Cytokines (aka monokines or lymphokines) are regulatory proteins which bind specific receptors and have pleiotropic (multiple) or redundant functions. Cytokines are important for: activation, such as stimulation proliferation of activated T cells; recruitment signals, such as brining cells to sites of inflammation; and differentiation, such as of lymphocytes in the thymus and bone marrow. There are three pathways in which cytokines operate: autocrine, where cells self-stimulate; paracrine, where cells interact with nearby cells; endocrine, where cells circulate to interact with far-away tissues.

Some cytokines are classified as chemokines. A chemokine is a small peptide released in response to injury or infection, with similarities to the antigen-binding domains of the major histocompatibility complex. Chemokines are released by macrophages, endothelial cells, keratinocytes, smooth muscle and T cells.

There are two kinds of chemokines: α chemokines, with a region bearing contiguous cysteins; and β chemokines, with conserved cysteins separated by another amino acid. α chemokines include MCAF, RANTES and MIP-1β β chemokines include IL-8 and SDF-1.

Phagocytic cells and T cells migrate towards concentrations of chemokines, following a chemokine gradient. Also, certain chemokines have multiple roles in fetal development. In addition, chemokine receptors for RANTES, MIP-1&alpha, MIP-1β and SDF-1 are accessory receptors for entry of HIV into a cell.

The inflammatory response is characterized by the following three events:

1. Vasodilation. Vasodilation is an increase in blood vessel diameter.
2. Permeability. Capillaries increase in permeability, allowing exudate to flow to and swell the site of inflammation.
3. Phagocyte influx. An influx of phagocytes consists of three steps: margination, where phagocytes adhere to the capillary endothelium; extravasation, where phagocytes exit the capillaries; and chemotaxis, where phagocytes migrate towards the area of inflammation.

Soluble mediators involved in the inflammatory response:

1. Acute phase proteins. Released by the liver, acute phase proteins bind polysaccharides and initiate the complement cascade.
2. Histamine. Released in response to injury, histamine leads to vasodilation and increased capillary permeability.
3. Kinins. Also released in response to injury, kinins lead to vasodilation, an increase in capillary permeability and stimulation of skin pain receptors.

The most important function of the human immune system occurs at the cellular level of the blood and tissues. The lymphatic and blood circulation systems are highways for specialized white blood cells to travel around the body. White blood cells include B cells, T cells, natural killer cells, and macrophages. Each has a different responsibility, but all function together with the primary objective of recognizing, attacking and destroying bacteria, viruses, cancer cells, and all substances seen as foreign. Without this coordinated effort, a person would not be able to survive more than a few days, before succumbing to overwhelming infection.

Infections set off an alarm that alerts the immune system to bring out its defensive weapons. Natural killer cells and macrophages rush to the scene to gobble up and digest infected cells. If the first line of defense fails to control the threat, antibodies, produced by the B cells, upon the order of T helper cells, are custom-designed to hone in on the invader.

Many disorders of the human immune system fall into two broad categories that are characterized by:

Innate immunity is a non-specific inherited defense system that provides a general response against all pathogens. Innate immunity provides the body’s first protection against invaders (on the other hand, acquired immunity — aka adaptive immunity — responds to a persisting infection). Innate immunity stimulates adaptive immunity, influencing its expression to optimize its response against the specific types of invading microbes. Also, innate immunity is so effective that its mechanisms are often included in acquired immunity. Innate immunity consists of the following:

Cells of the innate immune system recognize non-self antigens via pattern-recognition receptors. Pattern-recognition receptors are receptors on the cell surface that are encoded by the genome and can detect repetitive structures (or patterns) specific to pathogens. Important cells of the innate immune system include:

When a phagocyte recognizes a pathogen, there are four important consequences: phagocytosis of the pathogen; cytokine secretion by the phagocyte; induction of co-stimulatory molecules; and, in macrophages and dendritic cells, antigen uptake, processing and presentation. Thus, phagocytes play an important role in initiating the immune response. The steps of the immune system can be broken into the following:

The innate immune system, when activated, has a wide array of effector cells and mechanisms. There are several different types of phagocytic cells, which ingest and destroy invading pathogens. The most common phagocytes are neutrophils, macrophages, and dendritic cells. Another cell type, natural killer cells are especially adept at destroying cells infected with viruses. Another component of the innate immune system is known as the complement system. Complement proteins are normally innactive components of the blood. However, when activated by the recognition of a pathogen or antibody, the various proteins are activated to recruit inflammatory cells, coat pathogens to make them more easily phagocytosed, and to make destructive pores in the surfaces of pathogens

At this point, please study acquired immunity and its suggested reading. After, please continue to pattern-recognition receptors.

Note: This author uses the phrases ‘acquired immunity’ and ‘adaptive immunity’ interchangeably. ‘Immune response’ refers to aspects of the immune system which are antigen-specific.

Adaptive immunity is triggered when an infection eludes innate defenses and generates a threshold of antigen. Acquired immunity is effective only after several days, the time required for antigen-specific T and B cells to proliferate and differentiate into effector cells. Also, adaptive immunity ensures that mammals surviving an initial infection by a pathogen are generally immune to further illness from by that same pathogen. The adaptive immune system is based on T and B cells (aka leukocytes, a kind of white blood cell) that are produced by bone marrow stem cells and mature in the thymus and/or lymph nodes. The fundamental steps of acquired immunity are:

Innate and adaptive immunity are quite intertwined. For example, antigen presentation by dendritic cells (part of the innate immune system) activates thymocytes to proliferate and differentiate (part of the adaptive immune system). In many species, including mammals, the adaptive immune system can be divided into two major sections:

In addition to determining self from non-self, the immune response has B and T cells which identify different forms of non-self. Distinguishing various forms of non-self is crucial. For example, effectively responding to a bacterial infection would have no effect on an intracellular pathogen. B and T cells rely upon subtle differences in the biochemical structures of foreign proteins, carbohydrates, lipids and other building blocks. Known as antigens, these non-self structures are the target of the immune response and cause it to produce antigen-specific antibodies in response.

The immune response can only detect bio-organic antigens, essentially limiting it only to chemicals encoded or controlled by genes. Thus, all antigens are based on carbon and the atoms which bond to carbon (hydrogen, oxygen, nitrogen, phosphorous and sulfur — aka CHONPS). The immune response ignores any chemical not based on the six atoms listed. This means that sand, mercury, minerals and other contaminants are not subject to the immune response.

B and T cells bear receptors to distinguish self from non-self. B cells present immunoglobulin (antibody molecule) and T cells present T cell receptor (TCR). Much like enzyme binds substrate, the function of both molecules is to bind antigen. Binding of antigen by both B and T cells leads to removal of antigen from the system. The configuration of CHONPS is the antigenic determinant.

At this point, please study Clonal Expansion Theory to understand how the body specifically detects countless antigens; next, memorize important cells of the immune system to grasp the team of cells that make up the immune system.

Macrophages process pathogens and present on their cell surface antigens from the pathogen. After antigen presentation, antigen-specific cells under clonal expansion. Antigen-specific cells with high antigen affinity (due to their particular antibody configuration) respond efficiently and preferentially expand over time. This explains why subsequent responses to an antigen are anamnestic (stronger and faster) after initial exposure.

Immunity requires a primary stimulus, meaning nobody is immune to an antigen until they have been exposed to that antigen. However, some people may be resistant to an antigen for genetic reasons or may have strong innate protection. Innate responses and acquired (anamnestic) responses evolved together to confer resistance and protection against microbial invasion and malignant cells. Activation of innate immunity cells (such as via TLRs) promotes antigen-presenting cells to not only present antigens but also activate B and T cells.

The immune system has evolved to deal with invasion by microbial pathogens. The main task of the immune system is to distinguish self from non-self. The immune system must not attack and destroy self, but it must eliminate whole organisms (such as bacteria and fungi) as well as intracellular pathogens (such as viruses). Connected by blood and lymph, the immune system is a concert of cells, tissues and organs working together to protect their host. These cells, tissues and organs communicate via direct surface interaction and via chemical communication. In chemical communication, cells release cytokines that flow through blood and lymph to initiate cells elsewhere throughout the body. The immune system is broken into two main components: innate immunity and acquired immunity.

Non-self structures are known as antigens and are the target of the immune response. Antigens must be based on carbon and the atoms which bond to it (hydrogen, oxygen, nitrogen, phosphorous and sulfur). This means the immune response detects only bio-organic antigens, essentially limiting it to chemicals encoded or controlled by genes. The immune response consists of B and T cells that detect subtle protein, carbohydrate, lipid and other structure differences to distinguish self from non-self microbes (and the kind of non-self microbe).

Fundamentally, the immune system relies upon two methods for protecting the host: antigen elimination and inflammation. Antigen elimination is basically a three-step process: antigen recognition, antigen binding and antigen elimination. The result is obliteration of antigens and their corresponding foreign microbes. Antigen elimination involves B cells, T cells, macrophages and antibody. Chemicals released during antigen elimination lead to inflammation. Inflammation includes: fever; vascular permeability; fluid build-up in tissues (edema); and even tissue damage, which initiates healing.

There are five families of cytokine receptor, described below with their accompanying ligands.

A thoroughly-studied disease of cytokine receptors is SCID, a genetic disorder which has helped reveal the roles that cytokines play.

