Todar's Online Textbook of Bacteriology

Immune Defense against Microbial Pathogens: Adaptive Immunity

© 2008 Kenneth Todar University of Wisconsin Department of Bacteriology

An antigen (Ag) is a foreign substance (i.e., not part of the animal's tissues) of relatively high molecular weight (>12,000 daltons) that induces a specific immunological response in the form of AMI or CMI or both. Because of their complex macromolecular structure, a single microorganism consists of multiple antigens (e.g. surface structures such as cell wall components, fimbriae, flagella, etc., or extracellular proteins, such as toxins and extracellular enzymes). The coat proteins and some of the envelope proteins of animal viruses are also antigenic. The animal host is able to respond specifically to each and every antigen to come into contact with the components of the immunological system.

Adaptive immunity is a function of the immunological system (Figure 1). The immunological system is able to recognize specific antigens and react in such a way that the host generates  antibody-mediated immunity (AMI), cell-mediated immunity (CMI), or both. AMI and CMI are the two great arms of the immunological response discussed below.

Active vs passive immunity

2. Passive immunity is the acquisition by an animal of immune factors which were produced in another animal, i.e., the host receives antibodies and/or immuno-reactive lymphocytes originally produced during an active response in another animal. Passive immunity is typically short-lived and usually persists for only a few weeks or months.

Furthermore, either active or passive immunity may be acquired by natural means (e.g. self production of antibodies during infection or transfer of antibodies from mother to offspring) or by artificial means (i.e., vaccination and other immunization procedures). Some familiar examples of active and passive immunity are given in the table below.
 
 

Table 1. Examples of Active and Passive Immunity

Type of Immunity	How Acquired by Host	Examples
Active Immunity	As a result of exposure to an infectious agent or one of its products (antigens)	Natural: Antibodies are produced by the host in response to the infectious agent itself (e.g. recovery from the disease).  Artificial: immunization (vaccination) with some product derived from the infectious agent (e.g. toxoid, killed cells, structural components of cells, inactivated or attenuated viruses, etc.).
Passive Immunity	As a result of the acquisition of antibodies which have been produced in another animal (by active means) or derived from cells grown in tissue culture (e.g. monoclonal antibodies)	Natural: Transplacental transfer of antibodies from mother to fetus; transfer of antibodies from mother to infant in milk by nursing. Artificial: Injection of immune serum from an individual previously immunized or recovered from disease, e.g. hepatitis; injection of serum from an animal hyperimmunized with tetanus toxoid.

The immunological System

The immunological system is comprised of the lymphoid tissues and organs of the body. Lymphoid tissues are widely distributed: they are concentrated in bone marrow, lymph nodes, spleen, liver, thymus, and Peyer's patches scattered in linings of the GI tract. The lymphoid system is encompassed by the system of mononuclear phagocytes. Lymphocytes are the predominant cells, but macrophages, dendritic cells, and plasma cells are also present. Lymphocytes are cells which circulate, alternating between the circulatory blood stream and the lymphatic channels. The distribution of lymphatic tissues that make up the immunological system in humans is illustrated in the figure below.

Figure 1. Anatomy of the Immunological System. (A): The major components of the immunological system are lymph nodes connected by lymph ducts, Peyer's patches (masses of lymphocytes in the lower gastrointestinal tract), thymus, spleen, and bone marrow. (B): A lymph node. Afferent lymph ducts bring lymph-containing antigens into the lymph node. Macrophages, dendritic cells and  B-cells in the cortical region make contact with the antigen and process it for presentation to immunocompetent B-cells and T- cells, thereby initiating an immune response. As a result, B-cells are stimulated to develop into antibody-secreting plasma cells, and T-cells are stimulated to develop into effector T cells of various classes. Antibodies leave the lymph node by the efferent ducts that empty into the blood stream. Lymphocytes can also leave the node by the efferent duct and travel to other sites in the lymphatic system or enter into the blood circulation. A single lymphocyte completes a circuit through the circulating blood and lymphatic systems once every 24 hours.

Organs comprising the immune system

Bone Marrow  All cells of the immune system are initially derived from the bone marrow. During hematopoiesis, bone marrow stem cells develop into either mature cells or precursors of cells that migrate out of the bone marrow to continue their maturation elsewhere. The bone marrow produces lymphocytes  (B-cells, immature T-cells, and natural killer cells), granulocytes (including neutrophils, monocytes and dendritic cells), in addition to red blood cells and platelets.

Thymus T-cells mature in the thymus. Immature T-cells, also known as pre-T cells (or prothymocytes), leave the bone marrow and migrate into the thymus. In a maturation process sometimes referred to as "thymic education", T cells that are beneficial to the immune system are spared, while T cells that might evoke a detrimental autoimmune response are eliminated. The mature T cells are then released into the bloodstream.

Spleen The spleen is is made up of B cells, T cells, macrophages, dendritic cells, natural killer cells and red blood cells. The spleen filters antigens directly from the blood that passes through it, and migratory macrophages and dendritic cells bring antigens to the spleen via the bloodstream. An immune response is initiated when a macrophage or dendritic cell "presents antigen" to appropriate B or T cells. In the spleen, B cells become activated and produce large amounts of antibody.

Lymph Nodes The lymphatic system parallels the circulatory blood system. It is periodically guarded by  lymph nodes, which are found throughout the body. Composed mostly of T cells, B cells, dendritic cells and macrophages, the nodes drain fluid from most tissues. Antigens are filtered out of the lymph in the lymph node before returning the lymph to the circulation. In a similar fashion as the spleen, macrophages and dendritic cells capture antigens and present them T and B cells, consequently initiating an immune response.


Figure 2. Origin and differentiation of cells of the immune system.

