned immune responses result in the destruction or agglutination of foreign pathogen. The goal of the immune response is three-fold:

1. Destroy foreign antigen via innate or humoral responses such as complement fixation or endocytosis by macrophages.
2. Attract immune cells to the site of infection so they can engulf, process and present the antigen to T cells
3. Move the antigen to the site of differentiation (e.g. lymph nodes, thymus) via the lymphatic system, so that it can interact with naive B cell. This process creates new effector and memory cells which can return to the site of infection and destroy the foreign cells in a highly effective fashion via the adaptive response.

The immune responses listed above show the intimate interaction between the innate and adaptive immune systems, as well as the subclasses of adaptive responses. The clonal selection theory of immune response, introduced above, is clear in the action of lymphocytes. Clonal selection simply means that antigen is presented to many circulating naive B and (via MHC) T cells, and the lymphocytes that match the antigen are "selected" to form clones of themselves, both memory and effector. This mass production of daughter cells is termed clonal expansion, and it is essential in the understanding of the theoretical basis of immunology. Not only this; clonal selection is used negatively in the lymphoid organs. Here, the body's own epitopes are presented to the infant lymphocytes; those that react are recognized as traitors and destroyed before they (and their future cloned daughters) can leave and wreak havoc in the body.

Organs of the Immune System

There are two groups of immune system organs.

• Primary (central)--organs where immature lymphocytes develop
  • Thymus
  • Bone marrow
• Secondary (peripheral)--tissues where antigen is localized so that it can be effectively exposed to mature lymphocytes
  • Lymph nodes
  • Appendix
  • Peyer's Patches (of GI tract)
  • Tonsils
  • Adenoids
  • Spleen
  • MALT (Mucosal-Associated Lymphoid Tissue)
    • GALT (Gut-Associated Lymphoid Tissue)
    • BALT (Bronchial/Tracheal-Associated Lymphoid Tissue)
    • NALT (Nose-Associated Lymphoid Tissue)
    • VALT (Vulvovaginal-Associated Lymphoid Tissue)

Note: Some sources place the vermiform appendix, small intestine Peyer's Patches, tonsils, and adenoids within the GALT.

Thymus

The thymus (from θυμος, thumos, Greek for warty outgrowth) is the site of T cell maturation. T cells become immunocompetent here; that is, they develop their ability to mount an effective immune response against foreign invaders without attacking the host's own tissues. The thymus lies just above the heart in the mediastinum. It is largest in childhood, and it begins to shrink significantly as a person ages. The organ itself contains two lobes, and each lobe contains numerous lobules, separated from each other by connective tissue septa known as trabeculae. Each lobule is separated into an inner medulla (with few immature thymocytes) and an outer cortex (with large numbers of immature thymocytes). In the thymus, the many different T cells (produced via somatic genetic mutation, discussed below) are exposed to MHC/Ag and MHC/self-Ag. If they do not react to MHC/Ag, they are destroyed due to their ineffectiveness (positive selection). On the other hand, if they do react to MHC/self-AG, they are destroyed in order to stop them from becoming traitorous autoimmune responses against the body's own tissue(negative selection). Only 1 out of 20 immature thymocytes will pass successfully through this vetting process and become functional T cells. Dendritic cells, macrophages, and epithelial cells are interspersed throughout both the medulla and cortex; special epithelial nurse cells surround clusters of thymocytes in the cortex.

Myasthenia gravis is an autoimmune disease where the body creates antibodies against acetylcholine receptors at post-synaptic neuromuscular junctions. In up to 25% of MG cases, there is a tumor of the thymus present (thymoma); the exact reasons for this tumor development is unknown. Additionally, removal of the thymoma will halt the disease's progress; this deserves further study as antibodies are released from plasma B cells rather than T cells, yet T helper cells may mediate the autoimmunity.

DiGeorge's Syndrome is a congenital lack of a thymus, and causes in increase in infections and a depressed immune system (especially the cell-mediated response is retarded by the lack of a thymus).

Bone Marrow

Bone marrow (medulla ossea) is the site of B cell maturation in mice and humans. B cells undergo both positive and negative selection, similar to T cell maturation in the thymus. Bone marrow is also the site of hematopoiesis, the development of the myriad blood cells from progenitor cells. The site of B cell maturation in birds is the bursa of Fabricius, after which B cells are named. The tissue of bone marrow where leukocytes, red blood cells, and platelets develop (i.e., the site of hematopoiesis) is known as myeloid tissue.