The major histocompatibility complex (MHC) is present in all vertebrates, and is encoded by a group of genes called HLA in humans and H-2 in mice. Products of these genes are mostly cell surface glycoproteins involved in antigen presentation. MHC molecules must be bound to a peptide (it does not even have to be pathogenic) in order to be brought to the cell surface by the endoplasmic reticulum. The peptide-binding residues within the MHC determine what kinds of peptides it can bind. While B cells interact with free antigens, T cells interact only with antigens that are associated with a major histocompatibility complex, thus limiting T cells to interaction with antigens at cell surfaces.

Class I Major Histocompatibility Complex

Class I MHC molecules bind peptides and present them to cytotoxic T cells. A mammalian cell can express all Class I MHC alleles (up to six) at a time. Class I MHC molecules contain a 45kD transmembrane glyocoprotein α chain associated noncovalently with a 12kD β₂-microglobulin. The three Class I MHC genes — A, B and C in humans; K, D and L in mice — are polymorphic and each encode a different α chain (although be clear that each chain contains α1, α2 and α3 subunits). α chains contain α1, α2 and α3 domains; the α3 region contains a cytoplasmic tail that goes through the lipid bilayer and into the cytoplasm. To summarize, the Class I region of HLA contains HLA-A, HLA-B and HLA-C, which each encode different α chains. Lastly, the noncovalently bound β-2 microglobulin is not encoded within the MHC, but is critical for getting the MHC protein to the cell surface.

Between the α1 and α2 regions is the peptide binding groove (aka antigen binding groove), which presents antigenic peptides to CD8 (cytotoxic) T cells. Antigenic peptides — usually about nine amino acids — anchor into the groove at both ends via hydrogen bonds; without this anchoring, the Class I MHC glycoprotein is not brought to the cell surface. Longer peptides bulge out in the middle, while shorter peptides are taught. The middle of the peptide makes negligible contact with the MHC molecule, and is instead available for direct T cell receptor contact.

In the absence of β₂-microglobulin, Class I MHC molecules are not expressed on the cell surface. A cell that does not express β₂-microglobulin will have Class I MHC molecules floating in the cytoplasm but completely absent from the cell surface; if these same cells are transfected with a functional β₂-microglobulin gene, then they begin to express Class I MHC molecules on their cell surface. The enzyme papain cleaves Class I MHC molecules near the transmembrane domain, thus releasing just the extracellular portion (α1, α2, α3 and β₂-microglobulin).

Class II Major Histocompatibility Complex

Class II MHC molecules bind peptides and present them to helper T cells. Encoded by the centromeric end of HLA, the Class II MHC protein is (under normal conditions) found on macrophages, dendritic cells, B cells and activated T cells; however, in inflamed tissue, other cells can also express Class II MHC proteins. Class II MHC genes encode 32kD α and 27kD β transmembrane chains, and an intracellular invariant and non-polymorphic Ii chain. The α chain consists of α₁ and α₂ domains; the β chain consists of β₁ and β₂ domains. Just as with Class I MHC molecules, the α chain and β chain are noncovalently bound transmembrane glycoproteins with a cytoplasmic anchor.

Between the α₁ and β₁ domains is the peptide-binding cleft. Just as with the Class I cleft, the Class II cleft presents a peptide antigen; however, the Class II cleft binds antigens of at least 13 peptides (sometimes much longer) that are held in place along their backbone (instead of anchored at the ends) by interactions between cleft residues and the amino acids of the antigen. CD4 (helper) T cells recognize antigens presented by the Class II MHC protein.

The Class II region of HLA contains three genes (HLA-DR, HLA-DP and HLA-DQ) which each encode multiple different α and β chains. This allows a single translation of the HLA Class II genes to produce multiple different Class II MHC complexes. HLA-DR encodes three β chains and a single α chain; HLA-DP encodes one each of β1, β2, α1 and α2; HLA-DQ encodes one each of β1, β2, β3, α1 and α2. Please note, however, that α and β chains from different genes never mix; individual MHCs are always all HLA-DP, HLA-DR or HLA-DQ. Also, mice carry just two Class II Genes (IA, or AαAβ, and IE, or EαEβ).

Cell-surface peptide-bound MHCs bind to T Cell Receptors (TCRs). TCRs have a combining site which interacts the the α helices of α1 and α2, as well as the bound peptide. The center of the MHC-bound peptide nestles into a hydrophobic pocket between the CDR3α and CDR3β regions of the TCR. CDRs 1 and 2 of the Vα domain interact with the NH₂-terminus of the MHC-bound peptide; CDR2 1 and 2 of the Vβ domain interact with the C-terminus of the MHC-bound peptide, as well as some of the MHC helices.

Peptide Binding Cleft Detail

The binding of a peptide to an MHC molecule is very stable under physiologic conditions. Thus, most MHC molecules on a cell surface are associated with a peptide. Each cell expresses ∼10⁵ copies of each Class I molecule, with 2,000 different peptides being presented 100 to 4,000 times on each cell.

There are three different Class I MHC molecules — A, B and C in humans; K, D and L in mice — which each bind a different kind of peptide; within each gene, each allele delivers more specificity. Class I MHC molecules usually bind hydrophobic nonameric (nine amino acid) peptides, with specificity defined by same or similar amino acids at certain positions. Different alleles encode different peptide binding cleft residues at these positions.

Class II MHC molecules bind longer peptides than Class I MHC molecules, but only the central 13 amino acids actually interact with the molecule. These central 13 amino acids are conserved, meaning that certain patterns will specifically bind to certain Class II MHC molecule alleles. Peptides binding to Class I MHC molecules are bound primarily at their ends, while peptides binding to Class II MHC molecules are hydrogen-bound along their core amino acids (as opposed to being anchored at their ends).

Major Histocompatibility Complex Diversity

Class I and Class II MHC genes — present on Chromosome 6 in humans (Chromosome 17 in mice) — are polygenic, polymorphic and codominant. Polygenic DNA encodes multiple proteins with similar structure and function. A polymorphic gene has many different alleles which are all common within a population. Codominance means that both alleles of a gene — the maternal and the paternal copy — are equally expressed.

Antibodies and T Cell Receptors (TCRs) use mutation, recombination and other techniques to generate diversity. However, MHC Class I and II molecules just use promiscuity to bind the vast population of peptides. Class I and Class II molecules have low specificity, allowing a single molecule to bind many different kinds of peptide. In addition, Class I and Class II genes are highly polymorphic; for some genes there are over 100 common alleles. Within these alleles, the region encoding the peptide binding cleft has the highest variability.

With so many alleles, most individuals are heterozygous for MHC Class I and MHC Class II genes. However, inbred populations sometimes are homozygous for alleles that encode MHCs able to bind no protein. Although rare, this condition drives home the point that MHC diversity is at the population level, not the individual level (unlike with antibodies and TCRs, whose diversity is generated during hematopoiesis).

Class I genes are expressed co-dominantly. This means that all six alleles (three on each chromosome) are expressed together. As a result, a single cell can have up to six different types of Class I MHCs on its surface. Class II genes are expressed only on proliferating antigen-presenting cells (macrophages, dendritic cells, B cells and activated T cells. Because each class II molecule consists of two proteins encoded by two genes, an individual not only has combinations of individual α and β alleles, but also hybrid Class II MHCs contain maternal and paternal α and β chains (heterozygote complementation).

Major Histocompatiblity Complex Genetics

The major histocompatibility complex is encoded by HLA genes in humans and H-2 genes in mice. HLA and H-2 genes encoding the MHC itself are classical genes; HLA and H-2 genes which do not encode the MHC are non-classical genes). Non-classical genes are described in the table below, with non-classical genes without important immune functions are marked with a degree symbol. Although most non-classical genes have mysterious functions, it is suspected that some have MHC-like function.

Each region is highly polymorphic, meaning that it can have one of many different alleles. There are several common mouse strains, and for the sake of efficiency their haplotypes (their set of K, IA, IE, S and D alleles) have been compressed into shorthand. For example, H-2^k mice have all k alleles, H-2^d mice have all d alleles and H-2^b mice have all b alleles. Mice obviously have two chromosomes, so a H-2^k/k mouse is one whose two chromosomes have both have all k alleles, and a H-2^k/b is a heterozygote with one all-k-allele and one all-b-allele chromosome. Haplotypes are closely linked and new recombinant haplotypes rarely arise. If a patient receives an organ transplant from a donor with a different MHC haplotype (for example, H-2^b/b vs H-2^k/k) then that organ is rejected as non-self; the only exception is if a heterozygote (for example, H-2^b/k) receives an organ homozygous for one of its haplotypes (for example, H-2^b/b or H-2^k/k) because it recognizes that haplotype as self. Also, note that the alleles of individual regions can be described — for example, an H-2^k/b mouse has K^k and K^b regions.

The Class I region is ∼2,000kb long and contains ∼20 genes. In humans, the Class I region is at the telomeric end of the HLA complex. In mice, the Class I region is split in two, with Class II and III genes in the middle. The Class I region contains HLA-A, HLA-B and HLA-B in humans, and H-2K, H-2D and sometimes H-2L in mice. In humans, there is a litany of Class I non-classical proteins: HLA-E, -F and -G; HFE; HLA-J and -X; and MICA, B, C, D and E. In mice, Class I non-classical proteins are encoded in H-2Q, -T and M regions.