Cells of the immune system

B-cells The major function of B lymphocytes is to develop into antibody-secreting plasma cells following stimulation by foreign antigens of bacteria, viruses and tumor cells. Antibodies are specialized proteins that specifically recognize and bind to specific antigens that caused their stimulation. Antibody production and binding to foreign antigens is often critical as a means of signaling other cells to engulf, kill or remove that substance from the body.

T-cells T lymphocytes are usually divided into two major subsets that are functionally and phenotypically different. T helper (TH) cells, also called CD4+ T cells, are involved in coordination and regulation of immunological responses. They function to mediate responses by the secretion of lymphokines that stimulate or otherwise affect other cells involved in the immune responses.

The second subset type of T lymphocytes are cytotoxic T lymphocytes ( Tc cells or CTLs) or CD8+ T cells. These cells are involved in directly killing certain tumor cells, virus-infected cells, transplant cells, and sometimes eucaryotic parasites. CD8+ T cells are also important in down-regulation of immune responses.

Both types of T cells can be found throughout the body, most conspicuously in lymphoid organs (lymph nodes and spleen) but also the liver, lung, blood, and the intestinal tract.

Natural Killer cells Natural killer cells, known as NK cells, are similar to CTLs (CD8+ T cells). They function as effector cells that directly kill certain tumors such as melanomas, lymphomas and virus-infected cells, most notably herpes and cytomegalovirus-infected cells. However, NK cells, unlike the CD8+ (Tc) cells, kill their target cells without need for recognition of antigen in association with MHC molecules.  NK cells that have been activated by secretions from CD4+ T cells will kill their tumor or viral-infected targets more effectively.

Macrophages Macrophages are important in the regulation of immune responses. Besides their role in phagocytosis, they may function as antigen-presenting cells (APCs) because they ingest foreign materials and present these antigens to other cells of the immune system such as T-cells and B-cells. This is one of the important first steps in the initiation of an immunological response. Macrophages, stimulated by certain lymphokines, exhibit increased levels of phagocytosis and are also secrete cytokines that modulate immune responses.

Dendritic cells Dendritic cells also originate in the bone marrow and function as antigen presenting cells (APCs). In fact, the dendritic cells are more efficient APCs than macrophages. These cells are usually found in structural compartments of the lymphoid organs such as the thymus, lymph nodes and spleen. However, they are also found in the bloodstream and other tissues of the body. It is believed that they capture and process antigens in lymphoid organs where an immunological response is initiated. Of particular interest is the recent finding that dendritic cells bind large numbers of HIV particles, and may be a reservoir of virus that is transmitted to CD4+ T cells.

The Immunological Response

Immunological responses are associated with macrophages or dendritic cells and two subpopulations of lymphocytes, B-cells and T-cells.

Under antigenic stimulus, B-lymphocytes become transformed into antibody-secreting plasma cells. The plasma cells synthesize large amounts of immunoglobulins (antibodies) which will react stereochemically with the stimulating antigen.

Under antigenic stimulus, pre T-lymphocytes differentiate into several classes of effector T cells which are committed to various activities upon recognition of the specific antigen that induced their formation. T cells have many activities relevant to immunity including (1) mediation of the B-cell response to antigen; (2) ability to recognize and destroy cells bearing foreign Ag on their surface; and (3) production of a variety of diffusible compounds called cytokines and/or lymphokines, which include substances that are activators of macrophages, mediators of inflammation, chemotactic attractants, lymphocyte mitogens, and interferon. Cytokines and lymphokines are molecules (peptides, proteins) produced by cells as a means of intercellular communication. Generally, they are secreted by a cell to stimulate the activity of another cell.

The overall aspects of the induction of an immunological response (AMI and CMI) are shown in the following schematic diagram.
 

Three important features of an immunological response relevant to host defense and/or "immunity" to pathogenic microorganisms are:

1. Specificity. An antibody or reactive T-cell will react specifically with the antigen that induced its formation; it will not react with other antigens. Generally, this specificity is of the same order as that of enzyme-substrate specificity or a receptor-ligand interaction. However, cross-reactivity is possible. The specificity of the immunological response is explained on the basis of the clonal selection hypothesis: during the primary immune response, a specific antigen selects a pre-existing clone of specific lymphocytes and stimulates exclusively its activation, proliferation and differentiation.

2.  Memory. The immunological system has a "memory". Once the immunological response has reacted to produce a specific type of antibody or reactive T-cell, it is capable of producing more of the antibody or activated T-cells rapidly and in larger amounts. This is referred to as a secondary or memory response.

3. Tolerance. An animal generally does not undergo an immunological response to its own (potentially-antigenic) components. The animal is said to be tolerant to self-antigens. This ensures that under normal conditions, an immune response to "self" antigens (called an autoimmune response) does not occur. Autoimmune responses are potentially harmful to the host. Tolerance is brought about in a number of ways, but basically the immunological system is able to distinguish "self" antigens from "non-self" (foreign) antigens; it will respond to "non-self" but not to "self". Sometimes in an animal, tolerance can be "broken", which may result in an autoimmune pathology.

The Two Types Adaptive Immunity: AMI and CMI

Antibody-mediated immunity (AMI) is the type of adaptive immunity that is mediated by soluble host proteins called antibodies or immunoglobulins. Because it is largely due to the presence of circulating antibody molecules in the serum, is also called circulating immunity or humoral immunity.

If a naive (unstimulated) B cell encounters an antigen, it is stimulated to develop into a plasma cell which produces the antibodies that will react with the stimulating antigen. They also develop into clones of identical reactive B-cells called memory B-cells.

Antibodies (Ab) are proteins (globulins) produced in response to an encounter with an antigen (Ag). There are several classes or types of antibodies (and subclasses of the types), but all antibodies are produced in response to a specific antigen react stereochemically with that antigen and not with other (different) antigens. An animal has the genetic capacity to produce specific antibodies to thousands of different antigens, but does not do so until there is an appropriate (specific) antigenic stimulus. Due to clonal selection, the host produces only the homologous antibodies that will react with that antigen. These antibodies are found in the blood (plasma) and lymph and in many extravascular tissues. They have a various roles in host defense against microbial and viral pathogens as discussed below.