Leukemia is a cancer of the bone marrow that causes abnormal production of leukocytes (WBCs).

• Acute leukemia is the biggest killer of children in the US; it results from the overgrowth of immature leukocytes in the bone marrow, retarding the production of functional WBCs, RBCs, and platelets.
• Chronic leukemia is the overgrowth of relatively mature (but abnormal) leukocytes in the bone marrow.
• Lymphocytic leukemia is the overgrowth of lymphoid cells (T cells, B cells, Natural Killer Cells, dendritic cells).
• Myeloid leukemia is the overgrowth of myeloid cells (all blood cell types that are not lymphocytic are produced from myeloid progenitor stem cells).

Lymph Nodes

Extracellular fluid flows from capillary beds into tissue; from this tissue it enters lymphatic capillaries that are "pumped" along with the movement of skeletal muscle towards lymph nodes. The paracortical areas of the nodes contain T cells, and the central areas contain germinal centers, where B cells are contained. APCs and antigen are sent from the tissue into the lymphatics, eventually reaching the lymph nodes where they can be exposed to the T and B cell populations. This allows a faster response, as the many combinations of T and B cell specificities are able to reside in several locations throughout the body (the lymph nodes) rather than relying on random meetings of antigen and lymphocytes throughout the tissues themselves.

Spleen

The spleen acts as a site of hematopoiesis during the second and third trimesters of development, before the long bones have fully developed. In the adult, the spleen acts as a site for breakdown of dying red blood cells (lifespan 120 days). For this reason, enlargement of the spleen (splenomegaly) can occur in sickle cell anemia or in certain infections. White pulp, near the arteriolar entry points into the spleen, is where lymphocytes reside and are degraded. The central red pulp is the site of RBCs breakdown. The white pulp region has a central part, with the T cells residing in the PALS, or PeriArteriolar Lymphoid Sheath and a B cell ring (or corona) surrounding the PALS.

Cells of the Immune System

Hematopoiesis

Stem cells, like the stem of a plant, are cells that branch off into other cell types (hence the name). Hematopoiesis is the generation of blood cells in the bone marrow. Hematopoietic Stem Cells (HSCs) are cells within the bone marrow that maintain their own population through cell division while branching off some daughter cells to form subspecialized sets of cell-types. During the embryonic stage however, humans do not yet have long bones but are still able to accomplish hematopoesis. The yolk sac acts as the site of hematopoiesis for the first several months of gestation, and in the second and third trimesters the fetal liver and spleen take over hematopoietic duties. Gradually, during the final trimester, the hematopoetic duties are handed over to the long bones of the fetus. By parturition, hematopoiesis has become localized to the bone marrow itself. Most cells can multiply via division into cells like themselves, and thus are known to be unipotent. If a cell can differentiate into any cell type, it is called totipotent; a zygote's first dividing cells, and some embryonic stem cells (ES cells) are totipotent. HSCs are known as pluripotent or multipotent, meaning they can reproduce themselves (creating a store of unending stem cells) while having other cell-types branch off of their foundation stem, becoming several other types of cells.

The following is a list of the final differentiated types of cells that HSCs can become:

• Hematopoietic Stem Cell (HSCs renew their stocks)
• Lymphoid Progenitor Cell
  • Natural Killer Cell (NK Cell)
  • Dendritic Cell
    • Monocyte-derived dendritic cell
    • Plasmacytoid dendritic cell
  • T Cell Progenitor
    • CD4+ T Helper Cell
    • CD8+ T Cytotoxic Cell
  • B Cell Progenitor
• Myeloid Progenitor Cell
  • Mast Cell
  • Dendritic Cell
  • Granulocyte-Monocyte Progenitor Cell
    • Monocyte
    • Neutrophil (leukocytosis is high neutrophils and is used clinically to indicate infection; also known as polys, segs, and PMNs, Polymorphonucleophages)
  • Eosinophil Progenitor Cell
    • Eosinophil
  • Basophil Progenitor Cell
    • Basophil
  • Megakaryocyte
    • Platelets
  • Erythoid Progenitor Cell
    • Erythrocyte (RBC)