Located on the centromeric end of the MHC region, the Class II MHC region is broken into HLA-DR, -DP and -DQ in humans and H-2IA and H-2IE in mice. Each region encodes multiple Class II α and β chains — for example, HLA-DR encodes as many as four functional β chains. The multiple α and β chains combine, thereby increasing the diversity of Class II MHC molecules. There are also several non-classical Class II genes, all of which have limited polymorphism. Examples of murine non-classical Class II genes are Oα, Oβ, Mα and Mβ; and two human non-classical Class II genes have been identified, DM and DO.

Class III genes are located between Class I and Class II genes, and encode a variety of proteins. Although not encoding proteins directly related to the major histocompatibility complex itself, Class III genes are present in all vertebrates. Mutations in Class III genes frequently lead to disease.

Antibody-Dependent Cell Mediated Cytotoxicity (ADCC) is when leukocytes bearing F(c) receptors bind to and destroy antibody-coated cells.

Cells of the innate immune system recognize non-self cells by detecting structures unique to microbial pathogens and not present in mammalian cells. This non-adaptive recognition is performed by cell-surface pattern-recognition receptors. Pattern-recognition receptors detect repetitive pathogen-associated molecular patterns (PAMPs). Common PAMPs include nucleic acids, proteins, lipids and carbohydrates, as shown in the table below.

Pattern-recognition receptors are: encoded by the genome; expressed by all cells of a particular type (as opposed to clonal expansion); immediately responsive once bound to their ligand; and optimized for a broad range of pathogens. The two tables below chronicle the four steps in which pattern-recognition receptors operate, and the different types of pattern-recognition receptors.

The complement cascade (aka complement system or just complement) has seven functions — shown in the table below — all of which are essential to the immune system. Complements consists of three different simultaneous pathways of activating the membrane attack complex: the classical pathway, part of adaptive immunity; and alternative and lectin pathways, part of innate immunity. Three different pathways are involved in complement activation: classical pathway, which is stimulate by antigen-antibody complexes; alternative pathway, which spontaneously activates on contact with pathogenic cell surfaces; and mannose-binding lectin pathway, which recognizes mannose sugars which are usually present only on pathogenic cell surfaces.

Classical Pathway

In the classical pathway of complement, the F(c) of an antigen-antibody complex binds to the C1q component of C1. The F(c) must be immobilized as part of an antigen-antibody complex — an antibody enough is not alone to bind C1q and activate C1q’s two protease components (C1r and C1s). C1q has low affinity for F(c), so multiple F(c)s must be bound to C1q before C1r and C1s are activated. Once these proteases are activated, they cleave C4 and then C2. Two of the cleavage products — C4b and C2a — form a new protease (C3 convertase, aka C4b2a) which cleaves C3 into C3a and C3b. C3 convertase binds to C3b to form a new protease (C5 convertase, aka C4b2a3b) that cleaves C5 into C5a and C5b. C5b is the first protein of the membrane attack complex (MAC).

Because IgM has multiple F(c)s, it is more effective than other antibodies at activating complement. However, IgM must first change its conformation to expose its C1q binding sites. Also, please note that C3a and C5a just float away.

Lectin Pathway

The lectin pathway of complement is identical to the classical pathway, except the lectin pathway begins with mannose-binding protein instead of C1. Mannose-binding protein (aka MBP, mannose-binding lectin or MBL) is a primitive and non-clonal form of humoral immunity (immunity mediated by antibodies). Mannose-binding protein binds carbohydrate antigens frequently found on pathogen cell surfaces, but infrequently expressed by host cells. MBP is structurally similar to C1, with a binding region and two proteases, mannan-binding lectin associated proteases (or MASPs for short). Once MBP binds its carbohydrate antigen, the MASPs are activated to cleave C4 and then C2, with subsequent events identical to the classical pathway.

Alternative Pathway

There are two concepts behind the alternative pathway of complement: what occurs when a non-self cell is absent; and what occurs when a non-self cell is present. When a non-self cell is absent (meaning the tissue is healthy) then there is fluid-phase activation. Fluid-phase activation occurs continuously, spontaneously and very slowly. In fluid-phase activation, C3 spontaneously activates via hydrolysis to form C3_H2O — since it is unstable, C3_H2O usually reverts to C3. However, if C3_H2O encounters Factor B, then the two molecules bind to form a more stable C3_H2OB molecule. Factor D then cleaves C3_H2OB molecule to yield the enzyme C3_H2OBb (aka fluid-phase C3 convertase). C3_H2OBb has an active site on Bb; to culminate fluid-phase activation, this active site cleaves C3 into C3a and C3b. Fluid-phase activation is depicted in the figure to the left.

When a non-self cell is present, then a much faster process occurs. C3b binds to the surface of the non-self cell, then Factor B binds to the C3b. This cell-surface C3b-B complex is cleaved by Factor D to form C3b-Bb, a potent C3 convertase which begins cleaving C3 at an enormous rate to yield lots of C3a and C3b. The C3b eventually smothers the cell-surface, marking it for phagocytosis by macrophages, and granulocytes with C3b receptors. As the concentration of Factor B is depleted, a C3b-Bb begins to bind C3b to form the enzyme C3b-Bb-C3b (C5 convertase). This C5 convertase cleaves C5 into C5a and C5b: as in the classical pathway of complement, C3a and C5a just float away; C5b, already stuck to the cell surface, binds other proteins to form the membrane attack complex.

Membrane Attack Complex

The membrane attack complex (MAC) is an extremely potent agent of cell lysis. After cleavage of C5 into C5a and C5b, C5b binds one molecule each of C6, C7 and C8. After this, C5b binds an additional 6-10 molecules of C9 to form the complete membrane attack complex. The large multi-subunit membrane attack complex inserts into the lipid bilayer, allowing fluid to rush into the cell and kill the cell. Just one membrane attack complex per cell is enough to destroy the cell.

Peptides of Complement

Discovery of Complement

Cell lysis — a function of complement — was identified in the early 1900s as critical to the immune system yet independent of antibodies. An experiment was devised to identify the agents of complement.

Circulating white blood cells are stimulated to cross out of blood circulation (extravasation) by sets of adhesion molecules present on both white blood cells and the vascular endothelium of post-capilarry venules (or PCVs, located downstream of every blood capillary). Extravasation only occurs when adhesion molecules are expressed in both white blood cells and the vascular endothelium. Adhesion molecules on white blood cells are selectins, while adhesion molecules on PCVs are addressins. The table below describes the steps of extravasation to the site of an infection.

Similarly, virgin lymphocytes must extravasate to specific organs to complete their maturation. These lymphocytes express selectin-like molecules specific for addressins presented on PVCs adjacent to the target tissue. For example, virgin T cells migrating from the bone marrow express selectin-like molecules which bind specifically to addressins expressed only at the thymus (but not at lymph nodes or in the bone marrow). Similarly, virgin B cells migrating from the bone marrow express selectin-like molecules specific for addressins only at lymph nodes and the spleen.

The table above lists some addressins, when during an infection they are expressed and their affinity for the selectins they bind.

Bound pathogen is surrounded by a membrane, forming the phagosome which becomes acidified. Lycososomes containing enzymes, proteins and peptides fuse with the phagosome to form the phagolysosome. Upon phagocytosis, macrophages and neutrophils produce toxic products which are directly toxic to bacteria. Pathogenic microbes must either avoid engulfment, grow inside the phagosome (as do mycobacteria) or grow a thick polysaccharide capsule not recognized by any phagocyte receptor.

1. Foreign microbe attaches to pseudopods on the surface of a phagocyte. Pseudopods are long extensions of the cell, like arms.
2. The phagocyte’s cellular membrane engulfs the microbe to forma phagosome organelle which floats into the cytoplasm.
3. The phagosome fuses with a lysosome organelle which contains the toxic chemical and enzymes (such as H₂O₂, O₂^- and NO).
4. Lysosomal contents digest the captured microbe; the phagolysosome refuses with the phagocyte cellular membrane to release its contents into surrounding tissue.

Type 1: Allergy

Type I hypersensitivity (most commonly allergies) stem from overproduction of IgE. Thus, it requires an initial encounter so that isotype switching can occur — in secondary responses, the IgE crosslinks the antigen (and F(c)εRI and causes mast-cell and basophil degranulation. Atopic allergies and asthmatics are becoming more common in economically developed nations due to changes in exposure to childhood infectious disease (atopy is negatively associated with measles, hepatitis A or tuberculosis infections), environmental pollution (diesel exhaust enhances T_H2 responses, leading to higher IgE production), allergen levels (crazy cat ladies) and dietary changes.

IL-4 is pivotal in regulating the IgE response: IgG1 and IgE account ∼2% of all antibodies secreted by splenic B cells incubated with LPS; IgG1 accounts for ∼50% and IgE accounts for ∼20% of all antibodies secreted by B cells incubated with LPS and IL-4. IL-4 knockout mice cannot mount an IgE response to parasites. IL-4 stimulates isotype switching by activating the promoters for Iε, and Iγ1 (the I regions for ε and γ1 isotype genes). T_H1 cells enhance response to IL-3, IL-4 and IL-10. T_H2 cells reduce response to IFN-γ.