Cell-mediated immunity (CMI) is the type of adaptive immunity that is mediated by specific subpopulations of T-lymphocytes called effector T-cells. In non immune animals precursor T-cells (pT cells) exist as "resting T cells". They bear receptors for specific antigens. Stimulation with Ag results in their activation. The cells enlarge, enter into a mitotic cycle, reproduce and develop into effector T-cells whose activities are responsible for this type of immunity. They also develop into clones of identical reactive T-cells called memory T-cells.

The biological activities of the antibody-mediated and cell-mediated immune responses are different and vary from one type of infection to another. The AMI response involves interaction of B lymphocytes with antigen and their differentiation into antibody-secreting plasma cells. The secreted antibody binds to the antigen and in some way leads to its neutralization or elimination from the body. The CMI response involves several subpopulations of T lymphocytes that recognize antigens on the surfaces of cells. TH cells (CD4⁺) respond to antigen with the production of lymphokines. A distinction between TH1 cells and TH2 is based on their lymphokine profiles. TH2 cells have previously been referred to as T-helper cells because they provide lymphokines (e.g. IL-2 and IL-4) which activate T cells and B cells at the start of the immune response. TH1 cells were formerly known as delayed type hypersensitivity cells (TDTH) because of their role in this allergic process. Tc cells (CD8⁺) or cytotoxic T lymphocytes (CTLs) are able to kill cells that are showing a new or foreign antigen on their surface (as virus-infected cells, or tumor cells, or transplanted tissue cells).

AMI and CMI are discussed separately in more detail below.

Membrane receptors on B-cells and T-cells

The nature of the membrane receptors for antigen on B-cells and T-cells is fairly well understood. Each B cell has approximately 10⁵ membrane-bound antibody molecules (IgD or IgM) which correspond in specificity to the antibody that the cell is programmed to produce. Each T cell has about 10⁵ molecules of a specific antigen-binding T cell receptor (TCR) exposed on its surface. The TCR is similar, but not identical, to antibody. In addition, T cell subsets bear some distinguishing surface markers, notably CD4 or CD8. T cells bearing CD4 always recognize antigens in association with class II major histocompatability complex (class II MHC) proteins on the surfaces of other cells. CD4⁺ T lymphocytes generally function as T helper cells or in modulation of immune responses. T cells bearing CD8 ( CD8⁺ ) always recognize antigen in association with class I MHC proteins and typically function as cytotoxic T cells. The important markers, actions and interactions of T cells, B cells and Antigen Presenting Cells (APC) are illustrated below.

Figure 4. Receptor interactions between B cells, T cells and Antigen Presenting Cells (APC)
 

Induction of primary immunological responses

Induction of a primary immunological response begins when an antigen penetrates epithelial surfaces. It will eventually come into contact with macrophages or certain other classes of Antigen Presenting cells (APCs), which include B cells, monocytes, dendritic cells, Langerhans cells and endothelial cells. Antigens, such as bacterial cells, are internalized by endocytosis and "processed" by the APC, then "presented" to immunocompetent lymphocytes to initiate the early steps of the immunological response. Processing by a macrophage (for example) results in attaching antigenic materials to the surface of the membrane in association with MHC II molecules on the surface of the cell . The antigen-MHC II complex is presented to a T-helper (TH2) cell which is able to recognize processed antigen associated with a class II MHC molecule on the membrane of the macrophage. This interaction, together with stimulation by Interleukin 1 (IL-1), produced by the macrophage, will activate the TH2 cell. Activation of the TH2 cell causes it to begin to produce Interleukin 2 (IL-2), and to express a membrane receptor for IL-2. The secreted IL-2 auto stimulates proliferation of the TH2 cells. Stimulated Antigen stimulated TH2 cells produce a variety of lymphokines including IL-2, IL-4, IL-6, and gamma Interferon, which mediate various aspects of the immune response. For example, IL-2 binds to IL-2 receptors on other T cells (which have bound the Ag) and stimulates their proliferation, while IL-4 causes B cells to proliferate and differentiate into antibody-secreting plasma cells and memory B cells. IL-4 activates only B cells in the vicinity which themselves have bound the antigen, and not others, so as to sustain the specificity of the immunological response.

As previously mentioned, B cells themselves behave as APCs. Antigens bound to antibody receptors on the surface of a B cell cause internalization of some of the antigen and expression on the B cell membrane together with MHC II molecules. The TH2 cell recognizes the antigen together with the Class II MHC molecules, and secretes the various lymphokines that activate the B cells to become antibody-secreting plasma cells and memory B cells. Even if the antigen cannot cross-link the receptor, it may be endocytosed by the B cell, processed, and returned to the surface in association with MHC II where it can be recognized by specific TH2 cells which will become activated to initiate B cell differentiation and proliferation. In any case, the overall B-cell response leads to antibody-mediated immunity (AMI).

The antigen receptors on B cell surfaces bear the specificity of antibodies that they are genetically-programmed to produce. Hence, there are thousands of sub-populations of B cells distinguished only by their ability to produce a unique (reactive) type of antibody molecule. A B-cell can react with a homologous antigen on the surface of the macrophage or with soluble antigens. When a B-cell is bound to Ag, and simultaneously is stimulated by IL-4 produced by a nearby TH2 cell, the B cell is stimulated to grow and divide to form a clone of identical B cells, each capable of producing identical antibody molecules. The activated B cells further differentiate into plasma cells which synthesize and secrete large amounts of antibody, and into a special form of B cells called memory B cells.

The antibodies produced and secreted by the plasma cells react specifically with the homologous antigen that induced their formation. Many of these reactions lead to host defense and to prevention of reinfection by pathogens. Memory cells a role in secondary immune responses.