All of these cell types are created in the bone marrow. Throughout the body's tissues, monocytes and B cells can specialize further. B cells will become

• plasma cells (effector B cells)
• memory B cells

Monocytes can become any of the following vimentin+ cells:

• circulating blood monocytes
• cardiac failure cells (in alveoli)
• alveolar macrophages
• epithelial macrophages
• Mesangial Cells (in the kidney glomerulus)
• Kupffer Cells (in liver)
• Ito Cells (in liver)
• histiocytes (in connective tissue)
• Microglial cells (in brain)
• Osteoclasts (in lamellar bone)

NK1-T cells are a special set of rapid-response cells that have TCRs but interact with CD1 rather than MHC-I or MHC-II. These cells are a sort of NK cell/T cell hybrid. When activated, NK1-T cells recruit both cytotoxic T cells and B cells.

Returning to the bone marrow itself, certain growth factors and cytokines must be present in cell microenvironments in order for these cells to go down one differentiation pathway or another. Much like the stroma (supportive tissue) of an organ, the stromal cells of the bone marrow assist in hematopoiesis both by creating a scaffolding upon which the stem cells grow and differentiate while also secreting factors that influence and encourage growth.

Apoptosis

Apoptosis is programmed cell death via disintegration of the cell into membrane-bound pieces which are then phagocytized. The purpose of apoptosis is to destroy cells in an ordered way without releasing the possibly toxic intracellular chemicals and components into the extracellular tissue. Necrosis is death of a cell based on injury or disease and does not employ similar safeguards. Several genes act to influence apoptosis. They include:

• Apoptosis Inhibitors
  • bcl-2
  • bcl-X long
• Apoptosis Enhancers
  • bax (inhibits bcl-2)
  • bcl-X short (inhibits bcl-X long)
  • caspases
  • fas

T Cell Responses

T cells can initiate several different responses.

• TH1 Response--mostly inflammatory, recruits T cells and macrophages
• TH2 Response--mostly antibody-mediated, recruits B cells

Certain cells act to supress a T cell-mediated response, and are known as suppressor T cells. It is unknown whether there is an actual Ts subpopulation or if Tc and Th cells simply act in a suppressor role in some cases.

Natural Killer Cells

NK cells act to attach to IgG antibodies on the surface of cancerous or virus-infected cells. In this way, they connect the humoral (B cell) and cell-mediated responses. For this reason, NK cells are central to something known as antibody-dependent cell-mediated cytotoxicity (ADCC).

Macrophage Responses

Macrophages phagocytize foreign invaders after chemotaxic factors lure the macrophages to sites of infection. Certain factors that attach to the invading cells (e.g. antibodies) or break open the invading cells' walls (e.g. complement system) act as opsonins (from a Greek word for "tasty'). Opsonization is thus the alteration of a foreign invader to make it more palatable to the macrophage's reactions. Macrophages release certain compounds call Reactive Oxygen Intermediates (ROIs) to kill invading cells in something known as the respiratory burst. ROIs include H2O2 and oxygen radicals. Melanocytes are important in the degradation of these compounds, which act to destroy invader DNA but also endanger host DNA at times; for this reason melanocytes are found in the skin, where they neutralize radicals caused by UV light; interestingly melanocytes are also found in the dark internal esophagus, where they function to destroy radicals produced by acid reflux out of the stomach. Macrophages also release certain compounds like lysozyme, defensin peptides (which form holes in cell membranes, much like complement) and tumor necrosis factor alpha (which can kill certain cancer cells). Macrophages also secrete certain complement proteins (although the liver produces most complement proteins) and interleukins, which help in the inflammatory response.

Chronic granulomatous disease is a genetic malfunction where the body does not produce NADPH oxidase. This retards the macrophages' ability to create Reactive Oxygen and Nitrogen Intermediates (ROIs & RNIs), predisposing afflicted patients to infections. TEST darshan

Antigens

Antigens are substances that react with naive B cells when free-floating or with T cells when processed and displayed on cell surfaces along with MHC molecules.

Both antigens and haptens (small molecules) can act as antigenic agents when faced with an active immune response in progress. But only antigens themselves can cause an immune response to begin; they are both antigenic and immunogenic. Anything that can start an immune response can react with an in-progess immune response, but some compounds cannot start an immune response (although we can make antibodies to them and an in-progress response will react with them). This is the difference between something that is immunogenic and antigenic.