Type 4: Delayed-Type Hypersensitivity

Delayed-type hypersensitivity (DTH) is a T_H1-mediated localized inflammatory reaction that effectively clears intracellular pathogens (such as viruses) and contact allergens (such as poison oak and poison ivy). DTH is characterized by large influxes of inflammatory cells (like mφs), tissue damage and delayed onset. DTH reactions functional antibodies and a functional complement system; it cannot occur without B1 cells nor C5. There are two phases related to delayed-type hypersensitivity:

Contact allergens (such as poison oak) are usually small molecules which complex with skin proteins; these complexes are internalized by Langerhans cells (APCs found in skin) and then presented to T_H cells. Also, when DTH is continuously activated, normal tissue is damaged due to a continuous assault of lytic enzymes. This can occur in the lungs of Mycobacterium tuberculosis patients, as well as lesions in Mycobacterium leprae patients.

The Theory

The discovery of B cells led to a modern theory of antibody production called Clonal Expansion Theory (sometimes still referred to as Clonal Selection Theory). In Clonal Expansion Theory, B and T cells are created with random antibodies, then screened for self-reactivity. When antigen enters the system, it eventually binds to any B cell displaying an antibody specific to that antigen. This binding event triggers the following three steps:

1. Each activated B cell reproduces, to create an expanding population of identical B cell clones. This population is called a clone.
2. Some members of the clone become plasma cells. Plasma cells produce and secrete copies of the antibody displayed on the B cell surface.
3. Other members of the clone are stored as memory cells. Thus, there is a large population of cells to create a strong response when the antigen enters the system again.

Clonal Expansion Theory explains:

1. Specificity, since only antigen-reactive clones are triggered and only antigen-specific antibody is produced. Diversity is not explained but incorporated into this theory.
2. Memory, since clonal expansion explains why subsequent responses to an antigen are exponentially stronger than the initial response.
3. Tolerance, since B and T cells with potential self-reactivity are destroyed or rendered anergic (unable to respond).

Generation of Diversity

An enormous number of specific antibodies and T cells must be generated to respond to the enormous and changing antigenic universe. Immunoglobulin (for B cells) and TCR (for T cells) genes have multiple and similar methods for creating diverse antigenic receptors:

1. Multiple genes encode different protein sequences with different specificities.
2. Multiple segments of genes differentially combine to create increased specificities.
3. Differential junctions following gene segment recombination yield different amino acid sequences.
4. Somatic (after clonal expansion has begun) mutation of receptor genes in various clones causes changes in germline sequences.

Neutrophils are commonly referred to as polymorphonuclear neutrophilic leukocytes (PMNs/PMLs) or polys, even though technically any granulocyte is a PMN. Neutrophils are intensely phagocytic and arrive quickly at infection sites (unhealthy tissues) to phagocytize pathogens. Neutrophils have an F(c) receptor (FCR) which detects antibodies bound to antigens on the surface of pathogens. The process of detecting antibody-antigen complexes is called opsonization. After opsonization, the neutrophil absorbs the infectious microbe and degrades it.

Neutrophils have a lobulated nucleus, meaning it is condensed and dead — hence the term polymorphonuclear. Their cytoplasm is filled with granules containing degradative enzymes such as lysozyme, collagenase, elastase and others. During phagocytosis, these granules combine with phagosomes to break down pathogens.

Neutrophils are abundant in blood, constituting 50-60% of circulating white blood cells. However, neutrophils are absent from healthy (uninfected) tissues and only live about six hours. After performing phagocytosis several times, the neutrophil dies and degranulates (thus releasing its degradative enzymes). These enzymes damage and inflame local tissues — making neutrophils important in inflammation — but these corrosive enzymes also initiate healing.

After understanding Clonal Expansion Theory, it is important to become familiar with the origin and nature of cells involved in the immune systems. Granulocytes and monocytes travel only in the blood. Lymphocytes circulate through both blood and lymph; lymphocytes can exit a lymph node via its efferent vessel, travel through the lymph and then enter the bloodstream via the neck. Cells go back and forth between blood vessels, lymph vessels and tissue via extravasation. Leukocytes (aka white blood cells) are just any cell in the blood other than red blood cells, meaning all the cells below can be white blood cells depending on their location. All blood cells arise via hematopoiesis.

Granulocytes

Granulocytes are a type of white blood cell containing granules. Granulocytes are aka polymorphonuclear neutrophilic leukocytes (PMNs/PMLs) or polys, although PMN can refer specifically to neutrophils since neutrophils are the most common granulocyte. The granules inside granulocytes are secretory vesicle containing a molecule (for example, basophil granules contain histamine). The granules are dormant in the cytoplasm until a cell signal instructs the granules to release their components. This signal is typically shock or distress. Degranulation is when granules release their contents from the cell.

Lymphocytes

Lymphocytes bind to specific antigens and generate from stem cells in primary lymphoid tissue via hematopoiesis. Mφs, dendritic cells and B cells originate in bone marrow; and T cells originate in the thymus. After maturing, lymphocytes migrate to secondary lymphoid tissues (lymph nodes, blood and other tissues) to fulfill their role.

Macrophages

Macrophages (aka mononuclear phagocytes or Mφs) have two main functions: phagocytosis and antigen presentation. In phagocytosis, the macrophage or monocyte cell-surface F(c) Receptor (FcR) binds to the antibody-antigen complex (an antibody bound to an antigen on the cell-surface of a pathogen). The pathogen, antigen and antibody are engulfed and degradative granules and enzymes break it down in the lysosome. Digested antigen fragments are bound to MHC molecules and presented on the macrophage or monocyte cell-surface so that effector cells (B and T cells) can recognize the antigen. Because macrophages present antigens, they are antigen presenting cells (APCs). Macrophages play a tremendous role in clearing tagged antigens and releasing degradative enzymes to damage tissues and initiate healing.

Macrophages: trap, engulf and destroy pathogens (phagocytosis); present antigens for the adaptive immune response (antigen presentation); produce cytokines (including IL-12); and induce co-stimulatory molecules. Also, macrophages also bear CD14 (and LPS receptor), CD11b/CD18 complex (binds C3b and C4b, complement byproducts), scavenger receptor (binds sialic acid), TLR and F(c)R (described above). Please read about the precursor to macrophages, the monocyte.

Miscellaneous Cells

Important Classes

Immune system cells can be organized many different ways. The categories above are based primarily on origin. However, other broad classes are frequently used which are important to understand.

Hematopoiesis is the development of blood cells from hematopoietic stem cells (HSCs). A stem cell is any cell which can differentiate into other cell types; stem cells maintain their population via asymmetrical division — one daughter cell differentiates (the progenitor cell), and the other daughter cell remains in the bone marrow to continue the cycle. HSCs are sensitive to chemotherapy and radiation because of their frequent divisions; this sensitivity is the main barrier to tumor eradication. Stanford researcher Dr. Weissman and his colleagues developed stem cell enrichment to separate stem cells from bone marrow tissue.

The daughter cell that will differentiate is the progenitor cell, meaning it cannot self-renew and is committed to a particular cell lineage. There are two kinds of hematopoietic progenitor cells: lymphoid progenitor cells, which give rise to B cells, T cells and NK cells; and myeloid progenitor cells, which give rise to red blood cells (erythrocytes), various other white blood cells (neutrophils, eosinophils, basophils, monocytes, mast cells and dendritic cells) and platelet-generating cells called megakaryocytes.

Most hematopoiesis occurs in the bone marrow within a hematopoiesis inducing microenvironment (HIM) that is composed of cytokine-secreting stromal cells. Stromal cells include fat, endothelial, fibroblast and macrophage cells. Some of these cytokines operate via diffusion, while others are membrane-bound to the stromal cells and require direct cell-to-cell contact. Mostly identified via knockout mice, to the left are a few of the genes critical in hematopoiesis. These genes can impact many lineages or just one.

The lifespan of a cell can range from one 1 day for neutrophils to 120 days for erythrocytes to several decades for some T cells. With most blood cells dying due to aging, hematopoiesis produces just enough cells to keep up with cell death. The average human being produces 3.7E11 daily of white blood cells alone. To prevent overpopulation or misbehavior, cells undergo apoptosis (programmed cell death) or necrosis (death in response to cell injury). In apoptosis, the cell shrivels and then is phagocytosed by a macrophage. In necrosis, the cell swells, bursts and (in a crucial difference between apoptosis and necrosis) releases its contents into the surrounding area. This release can trigger an inflammatory response in surrounding tissues.

A component of the innate immune system, natural killer cells (NK cells) have genomic (not needed recombination, or RAG-independent) cell surface receptors which recognize classical Class I MHC molecules (and structural relatives like MICA, RAE-1 and H-60). Instead of directly recognizing pathogens, natural killer cells monitor cell surface molecules indicative of pathogenesis. This sensitivity allows natural killer cells to vigorously initiate natural killer cytotoxicity (by emptying granules of porforin and granzyme) and inflammation as soon as pathogenesis is detected, and is essential to protection against viruses and tumors.