Plasma cells are relatively short-lived (about one week) but produce large amounts of antibody during this period. Memory cells, on the other hand, are relatively long-lived and upon subsequent exposure to Ag they become quickly transformed into Ab-producing plasma cells.

Generation of cell mediated immunity (CMI) begins when (for example) a Tc cell recognizes a processed antigen associated with MHC I on the membrane of a cell (usually an altered self cell, but possibly a transplanted tissue cell or a eucaryotic parasite). Under stimulation by IL-2 produced by TH2 cells the Tc cell becomes activated to become a cytotoxic T lymphocyte (CTL) capable of lysing the cell which is showing the new (foreign) antigen on its surface, a primary manifestation of CMI.

The interaction between an antigen-presenting macrophage and a TH cell stimulates the macrophage to produce and secrete a cytokine called Interleukin-1 (IL-1) that acts locally on the TH cell. The IL-1 stimulates the TH cell to differentiate and produce its own cytokines (which in this case might be called lymphokines because they arise from a lymphocyte). These lymphokines have various functions. Interleukin-4 has an immediate effect on nearby B-cells. Interleukin-2 has an immediate effect on T cells as described above.

Time is required before a primary immunological response to be effective as a host defense. Antigens have to be recognized, taken up, digested, processed and presented by APCs; a few select TH cells must react with Ag and respond; preexisting B or T lymphocytes must encounter the Ag and proliferate and differentiate into effector cells (plasma cells or CTLs). In the case of AMI, antibody level has to build up to an effective physiological concentration to render its host resistant. It may take several days or weeks to reach a level of effective immunity, even though this immunity may persist for many months, or years, or even a lifetime due to the presence of the antibodies. In natural infections, the inoculum is small, and even though the antigenic stimulus increases during microbial replication, only small amounts of antibody are formed within the first few days, and circulating antibody is not detectable until about a week after infection.

Induction of a secondary immunological response

On re-exposure to microbial antigens (secondary exposure to antigen), there is an accelerated immunological response, the secondary or memory response. Larger amounts of antibodies are formed in only 1-2 days. This is due to the activities of specific memory B cells and memory T cells which were formed during the primary immune response. These memory cells, when stimulated by homologous Ag, "remember" having previously seen the Ag, and are able to rapidly divide and differentiate into effector cells. Stimulating memory cells to rapidly produce very high (effective) levels of persistent circulating antibodies is the basis for giving regular "booster"-type vaccinations to humans and pets.
 

Figure 4. Primary and Secondary Immunological Responses. Following the first exposure to an antigen the immune response (as evidenced by following the concentration of specific antibody in the serum, called "titer") develops gradually over a period of days, reaches a low plateau within 2-3 weeks, and usually begins to decline in a relatively short period of time. When the antigen is encountered a second time, a secondary (memory) response causes a rapid rise in the concentration of antibody, reaching a much higher level in the serum, which may persist for a relatively long period of time. This is not to say that a protective level of antibody may not be reached by primary exposure alone, but usually to ensure a high level of protective antibody that persists over a long period of time, it is necessary to have repeated antigenic stimulation of the immune system.
 
 

As mediators of immunity, it was discovered at the turn of the century that antibodies were contained within the serum fraction of blood. It was demonstrated in 1939 that antibodies were specifically located in the gamma fraction of electrophoresed serum, thus the term gamma globulin was coined for serum containing antibodies. Antibodies themselves, were called immunoglobulins.

The Classes of Antibodies

There are a number of types of antibodies or immunoglobulins that react stereochemically and specifically with an antigen that induced their formation. Each of these classes of immunoglobulins (abbreviated Ig) is produced by a specific clone of plasma cells. Five immunoglobulin classes are defined on the basis of their heavy chain composition, named IgG, IgM, IgA, IgE, and IgD. IgG and IgA are further divided into subclasses.

The classes of immunoglobulins have different physical and chemical characteristics and they exhibit unique biological properties. Their synthesis occurs at different stages and rates during an immunological response and/or during the course of an infection. Their importance and functions in host resistance (immunity) are different.

IgG. Immunoglobulin G is the predominant Ig in the serum; it makes up about 80% of the total antibody found in an animal at any given time, being 75% of the total serum antibody. It can diffuse out of the blood stream into the extravascular spaces and it is the most common Ig found there. Its concentration in tissue fluids is increased during inflammation. It is particularly effective at the neutralization of bacterial exotoxins and viruses. It also has opsonizing ability and complement-fixing ability. IgG crosses the placental barrier, and thereby provides passive immunity to the fetus and infant for the first six months of life.

IgG is the model for our understanding the structure and function of antibody molecules, so examination of its biochemical properties is appropriate before discussion of the other types of immunoglobulins.

Figure 5. Model of an Immunoglobulin: the Structure of IgG (see discussion below)
 

IgG is a protein with a molecular weight of about 150,000 daltons. The protein consists of two identical heavy (H) chains (each with a mw of about 50kDa) and two identical light (L) chains (mw about 25kDa). Each L chain is connected to a H chain and the two H-chains are connected to one another by disulfide bridges. The molecule is drawn to look like a Y. The stem of the Y is called the Fc region and it consists mainly of two halves of the identical H chains. Each of the "arms" of the Y contains one complete L-chain and half of one of the H-chains. The Y stem stands on the carboxy termini of the H chains; the tips of the arms contain the amino termini of the H and L-chains. Each arm is sometimes referred to as the Fab region of the molecule. The Fab region is the antigen binding fragment of the antibody molecule. A specific region of the antigen (called the antigenic determinant) will react stereochemically with the antigen-binding region at the amino terminus of each Fab. Hence, the IgG molecule, which has two antigen binding fragments [(Fab)2] is said to be divalent: it can bind to two Ag molecules. The polypeptide composition of the Fc region of all IgG1 antibody molecules is relatively constant regardless of antibody specificity; however, the Fab regions always differ in their exact amino acid sequences depending upon their antigenic specificity. Even though the antigen does not react with the Fc region of the IgG molecule, this should not be taken to mean that the Fc region has no importance or biological activity. On the contrary, specific amino acid regions of the Fc portion of the molecule are recognized by receptors on phagocytes and certain other cells, and the Fc domain contains a peptide region that will bind to and activate complement, which is often required for the manifestation of AMI.