What makes something immunogenic (capable of creating a full immune response)?

• heterogeneity (different amino acids, rather than synthetic monopolymers)
• foreigness (must be different from host's proteins)
• size (must be relatively large, e.g. 100 kDalton)

Because of the nature of antibody molecules, proteins and polysaccharides are the most common humoral antigens. Proteins are broken down and presented with MHC molecules to produce the cell-mediated immune response, and lipids/glycolipids are similarly presented (although they are presented on CD1 at the cell surface). Leprosy and tuberculosis are presented to the T cells in this CD1/lipid manner.

Immunogens are often given deliberately, as in the case of vaccines. Often they are complexed with adjuvants, compounds that enhance the vaccine. Adjuvants can include broken-up bacterial cell walls, oil (to slow the dispersion of the vaccine into the body), and water. The addition of bacterial cell wall components induces the innate immune system to actuate, enhancing the adaptive system due to a higher concentration of MHC-II receptors on the APCs, resulting in more activated B cells.

Remember that epitopes are the parts of an antigen that elicit an immune response. It makes sense that B cell-recruiting epitopes are more likely to reside on the surface of foreign invaders, and these epitopes are likely to be hydrophilic. T cell epitopes are more likely to be larger peptides, cleaved and presented with MHC-I and MHC--II on the surface of body cells and APCs. Thus, the outer hydrophilic parts of an invader elicit B cell responses, and the relatively hydrophobic inner parts elicit T cell responses. Also, denatured proteins are less likely to create a B cell response, but T cells are used to interacting, via their CD4 and CD8 receptors and TCRs, with broken-up peptides (antigenic peptides).

Haptens must join with carrier proteins in order to produce an immune response. Drug allergies are almost always caused by drugs that are, by themselves, too small to be immunogenic. Penicillin and other drugs will bind with nucleophilic sidechains on circulating body proteins, forming a hapten-carrier conjugate. This causes antibodies (IgE) to the drug to be produced, and an allergic reaction begins when IgE-receptors on mast cells are activated. Treatment with epinephrine will reverse the anaphylactic shock, diverting blood from the periphery and dilating the airways in a classical sympathetic response. For this reason, persons with life-threatening allergies will often carry epinephrine (adrenaline) injections with them in case of exposure to the hapten.

Antibodies

Antibodies, also known as immunoglobulins, are Y-shaped glycosylated proteins that are secreted by plasma B cells and reside in the membranes of activated effector B cells. The two arms of the Y are formed from the Variable regions of the two Heavy chains (which form both the stalk and the medial portions of both Y arms) and the entire Light chains (which form the lateral portions of both Y arms). The two arms of the antibody are known as the Fab fragments, as they are the portion that acts as an antibody, binding to antigen. The parallel heavy chain stalks of the antibody are known as the Fc fragments, which are relatively constant across all antibody (immunoglobulin) subtypes. There are certain hypervariable, or Complementarity-Determining Regions (CDRs) at the very ends of the arms, that is the Variable regions, of the antibody. The remainder of the Variable regions are known as Framework Regions. Genetic shuffling will give each antibody (immunoglobulin) a specificity to certain antigenic epitopes. Light chain proteins, secreted in excess in patients with multiple myeloma cancer of the plasma B cells, are known as Bence-Jones proteins, and are one of the few large proteins in the body with a notable positive charge. Waldenström macroglobulinemia (WM) is a non-Hodgkin lymphoma that also causes increased B-J proteins; it is a rare condition that also results in large numbers of IgM proteins throughout the serum.

Any of the Ig isotypes can contain one of two light chain subtypes, kappa κ (60%) or lambda λ (40%, with subtypes λ1, λ2, λ3).

