Some of these responses are specific, others are non-specific. This page will introduce host defense mechanisms by defining some commonly used terms and describing the specific cells and tissues involved in these immune responses.

Antigen (Ag): A molecule which elicits a specific immune response when introduced into an animal. More specifically, antigenic (immunogenic) substances are:

Antibody (Ab): A glycoprotein produced in response to an antigen that is specific for the antigen and binds to it via non-covalent interactions. The term "immunoglobulin" is aften used interchangeably with "antibody". We will use the term "immunoglobulin" to describe any antibody, regardless of specificity, and the term "antibody" to describe an antigen-specific "immunoglobulin". Immunogloblins (Igs) come in different forms (IgA, IgD, IgE, IgG, IgM) that reflect their structure. More information can be found here.

Clonal selection hypothesis (Jerne and Burnet): The clonal selection hypothesis attempts to explain the findings described above by suggesting the following:

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

Immunoglobulins

Immunoglobulins generally assume one of two roles: immunoglobulins may act as i) plasma membrane bound antigen receptors on the surface of a B-cell or ii) as antibodies free in cellular fluids functioning to intercept and eliminate antigenic determinants. In either role, antibody function is intimately related to its structure and this page will introduce immunoglobulins (antibodies) and relate their structure to their function in host defense.

Immunoglobulins are composed of four polypeptide chains: two "light" chains (lambda or kappa), and two "heavy" chains (alpha, delta, gamma, epsilon or mu). The type of heavy chain determines the immunoglobulin isotype (IgA, IgD, IgG, IgE, IgM, respectively). Light chains are composed of 220 amino acid residues while heavy chains are composed of 440-550 amino acids. Each chain has "constant" and "variable" regions as shown in the figure. Variable regions are contained within the amino (NH₂) terminal end of the polypeptide chain (amino acids 1-110). When comparing one antibody to another, these amino acid sequences are quite distinct. Constant regions, comprising amino acids 111-220 (or 440-550), are rather uniform, in comparison, from one antibody to another, within the same isotype. "Hypervariable" regions, or "Complementarity Determining Regions" (CDRs) are found within the variable regions of both the heavy and light chains. These regions serve to recognize and bind specifically to antigen. The four polypeptide chains are held together by covalent disulfide (-S-S-) bonds.

Structural differences between immunoglobulins are used for their classification. As stated above, the type of heavy chain an immunoglobulin possesses determines the immunoglobulin "isotype". More specifically, an isotype is determined by the primary sequence of amino acids in the constant region of the heavy chain, which in turn determines the three-dimensional structure of the molecule. Since immunoglobulins are proteins, they can act as an antigen, eliciting an immune response that generates anti-immunoglobulin antibodies. However, the structural (three-dimensional) features that define isotypes are not immunogenic in an animal of the same species, since they are not seen as "foreign". For example, the five human isotypes, IgA, IgD, IgG, IgE and IgM are found in all humans and a result, injection of human IgG into another human would not generate antibodies directed against the structural features (determinants) that define the IgG isotype. However, injection of human IgG into a rabbit would generate antibodies directed against those same structural features.

Another means of classifying immunoglobulins is defined by the term "allotype". Like isotypes, allotypes are determined by the amino acid sequence and corresponding three-dimensional structure of the constant region of the immunoglobulin molecule. Unlike isotypes, allotypes reflect genetic differences between members of the same species. This means that not all members of the species will possess any particular allotype. Therefore, injection of any specific human allotype into another human could possibly generate antibodies directed against the structural features that define that particular allotypic variation.

A third means of classifying immunoglobulins is defined by the term "idiotype". Unlike isotypes and allotypes, idiotypes are determined by the amino acid sequence and corresponding three-dimensional structure of the variable region of the immunoglobulin molecule. In this regard, idiotypes reflect the antigen binding specificty of any particular antibody molecule. Idiotypes are so unique that an individual person is probably capable of generating antibodies directed against their own idiotypic determinants. This probability forms the basis of the Idiotypic Network Hypothesis to be described later.