Understanding the structure and properties of IgG is useful to discussion of its function in host defense. Since the IgG molecule is divalent, it can cross-link Ag molecules, which may lead to precipitation or agglutination of antigens; if IgG is bound to Ag on a microbial cell surface, its Fc region may provide an extrinsic ligand which will be recognized by specific receptors on phagocytes. Such microbial cells or viruses coated with IgG molecules are said to be opsonized for phagocytosis. Opsonized pathogens are taken up and destroyed much more readily by phagocytes than their non-opsonized counterparts. IgG, as well as IgM and IgA, will neutralize the activity of toxins, including bacterial exotoxins. Furthermore, cross-linked IgG molecules on the surface of a cell can bind and activate complement from the serum and set off a cascade of reactions that can lead to destruction of the cell. It is because of its relatively small size and its persistence in the serum of a mother that IgG is shared with the fetus in utero, such that an infant is born with the full complement of mother's IgG antibodies.

IgM is the first immunoglobulin to be synthesized by infants and the first to appear in the blood stream during the course of an infection. Mainly, it is confined to the bloodstream giving the host protection against blood-borne pathogens. IgM makes up about 10% of the total serum immunoglobulins. IgM is arranged to resemble a pentamer of five immunoglobulin molecules (mw = 900kDa) tethered together at by their Fc domains (Figure 6). In addition to covalent linkages between the monomeric subunits, the pentamer is stabilized by a 15kDa polypeptide called the J chain. IgM, therefore, has a theoretical "valence" of 10 (i.e., it has exposed 10 Fab domains). Probably, the most important role of IgM is its ability to function early in the immune responses against blood-borne pathogens. As might be expected, IgM is very efficient at agglutinating particulate antigens. Also, IgM binds complement strongly and such IgM antibodies bound to a microbial surface act as opsonins, rendering the microbe more susceptible to phagocytosis. In the presence of complement and IgM whole microbial cells may be killed and lysed. IgM also appears on the surfaces of mature B cells as a transmembranous monomer where it functions as an antigen receptor, capable of activating B cells when bound to antigen.

Figure 6. Schematic representation of the various Classes of Immunoglobulins

IgA exists as a 160kDa monomer in serum and as a 400kDa dimer in secretions. As in the case of IgM, polymerization (dimerization) is via a J-chain (Figure 6). There are two subclasses based on different heavy chains, IgA1 and IgA2. IgA1 is produced in bone marrow and makes up most of the serum IgA. Both IgA1 and IgA2 are synthesized in GALT (gut associated lymphoid tissues) to be secreted onto the mucosal surfaces. Since IgA may be synthesized locally and secreted in the seromucous secretions of the body, it is sometimes referred to as secretory antibody or sIgA. Quantitatively, IgA is synthesized in amounts greater than IgG, but it has a short half life in serum (6 days), and it is lost in secretory products. The concentration of IgA in serum is about 15% of the total antibody. Secretion of dimeric IgA is mediated by a 100kDa glycoprotein called the secretory component. It is the addition of the secretory component to the IgA molecules that accounts for their ability to exit the body to mucosal surfaces via the exocrine glands. IgM can be transported similarly but makes up a small proportion of secretory antibodies.

Secretory IgA is the predominant immunoglobulin present in gastrointestinal fluids, nasal secretions, saliva, tears and other mucous secretions of the body. IgA antibodies are important in resistance to infection of the mucosal surfaces of the body, particularly the respiratory, intestinal and urogenital tracts. IgA acts as a protective coating for the mucous surfaces against microbial adherence or initial colonization. IgA can also neutralize toxin activity on mucosal surfaces. Fc receptors for IgA-coated microbes are found on monocytes and neutrophils in the lower respiratory tract, suggesting that IgA may have a role (in the lung, at least) in opsonization of pathogens. IgA does not activate complement, however.

Secretory IgA is also transferred in milk, via the colostrum, from a nursing mother to an infant. This provides passive immunity to many pathogens, especially those that enter by way of the GI tract. The transfer of IgA via the milk lasts about six months in the mother, during which time the nursing infant is protected from many infectious agents. Under these circumstances, the infectious agent might multiply to a limited extent, which stimulates the infant's own immune response without causing significant disease. Thus, as in the case with transplacental IgG, the infant acquires active immunity while undergoing protection by passive immunity.

IgE  is a 190kDa immunoglobulin that accounts for merely 0.002% of the total serum immunoglobulins. It is produced especially by plasma cells below the respiratory and intestinal epithelia. The majority of IgE is bound to tissue cells, especially mast cells. If an infectious agent succeeds in penetrating the IgA barrier, it comes up against the next line of defense, the MALT (mucosa-associated lymphoid tissues) system which is manned by IgE. IgE is bound very firmly to specific Fc receptors on the surface of mast cells. Contact with Ag leads to release of mediators of inflammation from the mast cells, which effectively recruits various agents of the immune responses including complement, chemotactic factors for phagocytes, T-cells, etc. Although a well-known manifestation of this reaction is a type of immediate hypersensitivity reaction called atopic allergy (e.g. hives, asthma, hay fever, etc.). However, the MALT responses are an important defense mechanism because they amplify the local inflammatory response that facilitates rejection of a pathogen.