There are several classes of immunoglobulin, each of which has a similar but distinct basic Fc structure, although several Ig subtypes form oligomers. The different subtypes of Ig are: Short (350 aa Fc) Ig isotypes with a hinge region

• IgA (α Fc stalk)
  • IgA1, IgA2
  • Can be found in monomer, dimer, trimer, or tetramer form with joining (J) chain between the monomers
• IgG (γ Fc stalk)
  • IgG1, IgG2, IgG3, IgG4
  • Monomer only
• IgD (δ Fc stalk)
  • Monomer only

Long (440 aa Fc) Ig isotypes with no hinge region

• IgE (ε Fc stalk)
  • Monomer only
• IgM (μ Fc stalk)
  • Monomer or pentamer only, with joining (J) chain at center of pentamer

Ig can be in a secretory sIg form or a membrane-bound mIg form. Naive B cells have only mIgM; as they mature, they express both mIgM and mIgD. Memory B cells can express mIgA, mIgG, mIgE, or mIgM (but not mIgD). Thus, as some point between naive and memory status, B cells stop expressing mIgD. Every B cell, be it memory or plasma, reacts against one specific epitope, even though a single cell can secrete or express on their membrane several different isotypes of Ig at once.

Although the arms of the Ig Y structure are important for binding Ag, macrophages, NK cells (mediators of antibody-dependent cell-mediated cytotoxicity, ADCC), and neutrophils have Fc receptors on their surface. Thus, antibodies will attach to pathogens, and passing neutrophils and macrophages will attach to the Y stalk pointing outwards, eating up the pathogens with greater fervor. Thus, Ig is an opsonizing agent.

The Complement System can be activated by both IgM and IgG. The complement system both

• forms pores in the cell membranes of target pathogen cells and
• attaches certain sticky proteins, such as C3b, which attach to RBCs and APCs, causing complement-attacked pathogens to be carried along with other blood cells to the liver and spleen for destruction

Immunoglobulin A (IgA)

• Secreted as dimer, with J (joining) chain between mononer substituents
• Most important secretory antibody, capable of passing across epithelial cell layers (transcytosis)
• Secreted in mother's milk to child
• Huge amounts are secreted in the GI tract each day (~10g per day)
• sIgA-secreting plasma B cells interact with poly-Ig receptors on basalateral surfaces of mucosal cells, especially those close to the GI tract lumen

Immunogloublin G (IgG)

• Passes through mucous membranes, and thus is partially secretory
• Crosses placenta from mother to fetus in third trimester (although IgG2 may not) via a neonatal Fc receptor
• Partial activator of the Complement System (IgG3>IgG1>IgG2; IgG4 does not activate complement)
• Fc regions bind very well to Fc receptors on neutrophils, NK cells, and macrophages (IgG1=IgG3>IgG4>IgG2)

Immunoglobulin D (IgD)

• Expressed on membrane of mature B cells

Immunoglobulin E (IgE)

• Fc regions bind very well to high affinity IgE receptors on Mast Cells (tissue) and basophils (blood), mediating their activation and release of histamine/heparin, and the production of multiple mediatiors inducing an allergic response. Low affinity IgE receptors, CD23, are also found on other cell types.

Immunoglobulin M (IgM)

• Expressed on membrane of mature B cells as monomer
• Expressed as a secretory pentamer, with monomer substituents joined by J (joining) chains
• First Ig produced by infant
• First Ig produced during primary (non-memory) immune response
• Major activator of the Complement System
• Shows some secretion across epithelial layers

The B Cell Receptor

Ig has a short cytoplasmic tail and must be complexed with a small dimer in order to form, altogether, the B Cell Receptor (BCR). A small dimer with an Ig-β and Ig-α component. This Ig-α is not related to the α Fc region of the IgA protein. The relatively long cytoplasmic tails of this small dimer allow the different mIg isotypes to send signals within the B cell upon binding with antigen.

Ig Classifications

• Isotype--within a species, all individuals share a given isotype of the Ig (this is concerned with the conserved regions of the Ig constant region)
• Allotype--within a species, each individual will have a given allele that codes for their Ig constant region (thus, individuals generally have a unique allotype)
• Idiotype--within an individual, each B cell will create a unique variable region, specific for a given antigen; this is the idiotype of each Ig/Ab, and can be similar only when B cells are reacting to a similar antigen

Antibody/Antigen Interactions

interaksi antigen-antibodi bisa menghasilkan presipitasi atau aglutinasi

T Cells

Differentiate in the Thymus

Cytotoxic T cells (CD8 cells)

• Binds to antigens on the membrane of target cells (virus infected, cancer and foreign cells) and directly destroys the cells.

T Helper/Helpeer T Cells (CD4 cells)

• Secrete cytokines that activate B cells, cytotoxic T cells, natural killer cells and macrophages.