Natural killer cells lack TcRs, CD4s and CD8; instead, they have: cell-surface activating receptors, which bind noncovalently to molecules with ITAMs; and on the cytoplasmic side, inhibitory receptors with ITIM(s) which — upon phosphorylation — recruit and activate SHP-1 & -2, which inhibit the activating receptors. The balance between activating signals and inhibitory signals is what determines whether a natural killer cell will destroy or bypass a microbe it encounters. There are many different inhibitory and activating receptors, but two well-studied ones are:

Macrophages (aka mononuclear phagocytes or mφs) have two main functions: phagocytosis and antigen presentation. In phagocytosis, the macrophage or monocyte cell-surface F(c) Receptor (FcR) binds to the antibody-antigen complex (an antibody bound to an antigen on the cell-surface of a pathogen). The pathogen, antigen and antibody are engulfed and degradative granules and enzymes break it down in the lysosome.

Digested antigen fragments are presented on the macrophage or monocyte cell-surface (in an MHC context) so other cells can recognize the antigen. Because macrophages present antigens, they are antigen presenting cells (APCs). Macrophages play a tremendous role in clearing tagged antigens and releasing degradative enzymes to damage tissues and initiate healing.

Since macrophages present antigens on their cell surface, they are known as antigen presenting cells (APCs). These antigens are then recognized by effector cells (B cells and T cells). Macrophages also activate cytokines, which stimulate differentiation and reproduction of lymphoid cells.

Macrophages are continuously maturing from circulating monocytes. Please read about the precursor to macrophages, the monocyte.

B cells (aka B lymphocytes) produce antibody when exposed to their complementary antigen. These antibodies can cause engulfment of infectious bacteria, neutralization of virions and induction of the complement cascade.

In the bone marrow, B cells complete their hematopoietic differentiation from stem cells into IgM⁺,IgD^weak immature virgin B cells. Next, in the medulla, B cells fully activate to become IgM⁺,IgD⁺ mature virgin B cells. Upon exposure to antigens, B cells in the medulla begin producing antibodies that flow through the lymph to the entire body. This helps create the body’s immune memory, with large amounts of antibody loaded in the bone marrow and at germinal centers. B cell activation occurs in the following steps:

1. An antigen flows through an afferent lymphatic vessel and into a node’s cortical sinus.
2. The antigen percolates through the node until getting trapped by reticular cells and dendritic cells.
3. Macrophages within the node trap and present the antigen. If it is a foreign antigen, either or both virgin B cells or memory B cells may react.

B-1 Cells

The B cells discussed so far are conventional B cells (aka B-2 cells). There is another subset of B cells known as B-1 cells (aka CD5 B cells, since some species’ B-1 cells display CD5). B-1 cells arise from stem cells during fetal life and self-renew via division of existing cells. While conventional B cells usually produce IgG, B-1 cells typically produce IgM and have little or no IgD. CD5 B cells secrete antibodies in response to TI-2 polysaccharides, leading to complement and removal of bacteria. This occurs within 48 hours of antigen exposure, as a bridge until the adaptive T cell response can activate. Unlike the T cell response, the B-1 response does not have memory. The table below (adapted from Immunology, 6^th edition) further compares and contrasts conventional (aka B-2) and B-1 cells.

After hematopoiesis in the bone marrow, granulocyte-monocyte progenitor cells differentiate into promonocytes. These promonocytes enter the bloodstream, where they mature further into monocytes; monocytes enlarge five- to ten-fold and become phagocytic while circulating for about up to three days in the blood. Next, these monocytes extravasate into tissues, where they become either fixed (tissue-specific) or free (wandering about the body) macrophages.

Monocytes move quickly to sites of infection in the tissues, and are one of the five major types of white blood cell. Monocytosis is the state of excess monocytes in the blood, and is indicative of disease. Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors. Splenocytes are monocytes found in the spleen; macrophages are monocytes which have migrated from blood circulation to other tissues.

Instructional theories postulate that antigens play a central role in determining antibody specificity. Conversely, selective theories state that an antigen reacts with an already-existing antibody. Selective theories better explains acquired immune responses. Below is a history of antigen-antibody theories.

B cells were discovered approximately fifteen years after Burnett’s Clonal Selection Theory, proving that antibodies all pre-exist and that the repertoire is independent of the antigenic universe. This validated Burnett’s theory as well as Ehrlich’s similar (although much older) proposal. However, two key facets of Ehrlich’s theory were also disproven: B cells are exclusively dedicated to antibody production; and antibodies are not from food. The Clonal Expansion Theory is the modern explanation for antibodies and their origin.

The T cell receptor (TCR) is a T cell surface receptor that recognizes antigens presented by MHC molecules. It is a heterodimer composed of either α and β chains or γ and δ chains, and which interacts with another T cell surface component CD3. αβ T cells are usually highly specific (adaptive immunity), while γδ T cells usually recognize broader antigen classes (innate immunity). TCR chains each contain two 60-75 amino acid domains: the amino-terminal variable (V) domain and the constant (C) domain. The V domain contains three hypervariable regions, similar to antibody complementarity determining regions.

The two chains are connected at a cysteine residue by a disulfide bond. The transmembrane region is 21 or 22 amino acids and positively charged, anchoring the TCR into the cell membrane and promoting TCR-CD3 interaction. Lastly, the cytoplasmic tail (carboxy-terminal end) is 5 to 12 amino acids. αβ and γδ TCR heterodimers resemble an Fab fragment attached to the cell membrane; due to this structural homology, they are included under the immunoglobulin superfamily.

αβ TCRs react only with peptide antigens. However, γδ TCRs can react to glycolipids and phospholipids (nonpeptides) presented by CD1. Furthermore, some γδ cells react with unprocessed peptide antigens not even presented by an MHC. Also, the most common γδ TCRs within a given species bind the most common pathogens that species encounters. These γδ TCRs directly binds microbes, and the γδ cells might directly kill pathogens.

Both innate and adaptive immunity rely heavily upon binding of the TCR to a peptide-MHC complex. γδ cells are characterized as part of innate immunity due to their rapid responses, low specificity and ability to bind free antigens. αβ cells, conversely, are clearly part of adaptive immunity. In addition to these functional differences, γδ cells and αβ cells are also structurally different. Allison, Garboczi and their coworkers found that a γδ receptor specific for non-presented microbial phospholipids has a deep binding cleft. Also, separate research found that γδ TCRs have a 111° bend between their V and C regions while αβ TCRs have a 147° bend.

T Cell Receptor Accessory Molecules

In B cells, membrane-bound antibodies associate with the Ig-α/Ig-β heterodimer to form the B cell antigen receptor. In T cells, the T cell receptor associates with its fellow membrane-bound component CD3 to form the TCR-CD3 membrane complex. Please note that CD3 is another component of the cell surface on T cells — TCR and CD3 are together on the T cell surface and interact together to transduce signals into the T cell. CD3 is essential for membrane expression of the TCR and for signal transduction. All CD3 chains contain a negatively charged amino acid (aspartic or glutamic acid) which interacts with one or two positively charged transmembrane TCR residues. Please note that B and T cell accessory molecules only participate in signal transduction after antigen interaction and do not actually influence antigen interaction.

Alone, the T cell receptor has low affinity for the peptide-MHC complex. For this reason, additional accessory molecules known as coreceptors or cell-adhesion molecules strengthen the bond between a T cell and a target or antigen-presenting cell. CD4 and CD8 are coreceptors which also distinguish cytotoxic T cells from helper T cells; CD4 binds Class II MHC molecules and CD8 binds Class I MHC molecules. Once bound, CD4 and CD8 also activate signal transduction. Additional coreceptors include CD2, LFA-1, CD28 and CD45R, all of which bind independently to other ligands on target or antigen-presenting cell. Once these coreceptors bind, the TCR scans for its appropriate peptide-MHC complex. Once TCR binds, membrane expression of cell-adhesion molecules is activated to tighten the intracellular bond. Cell-adhesion molecules play a role in tissue graft acceptance or rejection. If the grafted tissue bears MHC molecules which are similar to the host, then T cells will still respond to the MHC molecules.

T Cell Receptor Genomics

Genes encoding α, β, γ and δ chains are expressed only in T cells. Functional TCR genes are formed by rearrangements of V and J segments in α and γ genes, and of V, D and J segments (like IgH) in β and δ genes. The α, β, γ and δ genes also include C segments which do not rearrange. The C region contains a long exon encoding much of the C domain, followed by shorter exons encoding the connecting, transmembrane and cytoplasmic regions. In humans, α and δ gene segments are located on chromosome 14, while β and γ gene segments are located on chromosome 7. In mice, the α, β and γ gene segments are located on chromosomes 14, 6 and 13. In both mice and humans, δ gene segments are located between V and J α segments. The location of the δ gene segments is important: functional rearrangement of the α gene segments deletes C_δ, so that a T cell cannot co-express αβ TCRs with γδ TCRs. The table below describes human and murine TCR genes, and counts both pseudogenes (nonfunctional mutants) and functional genes.

Like the antibody L chain, the α chain is encoded by V, J and C segments. Like the antibody H chain, the β chain is encoded by V, D, J and C gene segments. Rearrangement of the TCR gene segments results in α VJ joining and β VDJ joining. After the rearranged TCR genes are transcribed, the α and β chains are expressed as a disulfide-linked heterodimer on the T cell membrane. Immunoglobulins can be bound or secreted, but TCRs are only membrane-bound. The TCR constant region, as shown in the figure above, includes constant (C), connecting, transmembrane and cytoplasmic sequences. Also, as opposed to the multiple different immunoglobulin C gene segments encoding different isotypes, α DNA has only one C segment and β DNA has duplicated J and C segments.