IgD is a 175kDa molecule that resembles IgG in its monomeric form. IgD antibodies are found for the most part on the surfaces of B lymphocytes. The same cells may also carry IgM antibody. It is thought that IgD and IgM function as mutually-interacting antigen receptors for control of B-cell activation and suppression. Hence, IgD may have an immunoregulatory function. Recall that only specific subclones of B-cells respond to a specific Ag upon stimulation. The B-cell bears a specific receptor (in the form of IgD) for the Ag that it specifically recognizes. It stands to reason that the basis of this specificity is mediated by a molecule with immunoglobulin characteristics. The T-cell receptor (TCR) is also a molecule with immunoglobulin-like characteristics.

Functions of Antibodies in Host Defense

The functions of  antibodies, and hence the AMI response, in host defense against pathogenic microbes is summarized below.

Opsonization Antibodies enhance phagocytic engulfment of microbial antigens. IgG and IgM Abs have a combining site for the Ag and a site for cytophilic association with phagocytes. Bacteria and viral particles are ingested with increased efficiency.

Steric hindrance Antibodies combine with the surfaces of microorganisms and may block or prevent their attachment to susceptible cells or mucosal surfaces. Ab against a viral component can block attachment of the virus to susceptible host cells and thereby reduce infectivity. Secretory IgA can block attachment of pathogens to mucosal surfaces.

Toxin Neutralization Toxin-neutralizing antibodies (antitoxins) react with a soluble bacterial toxin and block the interaction of the toxin with its specific target cell or substrate in the host.

Agglutination and Precipitation Antibodies combine with the surfaces of microorganisms or soluble antigens and cause them to agglutinate or precipitate. This reduces the number of separate infectious units and makes them more readily phagocytosed because the clump of particles is larger in size. Also, floccules or aggregates of neutralized toxin may be removed by phagocytes.

Activation of Complement Antibodies combined with the surface antigens of microbes activate the complement cascade which has four principal effects related to host defense:

1. induction of the inflammatory response

2. attraction of phagocytes to the site of immunological encounter

3. opsonization of cells which increases efficiency of phagocytosis

4. lysis of certain bacteria or viruses

Antibody-dependent cell cytotoxicity (ADCC): IgG can enable certain cells (Natural Killer or NK cells) to recognize and kill opsonized target cells. Certain other types of cells including monocytes and neutrophils also act this way. NK cells attach to opsonized target cells by means of an IgG Fc receptor and kill by an extracellular mechanism after attachment. ADCC will be discussed as part of cell-mediated immunity.
 
 

The CMI response

During the cell-mediated immune response, various subsets of T lymphocytes are activated and develop into effector T cells. These include cytotoxic T lymphocytes (CTLs or Tc cells) and T helper cells of the TH1 and TH2 subsets. TH1 cells secrete lymphokines that activate macrophages and mediate delayed type hypersensitivity responses. TH2 cells secrete lymphokines that stimulate B cell development and may help activate Tc cells to their full cytotoxic capacity.

T cells that generate CMI are present in lymphoid organs, blood and lymph nodes. Due to constant recirculation between blood and lymph nodes via lymphatics and back to the blood, one T-cell circulates once in about 24 hours. Each carries receptors for the specific Ag with which it can react. T-cell recognition of Ag only occurs when the Ag is associated with proteins of the MHC complex. T-cells have receptors (TCR) complementary to the complexed MHC determinant and the antigenic epitope. TH1 cells and TH2 cells recognize Ag in association with MHC II (as displayed by macrophages and other APCs); Tc cells recognize Ag on cells complexed with MHC I (as displayed by altered self cells).

Stepwise Activation of Tc cells

During a primary CMI response, antigen is presented to the precursor Tc lymphocytes (CD8⁺) in association with MHC Class I proteins. All nucleated cells express MHC I on their surfaces, so virtually any cell in the animal expressing a new ("nonself") Ag on its surface will activate the cytotoxic T lymphocytes. TH2 cells can augment activation of Tc cells, but they probably are not required.

Activation of TH cells

TH-cells (CD4⁺) reacting with Ag may produce a variety of lymphokines. Notably, Interleukin-2 (IL-2) stimulates T-cell activation and IL-4 stimulates B cells.

T-helper cells are composed of distinct subsets that are best distinguished on the basis of their patterns of lymphokine production. Both types of TH cells develop under most conditions but their ratios and the predominance of certain lymphokines can vary, and this may mediate the pathology and outcome of certain bacterial infections.

TH1 cells "see" foreign Ag on the surface of APCs in the context of MHC II. Mainly, TH1 cells produce IL-2, gamma interferon (IFN) and lymphotoxin. This results in macrophage activation and the delayed-type hypersensitivity reaction, as well as help for Tc cell activation.

TH2 cells also see foreign Ag on the surface of APCs in the context of MHC II. Their response is to secrete IL-4, IL-5, IL-6, IL-10 and IL-13 that help activate B cells, provide help for the production of IgE that attaches to mast cells, and promote mast cell and eosinophil activation.

The lymphokines produced by TH cells stimulate B cells and pTc cells, inducing them to proliferate and mature into effector cells. Gamma Interferon activates macrophages and Natural Killer (NK) cells to their full cytolytic potential. Lymphotoxins, such as tumor necrosis factor (TNF) cause fever and kill cells at a distance.

Function of cytotoxic T-lymphocytes

Tc cells (CTLs) can destroy cells bearing new antigens on their surfaces (as might result in a viral infection, a tumor cell, or an infection by a bacterial intracellular parasite). Tc cells exert their cytotoxic activity when they are in physical contact with cells bearing new Ag in association with MHC I protein. Contact between the Tc cell and the target cell is required for lysis, although the exact mechanism of lysis is not well understood. The target cell membrane is damaged at the site of contact (the "kiss of death") leaving a gaping hole about 40 nm in diameter that cannot be repaired. When the Tc cell moves away 30-60 seconds later, there is leakage of the cell components, an influx of H₂O, and the target cell swells up and dies. Apparently the Tc cell releases some of its cytolytic contents directly into the target cell, so that within a few minutes the target cell literally disintegrates. The Tc cell can move away and kill again.