B Cells

these cells diferentiate into antibody secreting plasma cells

Cytokines

These are signalling molecules that activate cells of the immune system

The Complement System

Complement serves to opsonize antigens, lyse pathogens, and mediate inflammation. There are three pathways by which complement can act: classical, alternative, and MB Lectin.

Inflammation

Inflammations is characterized by four key symptoms: 1)Redness-caused by warm blood flowing to the area 2)Pain-caused by damage to nerve fibers or irritation to toxic chemicals 3)Heat-also caused by warm blood flowing to the area 4)Swelling-caused by fluid moving from the blood to the tissue spaces of the injured area

Inflammation can cause a loss of function in an injured area. It traps microbes, toxins, and foreign material at the site of injury and prepares the site for tissue repair. For this reason, it helps to maintain tissure homeostasis.

Autoimmunity

Autoimmunity is a group of disorders in which the body's own immune system attacks the body's own antigens. Examples include rheumatoid arhtritis, systemic lupus erthymatosis, myasthenia gravis, Hashimoto's syndrome, etc.

Vaccines

Vaccines are a means of attaining artificial immunity.

Experimental Methods in Immunology

Stem Cells

The growth of stem cells in the bone marrow is the basis for cellular immunity. In vitro mimicry of this growth can be achieved by filling a semisolid medium with stromal cells. By adding different growth factors and cytokines, as well as stem cells of various differentiation level, the influence of chemical mediators of hematopoiesis can be understood. HSCs can be taken from a donor and injected into a person who has a defective or absent hematopoietic system. As little as 10% of the donor's bone marrow is removed and injected into the recipient, and the HSCs will find their way to the bone without direction, replenishing the hematopoietic system of the recipient. There are several types of stem cell grafts:

• autologous--donor is the recipient themself; the recipient can freeze HSCs prior to chemotherapy or radiation therapy for cancer; additionally, genetically engineered autologous HSCs can be injected back into the patient
• syngeneic--donor is genetically identical to recipient (identical twins)
• allogeneic--donor is genetically different from recipient
  • this can lead to graft-vs.-host disease (GVHD)
  • GVHD is based on MHC/HLA types, and so a bone marrow "match" occurs when an allogeneic member of the population is found to have relatively similar MHC/HLA types relative to the recipient

Knock-Out Mice

Genetic factors that produce certain cytokines (or certain cell types themselves) can be studied throught the use of knock-out mice. In these mice, a certain gene is inactivated and the animal is allowed to grow. The resulting phenotype is compared against other knock-out mice, in an attempt to piece together the genetic basis of different cellular functions and phenotypes. The process of gene knockout has become fine-tuned in recent years. Basically, a gene library is searched for a candidate gene to be knocked-out. A full mouse genome with the candidate gene knocked-out is developed and grown in a preliminary stem cell, and it is injected into a mouse stem cell via electroporation. Besides making the candidate gene inoperable, the altered genome will usually have two factors:

• A gene to make the cultured preliminary stem cell resistant to a certain antibiotic. Because not all of the stem cells will incorporate the altered genome, an antibiotic is applied to the treated cells, and only those that survive have therefore been injected with the altered genome (including both the antibiotic resistance gene and the knocked-out gene sequence)
• A gene to make the phenotype of a fully-altered knockout mouse obvious. For example, the altered mouse genome might include a gene to make the knockout mice pure white in color. The stem cells, before being treated, are collected from a black mouse. Thus, when the black fur mother mouse is injected with the treated stem cells, she will give birth to baby mice with varied coat colors, from black to white and grey in between. The nearly-white progeny are then cross-bred with each other, and the process is repeated until mice with pure white coats are created. These mice are most likely to have both the pure white genotype and the knocked-out gene. Thus, linked phenotypes are a marker for linked genotypes.