Pre-T cells expresses the recombination activating genes RAG-1 and RAG-2, as do pre-B cells. The RAG-1/2 recombinase recognizes conserved recombination signal sequences (RSSs) flanking each V, D and J gene segments in non-recombined TCR DNA. RAG-1/2 catalyzes V-J and V-D-J joining via the same deletions and inversions which occur in immunoglobulin genes. First, Rag-1/2 knicks one DNA strand between the coding and signal sequences and excises the resulting DNA loops. SCID mice (which lack B and T cells) are defective for a gene which repairs double-stranded DNA breaks; as a result, in SCID mice, the Ab and TCR D and J gene segments are not rejoined. RAG-1/2 only recombines TCR genes in T cells and Ig genes in B cells. This specificity is due to different recombinase regulatory systems, and uniquely configured chromatin in each lineage that allows recombinase to access only appropriate sites.

δ genes are located between the V and J segments of the &alpha gene. Thus, when the α gene recombines, the δ DNA is excised. In a phenomenon known as allelic exclusion, this mechanism prevents αβ TCRs and γδ TCRs from co-expressing. Another instance of allelic exclusion involves the two duplicate β gene J and C clusters. Having two duplicates means that at least one functional rearrangement is likely to take place. Once a functional rearrangement occurs, then rearrangement of the other cluster is inhibited. However, more than one α allele can undergo functional recombination; while this can lead to different α chains being expressed on the same cell surface, only one allele is MHC restricted and therefore functional. The mechanisms of TCR diversity are described below.

Isolating the T Cell Receptor

So Many Names For T Cells!

T cells — also known as T lymphocytes or thymocytes — are broken into two classes: CD8+ T cells, which are T cells which express CD8 cell surface proteins; and CD4+ T cells, which express CD4 cell surface proteins and are called TH cells. All T cells contain a T cell receptor (TCR), which is activated only by antigens embedded in an MHC complex. TcRs and antibodies are similar in that they both specifically bind antigens, but they are critically different because TcRs only bind MHC-associated antigens and an antibody will bind a free (floating) or membrane-bound antigen. When a TcR is activated by an antigen-MHC complex, its associated T cell will release a plethora of cytokines. Cytokines are used by the immune system to communicate within itself and with other tissues. To fully understand T cells, please read about T cell development upon completion of this article.

CD4 Cells    ·    TH Cells    ·    Helper T Cells

T cells expressing CD4 surface protein are called helper T cells (aka TH cells, T_H cells, effector T cells or any variation with the word ‘lymphocyte’ in place of ‘cell’. TH cells begin as TH₀ cells, but then differentiate into either TH1, TH2 or TH17 cells. TH1 and TH2 cells cross-regulate each other. However, an individual TH cell only produces one cytokine; thus, TH1 and TH2 effects are on the level of the entire body.

There are multiple diseases related to TH1 and TH2 cells. Experimental Allergenic Encephalomyelitis (EAE) is caused by a faulty TH1 response to myelin basic proteins of the central nervous system. Leprosey results from an inappropriate TH2 cell activity and is carried by a dominant allele. Allergies are causes by TH2 responses that lead to preferential IgE production. Also, as AIDS progresses, TH1 cells become TH2 cells, and TH17 cells rapidly disappear into the gut.

Virgin T cells migrate from the thymus through the blood, eventually flowing into capillaries at a lymph node and then through post-capillary venules (PCVs) into the node itself. T cells sometimes activate and exit lymph nodes to patrol the bloodstream. These circulating, activated T cells have two fates: extravasation to an infection site (detailed) below, following by reversion to a memory state and flow through the lymph to the nearest lymph node; or, if no extravasation occurs, reversion to a memory state after a few days of circulation, followed by crossing out of blood through PCVs and then flow to the nearest lymph node.

CD8 Cells    ·    TC Cells    ·    Cytotoxic T Cells

T cells expressing CD8 surface protein are called cytotoxic T cells (aka TC cells, T_C cells or any variation with the word ‘lymphocyte’ in place of ‘cell’). Cytotoxic T cells recognize surface markers on other cells in the body that label those cells for destruction. In this way, TCs help to keep virus-infected or malignant cells in check. Also, manipulation of antigen presenting TC cells is important for immune system targeting of tumor cells.

Traveling along chemical signals, thymocyte precursors migrate via blood from the bone marrow to the thymus. These cells have not yet rearranged their T cell receptor (TCR) genes and thus lack the T cell receptor (let alone CD3, CD4 or CD8); still lacking any characteristics of thymocytes, these immature T cells begin to divide furiously before individually undergoing four stages denoted double negative (DN) 1-4, named as such because the cells still lack CD4 and CD8 (CD4^-CD8^-). The four different DN steps — taking a total of ∼3 weeks — are described below, followed by the double-positive state (CD4⁺CD8⁺) and finally mature single-positive CD4⁺CD8^- or CD4^-CD8⁺ cells.

Thymic selection is a two-step process: positive selection, which induces apoptosis in thymocytes whose TCR cannot bind self MHC molecules; and negative selection, which induces apoptosis in thymocytes which bind self MHC molecules too well or in presence of a self peptide. Positive selection results in MHC restriction, and negative selection results in self-tolerance (meaning the thymocytes will not attack healthy self cells).

There are two proposed models as to how CD4⁺CD8⁺ cells mature into CD4⁺CD8^- or CD4^-CD8⁺ cells: the instructive model and the stochastic model. Neither model has been definitely proven nor disproven. The instructive model postulates that double-positive cells interact with either a Class I or a Class II MHC molecule, and are somehow signaled to differentiate into either CD4 or CD8 cells. The schotastic model postulates that repression of CD4 or CD8 is random and has nothing to do with TCR specificity. Only thymocytes whose TCR and coreceptor bind the same MHC molecule continue to mature.

The primary response is activation of naive thymocyte by a peptide-MHC complex. ∼48 hours after activation, the thymocyte enlarges into a blast cell and repeatedly divide to form a population of genetically identical cells (clonal expansion). Remember the G proteins described under transduction, and that G proteins help trigger the G₁ phase of the cell cycle). IL-2 concentration increases 100x in activated cells, helping induce up to 2-3 daily divisions for 4-5 days as well as thymocyte differentiation into either effector T cells or memory T cells.

T Cell Death

Over 98% of all thymocytes die during positive and negative selection, with the remaining cells entering the circulatory system to differentiate into effector or memory thymocytes. These T cells express two cell-surface proteins, Fas and Fas ligand (FasL), which are both essential for apoptosis via the Fas pathway. Upon activation, thymocytes increase Fas/FasL expression — the result is that over-stimulated cells are killed. This is essential for avoiding over-proliferation of thymocytes, and also for killing any self-reactive cells which avoided thymic selection.

One of the central mechanisms of the immune system is thymocyte activation, clonal expansion and differentiation (into either effector or memory cells). T cells are activated by binding of the TCR-CD3 complex to a processed antigen peptide bound to a Class I (CD8 cells, aka cytotoxic T cells) or a Class II (CD4, aka helper T cells) MHC molecule. A cascade of biochemical events is initiated, inducing the resting thymocyte to proliferate and differentiate. Induction occurs in two steps: initiation and signal generation, described below. This leads to expression of various gene products, listed below by how early they are expressed after initiation.

Naive thymocytes are those which have not yet encountered a peptide-MHC complex. Arrested in G₀, naive thymocytes have condensed chromatin, minimal cytoplasm and little transcriptional activity. They continuously recirculate through blood, lymph an lymph nodes. To activate, they need additional costimulatory signals to those described above. Signal 1 is the initial interaction between TCR-CD3 and peptide-MHC. Signal 2 is is provided by thymocyte CD28 and CD152 interaction with B7 proteins on the antigen-presenting cell. B7 proteins are constitutively expressed on dendritic cells, and in activated macrophages and activated B cells. B7 binds two proteins — CD28 and CD152 — which are both found on thymocyte membranes as disulfide-linked dimers. Binding of CD28 induces the cell to activate, while binding of CD152 represses activation. CD152 has a much higher B7 affinity than CD28, and its expression is activated by binding of CD28. Thus, CD152 is essentially a braking mechanism that maintains homeostasis; CD152 knockout mice have enlarged lymph nodes, enlarged spleen and die 3-4 weeks after birth. In the absence of a costimulatory signal, an unresponsive state called clonal anergy ensues (as opposed to clonal proliferation).

In essence, activation occurs when a dendritic cell simultaneously binds itself to a T_H’s antigen receptor (primary signal) and to its CD28 receptor (secondary signal). This signals to the dendritic cell that the antigen is foreign (dangerous) and that the next encountered cytotoxic thymocyte must be activated. Other times, dendritic cells are directly activated by an antigen via toll-like receptors and activate cytotoxic thymocytes — this is a critical example of how innate immunity activates adaptive immunity.

Before birth, the yolk sac, fetal liver and fetal bone marrow are the major sites of B cell maturation; after birth, B cells mature in the bone marrow. Many B cells are produced, but most die after a few weeks unless they encounter their specific antigen or nestle into a supportive lymphoid organ. An immature B cell bearing IgM in its membane leaves the bone marrow and matures to express both membrane-bound IgM and IgD (mIgM and mIgD) with a single antigenic specificity.