Antibodies function in a variety of ways designed to eliminate the antigen that elicited their production. Some of these functions are independent of the particular class (isotype) of immunoglobulin. These functions reflect the antigen binding capacity of the molecule as defined by the variable and hypervariable (idiotypic) regions. For example, an antibody might bind to a toxin and prevent that toxin from entering host cells where its biological effects would be acivated. Similarly, a different antibody might bind to the surface of a virus and prevent that virus from entering its host cell. In contrast, other antibody functions are dependent upon the immunoglobulin class (isotype). These functions are contained within the constant regions of the molecule. For example, only IgG and IgM antibodies have the ability to interact with and initiate the complement cascade. Likewise, only IgG molecules can bind to the surface of macrophages via Fc receptors to promote and enhance phagocytosis. The following table summarizes some immunoglobulin properties.

The immune system has the capacity to recognise and respond to about 10⁷ different antigens. This extreme diversity can be generated in at least three possible ways:

It is known that all three of these possiblities take place to produce antibody diversity. The following figures illustrate these possibilities:

The production of immunoglobulins by B-cells or plasma cells occurs in different stages. During differentiation of the B-cells from precursor stem cells, rearrangement, recombination and mutation of the immunoglobulin V, D, and J regions occurs to produce functional VJ (light chain) and VDJ (heavy chain) genes. At this point, the antigen specificity of the mature B-cell has been determined. Each cell can make only one heavy chain and one light chain, although the isotype of the heavy chain may change. Initially, a mature B-cell will produce primarily IgD (and some membrane IgM) that will migrate to the cell surface to act as the antigen receptor. Upon stimulation by antigen, the B-cell will differentiate into a plasma cell expressing large amounts of secreted IgM. Some cells will undergo a "class switch" during which a rearrangement of the DNA will occur, placing the VDJ gene next to the genes encoding the IgG, IgE or IgA constant regions. Upon secondary induction (i.e. the secondary response), these B-cells will differentiate into plasma cells expressing the new isotype. Most commonly, this results in a switch from IgM (primary response) to IgG (secondary response). The factors that lead to production of IgE or IgA instead of IgG are not well understood.

Class I molecules are composed of two polypeptide chains; one encoded by the BCA region and another (ß2-microglobulin) that is encoded elsewhere. The MHC-encoded polypeptide is about 350 amino acids long and glycosylated, giving a total molecular weight of about 45 kDa. This polypeptide folds into three separate domains called alpha-1, alpha-2 and alpha-3. ß2-microglobulin is a 12 kDa polypeptide that is non-covalently associated with the alpha-3 domain. Between the alpha-1 and alpha-2 domains lies a region bounded by a beta-pleated sheet on the bottom and two alpha helices on the sides. This region is capable of binding (via non-covalent interactions) a small peptide of about 10 amino acids. This small peptide is "presented" to a T-cell and defines the antigen "epitope" that the T-cell recognizes (see below). The following images illustrate the structure of the class I MHC as seen schematically, and three dimensionally from the side and from the top (T-cell perspective). The MHC-encoded polypeptide is shown in blue, the ß2-microglobulin is green and the peptide antigen is red.

Class II molecules are composed of two polypeptide chains, both encoded by the D region. These polypeptides (alpha and beta) are about 230 and 240 amino acids long, respectively, and are glycosylated, giving molecular weights of about 33 kDa and 28 kDa. These polypeptides fold into two separate domains; alpha-1 and alpha-2 for the alpha polypeptide, and beta-1 and beta-2 for the beta polypeptide. Between the alpha-1 and beta-1 domains lies a region very similar to that seen on the class I molecule. This region, bounded by a beta-pleated sheet on the bottom and two alpha helices on the sides, is capable of binding (via non-covalent interactions) a small peptide of about 10 amino acids. This small peptide is "presented" to a T-cell and defines the antigen "epitope" that the T-cell recognizes (see below). The following images illustrate the structure of the class II MHC as seen schematically, and three dimensionally from the side and from the top (T-cell perspective). The MHC-encoded polypeptides are shown in yellow and green, while the peptide antigen is shown in red.