Tc cells generally respond to Ag in association with MHC I proteins on the surface of a target cell. If they responded to Ag by itself, they could react with it when it was free in extracellular fluids, and their cytotoxic activity would be triggered with no purpose. As stated above, almost all host cells, including macrophages, display MHC I. Hence, an effector Tc cell can destroy a macrophage which is otherwise carrying out a useful function by presenting Ag to TH lymphocytes as part of the AMI or CMI responses. Usually, the time course of the response is such that TH cells have already developed and have carried out their (helping) function when Tc cells begin to become active.

Delayed Type Hypersensitivity

TH1-cells (CD4⁺) are a subset of T-lymphocytes that recognize Ag in association with Class II (and possibly Class I) MHC proteins. When TH1-cells are presented Ag in association with MHC II by a macrophage, their development is stimulated by macrophage Interleukin-1 (IL-1), and auto stimulated by IL-2, which the TH cell produces. They respond by differentiating and producing a variety lymphokines that induce a local inflammatory response, and which attract, trap, and activate phagocytes at the site. One aspect of this response is a state of delayed-type hypersensitivity in the host. This is usually evident in chronic infections wherein CMI is largely involved (e.g. tuberculosis).

Delayed-type hypersensitivity reactions usually present themselves as allergic reactions. Such allergic reactions generally require about 24 hours to develop following a secondary exposure to Ag. This time is required for the circulating TH cells (actually memory cells) to encounter the Ag and to produce cytokines that attract macrophages and Tc cells to the site wherein an allergic response is established. The phagocytic and cytolytic activities of these cells are responsible for the localized tissue destruction which occurs. Poison oak (ivy) rash is a familiar example of delayed hypersensitivity, but the reaction is also evident in several types of chronic or persistent bacterial infections including tuberculosis, leprosy and brucellosis, and in some fungal and protozoal infections.

One of the best known examples of the delayed-type hypersensitivity reaction is the Mantoux (tuberculin) test which is utilized to determine current or previous infection by the tubercle bacillus (Mycobacterium tuberculosis). A small amount of Ag called the purified protein derivative (PPD), derived from the cell wall of the bacterium, is injected subcutaneously under the skin of the forearm. The test is evaluated after 24-48 hours. A positive test is an allergic response (an "urticarial weal") at the site of the injection, which might look like a swollen reddened area about the size of a quarter. A negative test is no reaction. A positive test does not mean that the individual has an active case of tuberculosis, but that the individual has at least been exposed to the tubercle bacillus or one of its products sufficiently to have undergone a primary immune response. 

Other types of cells other than dermal macrophages have been proposed as antigen presenting cells (APCs) to initiate DTH reactions on the skin, including dendritic cells, epidermal Langerhans cells and venular endothelial cells. In humans, antigen presentation by Langerhans cells (which bear class II MHC), probably initiates sensitization, whereas antigen presentation by endothelial cells probably initiates DTH reactions upon secondary challenge.

5-hydroxytryptamine (5HT) has been shown to act as an adjuvant in the induction of the delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD). This supports the hypothesis that DTH reactions mediated by macrophages and dendrocytes require a cascade of both inflammatory and immunological signals.

Involvement of macrophages in mediation of CMI

During induction of the cell-mediated immune response, macrophages play their usual role in the presentation of Ag to T helper cells and in producing cytokines that are involved in the initiation of immune reactions. In addition, as in the case of DTH (above), macrophages play a role in the expression of CMI. Many of the lymphokines produced by TH cells are aimed at attraction, entrapment and activation of macrophages at the site of the reaction. One of these lymphokines, Gamma Interferon, causes the local macrophage population to develop an increased number of lysosomes and increased ability to secrete microbicidal products. Oxygen-dependent killing mechanisms of the macrophage are stimulated, and the macrophage develops increased power to ingest and kill microorganisms. Such lymphokine-stimulated macrophages are referred to as "angry" or activated macrophages.

Compared to normal macrophages, activated macrophages exhibit much greater ability to destroy intracellular pathogens. Activated macrophages may play an important role in the recovery from chronic bacterial infections and in resistance to certain tumors. Activated macrophages may be able to overcome bacterial intracellular parasites which are able to thwart the macrophage killing mechanisms before activation.

Macrophage involvement in CMI may be part of the pathology of certain diseases. Where there is difficulty in elimination an intracellular parasite (e.g. the tuberculosis bacillus) the chronic CMI response to local antigens leads to the accumulations of densely-packed macrophages which release fibrinogenic factors and stimulate the formation of granulation and fibrosis. The resulting structure, called a granuloma, actually represents an attempt by the host to isolate a persistent infection.

Other Aspects of cell-mediated immunity

Another class of cytotoxic lymphocytes distinct from Tc cells may be stimulated during the cell-mediated immune response. These are referred to as Natural Killer or NK cells. NK cells are found in blood and lymphoid tissues, especially the spleen. They do not bear T cell (or B cell) markers. Like Tc cells, they are able to recognize and kill cells that are displaying a foreign Ag on their surfaces, but unlike Tc cells, they do not display TCR and they are not MHC-restricted.

NK cells are present in an animal in the absence of antigenic stimulation, and it is for this reason that they are referred to as "natural" killers. They might also be considered part of the innate immune defenses; however, NK cells become activated in a CMI response by T-cell lymphokines, including Interleukin-2 and gamma interferon.