Naive B cells (have not encountered antigen) circulate in blood ad lymph and are carried to secondary lymphoid organs (notable the spleen and lymph nodes). If a B cell’s mIgM or mIgD interacts with its antigen, then the cell activates undergoes clonal expansion. This creates a population of genetically identical B cells (which express an identical antibody) that differentiates into memory B cells and plasma B cells. Also, some B cells undergo affinity maturation, whereby the average affinity of the antibodies they produce increases. Also, many B cells undergo class switching, whereby the B cells switch from producing µ isotype antibodies (IgMs) to produce γ, α or ε isotype antibodies.

When mature naïve B cell exit the bone marrow and begin recirculation, they are arrested in G₀ and typically die within a few weeks unless they are activated by their complementary antigen. An activated B cells undergoes proliferation and differentiation into memory and plasma cells, going from G₀ to G₁, S phase and then mitosis (cell division). There are two kinds of antigens, with each activating B cells along a unique pathway: thymus-dependent (TD) antigens and thymus-independent (TI) antigens. TD antigens requires direct contact with T_H cells (aka CD4 cells) and not just cytokines secreted by T_H cells. The humoral response to TI antigens is typically weaker than the TD response, does not form memory cells and predominantly leads to IgM secretion (indicating an absence of class switching). This is due to the critical role of T_H in affinity maturation, generating memory B cells and class switching to other isotypes.

The B cell response to thymus-independent antigens is split into two different pathways: the type-1 thymus-independent pathway (TI-1) is caused by lipopolysaccharide and other bacterial cell wall components; the type-2 thymus-independent pathway (TI-2) is caused by repetitive molecules such as bacterial flagellin and bacterial cell wall polysaccharides. Most TI-1 antigens are able to activate B cells regardless of their antigen specificity — they are polyclonal B cel activators or mitogens. TI-2 antigens activate B cells by binding mIg — however, cytokines secreted by T_H cells are required for full B cell proliferation and for class switching from IgM to other isotypes. The table below describes the TD, TI-1 and TI-2 antigens as well as their effects on the humoral response.

Activation By Thymus-Dependent Antigens

Two distinct signalling events are needed to push the resting naïve B cell into the cell cycle: signal 1 followed by signal 2. T_H cells are essential for activation of a B cell by thymus-dependent antigens. Binding of thymus-dependent antigens to a B cell’s mIg does not alone induce proliferation and differentiation without additional interaction with T_H membrane molecules as well as appropriate cytokines. The steps are described below:

Cross-linking of a membrane-bound immunoglobulin (mIg) with its complementary antigen initiates a signal transduction cascade that activates the attached B cell. Membrane-bound immunoglobulins have short cytoplasmic tails, rendering them unable to transduce activating signals on their own. However, each membrane-bound ligand-binding immunoglobulin associates with a single disulfide-linked signal-transducing heterodimer Ig-α/Ig-β to form the B cell receptor. Similarly, the pre-BCR consists of the Ig-α/Ig-β heterodimer associating with the surrogate light chain and µ heavy chains. Ig-α and Ig-β each contain a cytoplasmic tail with an 18-residue motif known as the immunoreceptor tyrosine-based activation motif (ITAM) which is also present in the T cell receptor (TCR). Also, just like the TCR, the BCR draws protein tyrosine kinases (PTKs) to its cytoplasmic tail upon cross-linking of the mIg by its complementary antigen.

Antigen-antibody crosslinking leads to phosphorylation of the tyrosines within the Ig-α and Ig-β ITAMs. This phosphorylation is performed by the receptor-associated PTKs Lyn, Blk and Fyn (similar to p56^Lck activity on TCRs). This ITAM phosphorylation creates docking sites for the critical proteins Syk (also a PTK, analogous to the TCR’s ZAP-70) and B cell linker protein (BLNK). These critical proteins provide docking sites for further proteins. Once BLNK has been phosphorylated by Syk, it recruits Bruton’s tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2) so Syk can activate Btk, and so that Btk can then phosphorylate PLCγ2. Once PLCγ2 has been phosphorylated, it activates early calcium signaling and the initiation of pathways dependent on protein kinase C (PKC). The pathways activated by the BCR include small G protein pathways (for growth), PKC-dependent pathways and NF-κB production pathways — note the similarities to T cell activation.

In addition to the BCR, there are two membrane-bound components which provide stimulation (the B cell coreceptor) or inhibition (CD22). The B cell coreceptor is a complex of three proteins: CD19, which provides a long cytoplasmic tail with docking sites; CD21 (aka CDR2), which is a receptor for C3d; and CD81. C3d is a byproduct of complement that coats antigens — while the immunoglobulin binds the antigen, CD21 cross-links with C3d. This forms a BCR-antigen-BCcoR complex, allowing CD19’s cytoplasmic tail to interact with Ig-α and Ig-β and undergo phosphorylation. CD19’s phosphorylated cytoplasmic tail then binds signaling molecules, including the protein tyrosine kinase (PTK) Lyn, and hugely amplifies the activating signal. This explains how naïve B cells with low antigen affinity are still able to respond to low concentrations of antigen.

CD22 delivers a negative signal that makes activation of B cells more difficult. Activation of B cells leads to phosphorylation of the immunoreceptor tyrosine inhibitory motif (ITIM) in the cytoplasmic tail of CD22. Tyrosine phosphatase then binds the CD22 ITIM, stripping phosphates from the ITAMs of neighboring signaling complexes. Since ITAM phosphorylation is the core of B cell activation, phosphate removal deactivates the cell. CD22 knockout mice develop autoimmune diseases as they age, illuminating the importance of negative regulation.

Antibody production by activated B cells is the core the humoral response: antibody effects, such as complement activation by IgM and certain IgGs, opsonization via F(c)Rs and pathogen/toxin neutralization by high-affinity IgG and IgA; and processes related to B cell activation, such as T_H2 activation and cytokine production, germinal center formation, isotype switching, affinity maturation and memory cell production. The F(c) region of IgG binds to F(c) receptors, playing a critical role (along with receptors for complement byproducts) in clearing extracellular bacteria. Intracellular bacteria are cleared by cell-mediated immunity.

Antigens are grouped into thymus-dependent antigens and thymus-independent antigens. Activation by thymus-dependent antigens requires two signals: first, binding of the antigen itself to the B cell; second, binding of of a thymocyte to the B cell. Thymus-independent antigens, conversely, activate B cells on their own; in some cases, however, T_H cytokine secretion (but not binding) is needed for maximum B cell activity.

The thymus-dependent response requires linked recognition (aka associative recognition) whereby T_H and B cells must both recognize a given molecule as an antigen for it to activate the B cell. This was demonstrated in the cell-transfer experiment. Antigens in the blood are concentrated in the spleen, while antigens accessible by lymph are concentrated in the nearest lymph nodes and nodules. Lymph nodes trap more than 90% of all antigens which flow through them, whether those antigens are bound to free-floating antibodies or antibodies bound to antigen-transporting cells (such as Langerhans or dendritic cells) or macrophages.

T and B cell epitopes are not necessarily identical; for example, T cells respond well to internal viral proteins while B cells produce neutralizing antibodies to viral coat proteins. (Once virus-infected cells have been killed and unassembled virus proteins released, B cells specific for internal proteins can also be activated to make opsonizing antibodies to those proteins.) Attaching a carbohydrate to a protein can convert the carbohydrate into a T-dependent antigen; the carbohydrate-specific B cell internalizes the complex and presents peptides to Th2 cells, which in turn activate the B cell to make antibodies specific for the carbohydrate.

Antibodies (aka immunoglobulin or Ab) are produced by B cells and specifically bind to antigens (aka Ag) in solution (as opposed to TCRs, which bind antigens on cell surfaces). An antigen is any substance that binds specifically to a T cell receptor or B cell receptor. This antibody-antigen binding can: cause the antigen to be engulfed by macrophages (opsonization); can neutralize a virus and prevent it from entering cells; and can induce the complement cascade (causes bacterial lysis). All antibodies exist in both secreted and membrane-bound (mIg) forms, differing in their carboxy terminal sequence. Secreted antibodies have a hydrophilic terminus, while membrane immunoglobulins have a hydrophobic sequence which inserts into the plasma membrane and a short cytoplasmic sequence. Antibodies have various functions, described below:

Antibodies consist of heavy chains and light chains. The different istotypes of antibody have structurally different heavy chains, leading to functional differences as well. IgG is the most common immunoglobulin isotype, and its relatively simple structure (consisting of two γ heavy chains, and two light chains) is shown to the right. There are five different types of heavy chain to differentiate the five antibody isotypes (IgA, IgD, IgE, IgG and IgM).

Antibody chains contain constant domains (same amongst antibodies) and variable domains (different amongst antibodies). Heavy chains have four or five constant domains at one end, a variable region at the other and also at least one carbohydrate moiety attached. There are many types of heavy chain variable regions. Light chains have a constant region at one end and a variable region at the other end. Light chain variable regions are either kappa (κ) isotype or lambda (λ) isotype. The two light chain isotypes have no known functional differences. Free light chains are known as Bence-Jones proteins.

Variable regions contain relatively conserved regions and hypervariable regions (aka complementarity determining regions). Complementarity determining regions (CDRs) of heavy and light chain variable regions group together to form a complex that directly interacts with antigens. X-ray crystallography has shown that CDRs are loops which stick out for accessibility, and that relatively conserved regions are merely a scaffold to make sure these CDR loops stay in place.