Class II MHC	Side view	Top view

CLASS I vs CLASS II MOLECULES

While class I and class II molecules appear somewhat structurally similar and both present antigen to T-cells, their functions are really quite distinct. First, class I molecules are found on virtually every cell in the human body. Class II molecules, in contrast, are only found on B-cells, macrophages and other "antigen-presenting cells" (APCs). Second, class I molecules present antigen to cytotoxic T-cells (CTLs) while class II molecules present antigen to helper T-cells (TH-cells). This specificity reflects the third difference, the type of antigen presented. Class I molecules present "endogenous" antigen while class II molecules present "exogenous" antigens. An endogenous antigen might be fragments of viral proteins or tumor proteins. Presentation of such antigens would indicate internal cellular alterations that if not contained could spread throughout the body. Hence, destruction of these cells by CTLs is advantageous to the body as a whole. Exogenous antigens, in contrast, might be fragments of bacterial cells or viruses that are engulfed and processed by e.g. a macrophage and then presented to helper T-cells. The TH-cells, in turn, could activate B-cells to produce antibody that would lead to the destruction of the pathogen.

T-CELL RECEPTOR (TCR) MOLECULES

The T-cell receptor molecule (TCR) is structurally and functionally similar to the B-cell immunoglobulin receptor. TCR is composed of two, disulfide-linked polypeptide chains, alpha and beta, each having separate constant and variable domains much like immunoglobulins. The variable domain contains three hypervariable regions that are responsible for antigen recognition. Genetic diversity is ensured in a manner analogous to that for immunoglobulins (click here for more information). Thus, just like the B-cell surface immunoglobulin provides antigen specificity to its B-cell, the TCR allows T-cells to recognize their particular antigenic moiety. However, T-cells cannot recognize antigen without help; the antigenic determinant must be presented by an appropriate (i.e. self) MHC molecule. Upon recognition of a specific antigen, the signal is passed to the CD3 molecule and then into the T-cell, prompting T-cell activation and the release of lymphokines. The following images illustrate the structure of the TCR as seen schematically, and three dimensionally from the side.

TCR	Side view

ANTIGEN RECOGNITION BY T-CELLS

The TCR provides the specificity for an individual T-cell to recognize its particular antigen. However, this recognition is "MHC-restricted" because the TCR also requires interactions with MHC. Also, interactions between the CD4 molecule (found on helper T-cells) and class II MHC or the CD8 molecule (found on cytotoxic T-cells) and class I MHC stabilize and consumate the antigen recognition process, allowing helper T-cells to respond to "exogenous" antigens (leading to B-cell activation and the production of antibody) or cytotoxic T-cells to respond to "endogenous" antigens (leading to target cell destruction). The following images illustrate these processes schematically, and three dimensionally.

TCR - APC (class II)		TCR - Target cell (class I)

Antigen Presentation by MHC-II to TCR

Humoral Immunity

The production of antibody involves three distinct phases:

Induction phase: Ag reacts with specific T and B cells
Expansion and Differentiation phase: Induced lymphocyte clones proliferate and mature to a functional stage (i.e. Ag receptor cells mature to Ag effector cells)
Effector phase: Abs or T cells exert biological effects either:
1) Independently or
2) Through the action of macrophages, complement, other non-specific agents

This page will discuss induction, differentiation and regulation of the humoral immune response, focusing on the production of Abs.

ANTIGEN PRESENTING CELLS (APCs)

Induction of the humoral immune response begins with the recognition of antigen. Through a process of clonal selection, specific B-cells are stimulated to proliferate and differentiate. However, this process requires the intervention of specific T-cells that are themselves stimulated to produce lymphokines that are responsible for activation of the antigen-induced B-cells. In other words, B cells recognize antigen via immunoglobulin receptors on their surface but are unable to proliferate and differentiate unless prompted by the action of T-cell lymphokines. In order for the T-cells to become stimulated to release lymphokines, they must also recognize specific antigen. However, while T-cells recognize antigen via their T-cell receptors, they can only do so in the context of the MHC molecules. This "antigen-presentation" is the responsibility of the antigen-presenting cells (APCs).