Several genes effect hematopoiesis, and have been developed via gene knockout. These include:

• GATA-2--regulates lymphoid and myeloid cell production, as well as RBC (erythrocyte) production
• Ikaros--regulates lymphoid cell production
• Oct-2--regulates differentiation of naive B cells into plasma cells

Hematopoietic Stem Cell Concentration

Irradiation of a mouse can wipe out the HSCs in the bone marrow, destroying the mouse's ability to produce red blood cells and leukocytes. These cells can be replaced with the bone marrow cells of an immunologically identical mouse, although in the injected replacement cells very few cells are actually HSCs. In order to concentrate the amount of actual HSCs in a sample, mice can be irradiated and replacement bone marrow cells are injected with fluorescent immunoglobulins that bind to mature RBCs and WBCs. A flow cytometry method can now be used to weed out these labelled cells, and the process can be repeated until only those that are most likely undifferentiated remain. The HSCs can be found in a subset of cells that contain the CD34 receptor on their surface; the CD34+ subpopulation is almost totally HSC with few differentiated cells.

Monoclonal Antibodies

A given antigen can have many epitopes, each one reacting with the immune system to create antibodies specific for each of the epitopes. When a given antigen is given to an experiment animal, the animal will produce antigens for each of the epitopes of the antigen, forming a polyclonal antibody response. Plasma B cells are then isolated from the animal's spleen, and these cells are fused with special cancerous (and therefore immortal) myeloma B cells, which do not generate any specific antibody themselves. The myeloma cells lack a certain enzyme, hypoxanthine-guanine phosphoribosyltransferase, or HGPRT, and therefore cannot grow under certain conditions (namely in the presence of HAT medium). The product of these B cell/myeloma cell hybridizations are known as hybridomas, and they secrete monoclonal antibody indefinitely. After hybridization, there will be three types of cells: 1) spleen B cells, 2) myeloma cells, and 3) successfully formed hybridomas. In order to select for the hybridomas, the mixture is grown on HAT medium (killing any unhybridized myeloma cells) and the spleen B cells (as they are not cancerous and will die of old age) are allowed to die. Only those cells that can live a very long time (myeloma component) and contain HGPRT (plasma cell component) are able to survive the selection process. This results only in hybridomas, each secreting a different antibody. The mixture of polyclonal hybridomas are now separated via differential dilution in order to isolate the hybridomas for each specific epitopes, finally resulting in functional, immortal, monoclonal B cells.

Monoclonal antibodies (mAbs) are incredibly useful. They can be radiolabelled or labelled with fluorescent molecules in order to detect proteins, such as cancer proteins in a tissue. The use of fluorescent antibodies has also allowed scientists to image cellular components, increasing the understanding of the cell's structure as well as its genetics. Additionally, the anticancer properties of immunotoxins result from a mAb bound to a certain toxin, which becomes localized to the cancer epitope that matches the mAb. Antibodies can also be formed with enzymatic function (abzymes), which hold the promise of cleaving proteins at specific points in order to study proteomes more successfully.

Affinity Chromatography

A column of beads contains monoclonal antibodies. A mixture of proteins (potential antigens) is then passed through the column, and the antigen in question binds with the monoclonal antibodies. The column is then washed, removing any antigen that is not captured by the monoclonal antibodies. The pH is then adjusted in order to break the Ab/Ag interactions, and the column is rinsed; the Ag in question will be eluted in this last wash.

Radioimmunoassay (RIA)

A radioimmunoassay is a scientific method used to test hormone levels in the blood without the need to use a bioassay. It involves mixing a radioactive antigen (frequently labelled with isotopes of iodine attached to tyrosine) with antibody to that antigen, then adding unlabeled or "cold" antigen in known quantities and measuring the amount of labeled antigen displaced.

Initially, the radioactive antigen is bound to the antibodies. When cold antigen is added, the two compete for antibody binding sites - and at higher concentrations of cold antigen, more binds to the antibody, displacing the radioactive variant. The bound antigens are separated from the unbound ones in solution and the radioactivity of each used to plot a binding curve.

Once this standard curve has been created, a sample can be taken from a patient. The sample is then added to the Ab/radioactive Ag mixture. The sample is allowed to mix with the radioactive Ag, and the unlabelled Ag (often a hormone being tested for in the body) will displace some of the radioactive Ag. The mixture is then "rinsed," and the amount of remaining radiolabelled antigen is compared with a standard curve, allowing an easy determination of unlabelled (patient-derived) Ag/hormone levels.

The technique is both extremely sensitive, and specific, if costly, and it is especially useful in diagnosing and treating autoimmune diseases such as Hashimoto's thyroiditis and Systemic Lupus Erythematosus.