Antigens are foreign bio-organic molecules that interact with B cells (via antibodies) and T cells (via T cell receptors). Blood-borne antigens are concentrated in the spleen; lymph-borne antigens are concentrated in nearby lymph nodes and nodules. Upon detection by the acquired immune response, antigens stimulate production of antigen-specific antibodies. Toxins, invading bacteria and viruses, and the cells of transplanted organs can all function as antigens. Bio-organic chemicals are those based on carbon and the atoms which bond to carbon (hydrogen, oxygen, nitrogen, phosphorous and sulfur — aka CHONPS). An antigen’s configuration of CHONPS is called its antigenic determinant. By ignoring any non-CHONPS chemical, the immune response does not recognize sand, mercury, minerals and other potentially hazardous contaminants. By just recognizing bio-organic antigens, the immune response has evolved to detect only antigens encoded or controlled by genes.

Antigens are any substance that binds specifically to B cell receptors or T cell receptors. There are two types of antigens: immunogens and haptens. An immunogen is a any substance that can elicit an innate or acquired response, and haptens are research-useful small molecules which must be attached to a carrier molecule to elicit a response.

The immune response has two cells that recognize antigens: B cells and T cells. B cells present immunoglobulin (antibody molecule) and T cells present T cell receptor (TCR). The function of these cells is to bind antigens and to remove them from the system. Antibodies and TCRs are specific to the antigenic determinant of a particular antigen. B cells respond to different antigens in different ways (thymus-dependent and thymus-independent) and this is described in the first two paragraphs of B cell activation.

Antigens are difficult for the immune response to detect due to the size of the antigenic universe and because the antigenic universe is constantly changing. The size of the antigenic universe is due to the billions of foreign microbes around. With each microbe containing a few foreign molecules, the number of antigens is inconceivably large. Also, genetic change causes this antigenic universe to constantly change. Bacteria replicate at a fast rate, meaning antigenic drift (change) is too fast for standard mechanisms that deal with diversity.

The various F(c) Receptors are organized by the type of antibody they bind — F(c)γ Receptors bind IgG (aka γ isotype antibodies) and F(c)ε Receptors bind IgE (aka ε isotype antibodies.

F(c)γ Receptors

One way antibodies stimulate inflammation and clearing of pathogens is by binding of their F(c) regions to F(c)γ receptors (FcγRs) on effector cells. There are three families of FcγRs: FcγRI, aka CD64; FcγRII, aka CD32; and FcγRIII, aka CD16. FcγRs also stimulate binding and uptake of antigens in human complexes, thus playing an important role in antigen presentation by macrophages and dendritic cells. There is a balance between activator FcγRs and inhibitor FcγRs, with many cells displaying both.

F(c)ε Receptors

High-affinity F(c)&epsilonRI is found on most mast cells and basophils. It is only activated by cross-linked antibodies, meaning only those antibodies which have already bound their complementary antigen. Once this cross-linking occurs, a Ca²⁺ flux occurs which triggers granules to swell, move to the membrane and burst out (degranulation). This leads to release of leukotrines and prostaglandins.

One problem with the study of antibodies was that they are heterogenous. For example, an electrophoresis pattern of an animal immunized against albumin (a homogenous protein) would show a spike of albumin and then several much smaller spikes of antibodies (meaning the albumin antibodies are polyclonal, or consisting of different subsets binding different sites on the same antigen). In multiple myeloma, tumorous plasma cells all secrete the tame type of immunoglobulin. This leads to a huge monoclonal spike of antibodies, since there will be huge amounts of a single antibody. The experiments below were all performed to determine the structure of antibodies. After all these experiments, the antibody structure shown above was determined.

There are several different isotypes of heavy chain constant regions, broken into classes and subclasses. Classes are differentiated by large structural differences correlated to large functional differences. Subclasses have small but significant differences, also corresponding to separate functions. Most functions of antibodies are mediated (determined) by the heavy chain constant region. However, all antibody functions are triggered only by binding of an antigen to the variable region. The two light chain isotypes (κ and λ) associate with all the different heavy chain isotypes. Each isotype is encoded by a separate gene, and all genes are present in normal individuals.

Antibodies bind to antigens in a reversable non-covalent manner via hydrogen bonds, ionic bonds, hydrophobic interactions and van der Waal’s interactions. Antibodies only react with antigens in solution — as opposed to TCRs, which react with antigens bound to cell surfaces. These forces operate at short distances, so the antibody CDR (accounting for most of the antigen-antibody interaction) and the antigen epitope (where the CDR binds) must fit together very well. The more precise the fit, the better the interaction.

Antibodies make contact with protein antigens, usually 15-22 amino acids on the antigen contact a similar number on the antibody giving a complementary surface of 650-900 Angstroms. The amino acids comprising the epitope are adjacent in 3D space, but not necessarily in linear sequence.

When exposed to an antigen, B cells producing antibodies reactive to that antigen will begin to produce vast amounts of antibody. Each B cell produces a monoclonal population of antibodies, meaning each antibody binds the same epitope (site) on the same antigen; however, the B cells together produce a polyclonal population of antibodies, consisting of different antibodies binding a different epitope (site) on the same antigen.

Interferon (IFN) is secreted by avirally infected cell, and induces an anti-viral state in surrounded cells. It is too late for the infected cell, which dies, but the surrounding cells might be saved. It is not a single protein, but rather a group of stable (acid pH) proteins of 17,000 d secreted by different cells types. Very potent, only a few molecules bound to surface of cell is sufficient. This initially made it very difficult to isoalte and purify. There are three categories:

Signalling pathway for IFNγ:

1. Infected cell secretes IFNγ
2. Binds to neighboring cell expressing IFN receptor (most cells express them with very high affinity)
3. Binding results in dimerizaion and activation of Jak kinase, which binds to cytoplasmic side of receptor
4. Activated Jak phosphorylates STAT1, which dimerizes and translocated to nucleus
5. Transcription factor which activates genes via gamma activated sequence.
6. Genes expressed by STAT1 act to block replication of incoming virus

There are 2 mechanisms by which STAT1 genes block replication of incoming virus:

Mechanism 1: dsRNA-dependent eIF2 kinase (PKR)

• First gene is PKR
• Blocks viral replicaiton by inactivating cellular translation factor called PKR, thereby inhibited viral replicatoin at protein synthesis level.
• eIF2 is important for translation
• PKR phosorylates eIF2, which brings first tRNA-methiionine to reibosome to initiate protien translation
• PKR phosphorylates eIF2, blocks recycling of eIF2-GDP to eIFS-GTP by GNEF, eIF2-GDP cannot bind tRNA-methionine.
• In IFN-treated uninected cells IFN → PKR (inactive)
• What activates it?
• In IFN-treated cells infected with RNA virus….dsRMA forms during course of replication especially in an RNA virus like influenza and these are necessary intermediates and these nucleic acids activate PKR and then it phosphorylates eiF2 and blocks protein synthesis.
• Most dsRNA appear in cell in high concentrations as a result of viral replication…cell doesn’t use dsRNA. ONly viral infections will activate PKR.
• But blocking proteint translation also blocks host cell protein synthesis. This protective mechanism preotects cell but also harms cell. However cell uses another mechnism to specifically block viral genome replication

Mechanism 2: 2′-5′ oligo A induced pathway

In this pathway IFN stimulates expression of 3 host cell enzymes:

• 2′-5′2 oligo A synthase (activated by dsRNA)
• Ribonuclease L (activated by 2′-5′ oligo A)
• 2′-5′ olido A phosphodiesterase (degrades 2′-5′ oligo A)

• This mechanism expalins specific inhibition of viral protein synthesis as opposed to host cell protein synthesis.
• oligo A synthase syntehsizes a few A’s…only adenine residues with 2′5′ linkage…if it were DNA, phosphate would be joined onto 3′ carbon instead of 2′ carbon…like PKR oligo A synthase activated by dsRNA expressed when cell receives signal from interferon but only actie when virus enters cell and starts to replicate
• 2^nd enzyme also expressed, sitting around in inactive form. RNAseL when bound to oligo A becomes active. Degrades ssRNA, including viral and cellular mRNA, rRNA, and tRNA. Of course this also effects cellular and viral RNA’s.
• The result of these 3 enzymes is that the RNAse is only activated in the microvicinity of dsRNA.

Concentrations of antibodies do not peak until long after the virus has cleared. Therefore, antibodies are probably not the reason for the fall in virus titre. The host factor which does seem responsible is called interferon. Conentrations of interferons much levels of virus very closely. Interferon is a protein which protects cells from infection. It is not specific for one particular virus. It protects the cell from a number of different viruses.

Discovering Interferon

Isaacs and Lindenman found in 1957 that: sometimes patients are infected by more than one type of bacteria; very rare for somebody to be infected by more than one type of virus; infection with one virus somehow protects body from infection by 2^nd virus. Use embryonated chicken eggs as host. Infect embryos with infleunza virus (common way of growing virus before development of tissue culture techniques). When infected with flue, and something in fluid other than virus itself protects Embryonic cells were secreted something into the chorioallantoic fluid which protected the 2^nd egg from viral infection. Interrfered with viral replication, interferon