Several types of cells may serve the APC function. Perhaps the best APC is, in fact, the B-cell itself. When B-cells bind antigen, the antigen becomes internalized, processed and expressed on the surface of the B-cell. Expression occurs within the class II MHC molecule, which can then be recognized by T-helper cells (CD4⁺).

Other types of antigen-presenting cells include the macrophage and dendritic cells. These cells either actively phagocytose or pinocytose foreign antigens. The antigens are then processed in a manner similar to that observed for the B-cells. Next, specific antigen epitopes are expressed on the macrophage or dendritic cell surface. Again, this expression occurs within the class II MHC molecule, where T-cell recognition occurs. The stimulated T-cells then release lymphokines that act upon "primed" B-cells (B-cells that have already encountered antigen), inducing B-cell proliferation and differentiation.

DIFFERENTIATION OF B-LYMPHOCYTES

B-cells begin their lives in the bone marrow as multipotential stem cells. These completely undifferentiated cells serve as the source for all of the cellular components of the blood and lymphoid system. The initial differentiation step that ultimately leads to the mature B-cell involves DNA rearrangements joining the D and J segments of the immunoglobulin heavy chain genes (click here for more information). Next, DNA rearrangements joining the variable (V) region to the DJ segments of the immunoglobulin heavy chain, as well as similar rearrrangements within the light chain genes gives rise to the pre-B-cell. Establishment of the B-cell specificity and consequent expression of surface immunoglobulin gives rise to the "virgin", fully functional B-cell. Each of these steps is entirely independent of antigen.

The antigen-dependent stages of B-lymphocyte differentiation occur in the spleen, lymph nodes and other peripheral tissue. These stages are, of course, initiated upon encounter with antigen and activation by T-cell lymphokines. The activated B-cell first develops into a B-lymphoblast, becoming much larger and shedding all surface immunoglobulin. The B-lymphoblast then develops into a plasma cell, which is, in essense, an antibody factory. This terminal differentiation stage is responsible for production of primarily IgM antibody during the "primary response". Some B-cells, however, do not differentiate into plasma cells. Instead, these cells undergo secondary DNA rearrangements that place the constant region of the IgG, IgA or IgE genes in conjunction with the VDJ genes. This "class switch" establishes the phenotype of these newly differentiated B-cells; these cells remain as long-lived "memory cells". Upon susequent encounter with antigen, these cells respond very quickly to produce large amounts of IgG, IgA or IgE antibody, generating the secondary response.

REGULATION OF THE HUMORAL RESPONSE

Regulation of the immune response is possibly mediated in several ways. First, a specific group of T-cells, suppressor T-cells, are thought to be involved in turning down the immune response. Like helper T-cells, suppressor T-cells are stimulated by antigen but instead of releasing lymphokines that activate B-cells (and other cells), suppressor T-cells release factors that suppress the B-cell response. While immunosuppression is not completely understood, it appears to be more complicated than the activation pathway, possibly involving additional cells in the overall pathway.

Other means of regulation involve interactions between antibody and B-cells. One mechanism, "antigen blocking", occurs when high doses of antibody interact with all of the antigen's epitopes, thereby inhibiting interactions with B-cell receptors.
A second mechanism, "receptor cross linking", results when antibody, bound to a B-cell via its Fc receptor, and the B-cell receptor both combine with antigen. This "cross-linking" inhibits the B-cell from producing further antibody.

Another means of regulation that has been proposed is the idiotypic network hypothesis. This theory suggests that the idiotypic determinants of antibody molecules are so unique that they appear foreign to the immune system and are, therefore, antigenic. Thus, production of antibody in response to antigen leads to the production of anti-antibody in response, and anti-anti-antibody and so on. Eventually, however, the level of [anti]_n-antibody is not sufficient to induce another round and the cascade ends.