Enzyme-Linked ImmunoSorbent Assay (ELISA)

A sample of antigen is applied to the surface of a well, and the spaces between the sample are filled with proteins that do not bind antibody. A large amount of antibody, linked to a certain enzyme that can create a color change, is added to the sample under conditions that allow only Ab/Ag binding; there must be enough Ab added to react with every possible Ag molecule in the sample--if you have more Ag than Ab, the extra Ag will not be detected by the test. The well is then washed, and the color-changing reaction is activated. The level of color change can be measured by spectrometry, and the concentration of antigen can be determined via comparison of color change with a standard curve.

• Sandwich (capture) ELISA--for Antigens of very low concentration, such as cytokines, binding to the well plate can be difficult. This method uses an Ab specific for one of the epitopes on the Ag, attaching these bottom antibodies to the plate well, where they bind the added small concentration of Ag. A second type of antibody, one that binds to a different epitope on the Ag, is added to the well. Thus, the Ag is sandwiched between a bottom "holding" antibody and an upper, "detector" antibody that is linked to a color-changing enzyme.

Competitive Inhibition Assay

Similar to the RIA, in this test a fixed amount of Ab is attached to the surface of a well. A known amount of radiolabelled Ag is added to the well, and the sample (ostensibly containing unlabelled Ag) is also added to the well. There must be enough labelled Ag added to react with every Ab, otherwise the test will not be accurate. This is because the unlabelled Ag must compete with the labelled Ag, and to truly compete every Ab binding site must be potentially filled with the labelled Ag. The well are rinsed, and the amount of unlabelled Ag that "out-competed" the labelled Ag is compared to a standard curve (thus, the amount of remaining labelled Ag is inversely proportional, via some relationship in the std curve, to the amount of Ag in the sample.

Blood Typing

The fact that each antibody has at least two (and, in the case of dimers and pentamers, more than two) identical binding sites for an epitope allows agglutination of antigens. Hemagglutination is the use of this property to test for blood types; anti-A Ab anti-B Ab, and a mixture of anti-A and anti-B Ab are added to aliquots of a blood sample, and the clumping (agglutination) indicates the blood type.

Coombs Test

The Coombs Test (also Coombs' Test) is a blood test used to determine whether there are red blood cell antibodies, which leads usually to hemolysis, especially in Rh disease. Coombs antibody is an anti-human globulin. It was first described in 1945 by Cambridge immunologists Robin Coombs, Arthur Mourant and Rob Race. The test is also used in screening blood prior to blood transfusion.

Two types of the test exist:

• Indirect Coombs test - also known as the indirect antiglobulin test (IAT). This is used in the matching of blood products. It detects immunoproteins present on red blood cell membranes, by adding a polyspecific antiserum which contains antibodies specific for human immunoglobulins and complement to agglutinate the cells.
• Direct Coombs test - also known as the direct antiglobulin test (DAT). It detects antibodies capable of attaching to normal red blood cells, by incubating normal red blood cells in the serum, washing the cells, and then using a polyspecific antiserum which contains antibodies specific for human immunoglobulins and complement to agglutinate the cells. The DAT is used to determine if the patient has immune-mediated hemolysis (antibody-mediated destruction of red blood cells), as occurs in Rh disease.

The Coombs Test is used to detect the presence of agglutinated red blood cells in a patient's blood. If positive, the interpretation is that an antigen-antibody reaction has taken place in vivo. The Coombs test is sometimes referred to as the Direct Antiglobulin Test. The indirect antiglobulin test is also called an antibody screen. In this test, a few drops of patient serum is placed in a small test tube with a drop of reagent red blood cells and checked to see if there is an antigen-antibody reaction. If there is such a reaction, it means the patient's serum contains antibodies to the known antigen on the reagent red blood cells.

References

• Goldsby RA, Kindt TK, Osborne BA and Kuby J (2003) Immunology, 5th Edition, W.H. Freeman and Company, New York, New York, ISBN 0-7167-4947-5
• Janeway CA, Travers P, Walport M, and Shlomchik M (2001) Immunobiology, 6th Edition, Garland Publishing, New York, New York, ISBN 0-8153-3642-X

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External Links

• The Merck Manual of Diagnosis and Therapy, Section 12: Immunology; Allergic Disorders
• Immunology notes, presentations and other academic materials
• Immunology

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