Antigen-Antibody Interactions

AFFINITY

Interactions between antigen and antibody involve non-covalent binding of an antigenic determinant (epitope) to the variable region (complementarity determining region, CDR) of both the heavy and light immunoglobulin chains. These interactions are analogous to those observed in enzyme-substrate interactions and they can be defined similarly. To describe the strength of the antigen-antibody interaction, one can define the affinity constant (K) as shown:

Affinity K =	[Ab - Ag] ——————— [Ab] × [Ag]	= 10⁴ to 10¹² L/mol

If the interaction between antigen and antibody were totally random, one would expect the concentrations of free antigen, free antibody and bound Ag-Ab complex to all be equivalent. In other words,

Affinity K =	1 ——————— 1 × 1	= 10⁰ L/mol

Therefore, the greater the K, the stronger the affinity between antigen and antibody. These interactions are the result of complementarity in shapes, hydrophobic interactions, hydrogen bonds and Van der Waals forces.

ANTIGEN-ANTIBODY RATIOS

Experimentally, if one adds a known concentration of antibody to a tube and then adds increasing amounts of the specific antigen, the Ag-Ab complexes will begin to precipitate. If one continues to add increasing amounts of antigen, the complexes will begin to dissolve and return to solution. The following graph illustrates this process.


	Tube #	1	2	3	4	5	6	7
	Amount of precipitate
	Amount of Ag (arbitrary units)	1	2	3	4	5	6	7
If one then measures the amount of antigen and antibody remaining in the supernatant, one sees the following:
	Excess Ab	+	+	+	–	–	–	–
	Excess Ag	–	–	–	–	+	+	+

The left portion of the graph (tubes 1-3) illustrates "Antibody Excess", since not all of the antibody that is available to bind to antigen has actually bound antigen. The right portion of the graph (tubes 5-7) illustrates "Antigen Excess", where there is not enough antibody to bind to all of the available antigen. In the middle (tube 4) is a region known as "Equivalence". Here, the ration of antigen to antibody is perfect, so that all the antigen molecules and all of the antibody molecules are part of a complex. These are interesting experimental observations that do have relevance to situations occurring in the human body. For example:

Antibody excess might occur when a person is exposed to a virus from which they have recently recovered. Hence, their body would contain a relatively large concentration of antiviral antibodies. These antibodies could quickly act to block cell receptors on the viral surface and prevent adsorption to host cells, thereby preventing disease.
Antigen excess might occur early in the first infection by a microorganism. A person would have relatively few antibodies and these would form complexes but they would be very small. Such small complexes probably would not be phagocytosed or removed by the kidneys and could become lodged near tissue surfaces. Later, when antibody becomes available, the size of the complexes can increase leading to effective elimination by phagocytes or tissue damage where the smaller complexes had become lodged. Click here for more information.
Equivalence would occur when a person is exposed to an agent to which they have circulating antibodies. The correct ratio of antigen to antibody would produce extensive lattice formation, leading to enhanced phagocytosis, opsonization or agglutination, effectively eliminating the foreign agent.

CROSSREACTIVITY

Crossreactivity can occur when two (or more) antigens share similar structural features. Consider three different antigens, as shown on the right. Antibody produced in response to Ag1 is very specific and would, therefore, have a large affinity constant (K) when combining with Ag1. However, Ag2 is similar in shape to Ag1 and is capable of interacting with anti-Ag1 antibody via two of three sites. The interaction between Ab and Ag2 is not as strong as the interaction between Ab and Ag 1 (i.e. K is much smaller) but is still strong enough to allow binding. Hence, Ag1 and Ag2 are said to cross-react. Ag3, in contrast, cannot interact very well with anti-Ag1 antibody and would have a K value so low that significant binding would not occur. Ag3, therefore, would not cross-react with Ag1. Would antibody produced in response to Ag2 bind Ag3? Would antibody produced in response to Ag2 bind Ag1?

Crossreactivity also forms the basis for several diagnostic tests. For example, infection with Treponema pallidum (syphilis) causes the production of antibodies that cross-react with a subsatnce found in cardiac muscle, cardiolipin. Since it is much easier to obtain pure cardiolipin than pure Treponemal antigens, this cross-reaction is used to test for syphilis (Wassermann test). Likewise, antibodies produced against certain Rickettsia cross-react with antigens from Proteus. Since the latter are much easier to obtain, they can be used to test for the former.

Cell Mediated Immunity

The second arm of the immune response is refered to as Cell Mediated Immunity (CMIR). As the name implies, the functional "effectors" of this response are various immune cells. These functions include:

Phagocytosis and killing of intracellular pathogens
Direct cell killing by cytotoxic T cells
Direct cell killing by NK and K cells

These responses are especially important for destroying intracellular bacteria, eliminating viral infections and destroying tumor cells. This page will discuss the cell-mediated immune response, focusing on the mechanisms involved.

MACROPHAGE ACTIVATION

While the production of antibody through the humoral immune response can effectively lead to the elimination of a variety of pathogens, bacteria that have evolved to invade and multiply within phagocytic cells of the immune response pose a different threat. The following graphic illustrates this dilemma:

Extracellular microorganisms

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

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

Intracellular microorganisms

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

IFN
TNF

Intracellular microorganisms

Cell group	Surface components	Function
B-lymphocytes	Surface immunoglobulin (Ag recognition) Immunoglobulin Fc receptor Class II Major Histocompatability Complex (MHC) molecule (Ag presentation)	Direct antigen recognition Differentiation into antibody-producing plasma cells Antigen presentation within Class II MHC
––––––––––––––	––––––––––––––	––––––––––––––
T-lymphocytes	CD3 molecule T-cell receptor (TCR, Ag recognition)	Involved in both humoral and cell-mediated responses
Helper T-cells (T_H)	CD4 molecule	Recognizes antigen presented within Class II MHC Promotes differentiation of B-cells and cytotoxic T-cells Activates macrophages
Suppressor T-cells (T_S)	CD8 molecule	Downregulates the activities of other cells
Cytotoxic T-cells (CTL)	CD8 molecule	Recognizes antigen presented within Class I MHC Kills cells expressing appropriate antigen
––––––––––––––	––––––––––––––	––––––––––––––
Accessory cells	Variable	Phagocytosis and cell killing
Macrophages	Immunoglobulin Fc receptor Complement component C3b receptor Class II MHC molecule	Bind Fc portion of immunoglobulin (enhances phagocytosis) Bind complement component C3b (enhances phagocytosis) Antigen presentation within Class II MHC Secrete IL-1 (macrokine) promoting T-cell differentiation and proliferation Can be "activated" by T-cell lymphokines
Dendritic cells	Class II MHC molecule	Antigen presentation within Class II MHC
Polymorphonuclear cells (PMNs)	Immunoglobulin Fc receptor Complement component C3b receptor	Bind Fc portion of immunoglobulin (enhances phagocytosis) Bind complement component C3b (enhances phagocytosis)
––––––––––––––	––––––––––––––	––––––––––––––
Killer cells	Variable	Direct cell killing
NK cells	Unknown	Kills variety of target cells (e.g. tumor cells, virus-infected cells, transplanted cells)
K cells	Immunoglobulin Fc receptor	Bind Fc portion of immunoglobulin Kills antibody-coated target cells (antibody-dependent cell-mediated cytotoxicity, ADCC)
––––––––––––––	––––––––––––––	––––––––––––––
Mast cells	High affinity IgE Fc receptors	Bind IgE and initiate allergic responses by release of histamine

Isotype	Structure	Placental transfer	Binds mast cell surfaces	Binds phagocytic cell surfaces	Activates complement	Additional features
IgM		–	–	–	+	First Ab in development and response.
IgD		–	–	–	–	B-cell receptor.
IgG		+	–	+	+	Involved in opsonization and ADCC. Four subclasses; IgG1, IgG2, IgG3, IgG4.
IgE		–	+	–	–	Involved in allergic responses.
IgA		–	–	–	–	Two subclasses; IgA1, IgA2. Also found as dimer (sIgA) in secretions.

Immunoglobulins

Histocompatibility

Humoral Immunity

Antigen-Antibody Interactions

Cell Mediated Immunity