Some NK cells are thought to be an immature form of a T-lymphocyte, but various other types of cells including macrophages, neutrophils and eosinophils, display NK activity. Some NK cells have surface receptors (CD16) for the Fc portion of IgG. They bind to target cells by receptors for the Fc portion of antibody that has reacted with antigen on the target cell. This type of CMI is called antibody-dependent cell-mediated cytotoxicity or ADCC. NK cells may also have receptors for the C3b component of complement, and therefore recognize cells that are coated with C3b as targets. ADCC is thought to be an important defense against a variety of parasitic infections caused by protozoa and helminths.

Summary: cells involved in expression of CMI

Cell mediated immunity (CMI) is carried out by several types of cells including macrophages, TH lymphocytes Tc lymphocytes, and NK cells. After an immunological encounter, these cells are activated to produce and/or respond to various classes of lymphokines that are the mediators of CMI. A summary of the role of these cells  in the expression of CMI is provided below.

Tc (cytotoxic) Lymphocytes (CTLs) kill cells bearing foreign Ag on surface in association with MHC I. Tc cells can kill cells that are harboring intracellular parasites (either bacteria or viruses) as long as the infected cell is displaying a microbial antigen on its surface. Tc cells kill tumor cells and account for rejection of transplanted cells. Tc cells recognize Ag-MHC I complexes on target cells, contact them, and release the contents of granules directly into the target cell membrane which lyses the cell.

TH Lymphocytes produce lymphokines that are "helper" factors for development of B-cells into antibody-secreting plasma cells. They also produce certain lymphokines which stimulate the differentiation of effector T lymphocytes and the activity of macrophages. TH1 cells recognize Ag on macrophages in association with MHC II and become activated (by IL-1) to produce lymphokines including gamma Interferon that activates macrophages and NK cells. These cells mediate various aspects of the CMI response including delayed type hypersensitivity reactions. TH2 cells recognize Ag in association with MHC II on an APC and then produce interleukins and other substances that stimulate specific B-cell and T-cell proliferation and activity.

Macrophages are an important as Ag-presenting cells (APCs) that initiate T-cell interactions, development and proliferation. Macrophages are also involved in expression of CMI since they become activated by gamma IFN produced in a CMI response. Activated macrophages have increased phagocytic potential and release soluble substances that cause inflammation and destroy many bacteria and other cells.

Natural killer (NK) cells are cytotoxic cells that lyse cells bearing new antigen regardless of their MHC type and even lyse some cells that bear no MHC proteins. Natural Killer cells are defined by their ability to kill cells displaying a foreign Ag (e.g. tumor cells) regardless of MHC type and regardless of previous sensitization (exposure) to the Ag. Some NK cells are probably derived from Tc cells (CTLs), but they do not display T cell markers. NK cells can be activated by IL-2 and gamma IFN. Natural Killers lyse cells in the same manner as CTLs. Some NK cells have receptors for the Fc domain of IgG and so are able to bind to the Fc portion of IgG antibody on the surface of a target cell and release cytolytic components that kill the target cell. This mechanism of killing is referred to as antibody-dependent cell-mediated cytotoxicity (ADCC).
 

Summary: Lymphokines involved in expression of CMI

Extracellular factors that affect cell proliferation and differentiation have been defined as cytokines. These include the lymphokines, which are proteins produced by T-lymphocytes that have effects on the differentiation, proliferation and activity of various cells involved in the expression of CMI. In general, lymphokines function by (1) focusing circulating leukocytes and lymphocytes into the site of immunological encounter; (2) stimulating the development and proliferation of B-cells and T-cells; (3) stimulating and preparing macrophages for their phagocytic tasks; (4) stimulating natural killer (NK) cells; (5) providing antiviral cover and activity. The names and functions of some of the important lymphokines are described below.

IL-1 (Interleukin-1): Initially called lymphocyte activation factor. Mainly a product of macrophages, IL-1 has a variety of effects on various types of cells. It acts as a growth regulator of T-cells and B-cells, and it induces other cells such as hepatocytes to produce proteins relevant to host defense. IL-1 forms a chemotactic gradient for neutrophils and serves as an endogenous pyrogen which produces fever. Thus, IL-1 plays an important role in both the immunological responses and in the inflammatory response.

IL-2 (Interleukin-2): stimulates the proliferation of T-cells and activates NK (natural killer) cells.

IL-3 (Interleukin-3): regulates the proliferation of stem cells and the differentiation of mast cells.

IL-4 (Interleukin-4): causes B cell proliferation and enhanced antibody synthesis.

IL-6 (Interleukin-6): (same as beta Interferon) has effects on B cell differentiation and on antibody production and on T cell activation, growth, and differentiation. Probably has a major role in the mediation of the inflammatory and immune responses initiated by infection or injury.

IL-8 (Interleukin-8): chemotactic attractant for neutrophils.

IL-13 (Interleukin-13): shares many of the properties of IL-4, and is a potent regulator of inflammatory and immune responses.

Interferons:  Gamma-Interferon (gamma IFN) is produced by T cells and may be considered a lymphokine. It is sometimes called "immune interferon" (alpha-Interferon is "leukocyte interferon"; beta-Interferon is "fibroblast interferon"). Gamma-interferon has several antiviral effects including inhibition of viral protein synthesis (translation) in infected cells. It also activates macrophages and NK cells, and stimulates IL-1, IL-2, and antibody production.

Lymphotoxins: (Tumor Necrosis Factor-Beta): (TNF-beta is produced by T cells; TNF-alpha is produced by T cells, as well as other types of cells.) TNF kills cells, including tumor cells (at a distance). It is also a pyrogen.

Colony Stimulating Factor (CSF): several, including GMCSF, cause phagocytic white cells of all types to differentiate and divide.
 

Contrasting Roles of the AMI and CMI Responses in Host Defense

AMI and CMI responses are generated during almost all infections, but the relative magnitude and importance of each type of response shows great variation in different hosts and with different infectious agents.

In some types of infections antibody plays a major role in immunity or recovery. For example, viruses producing systemic disease with a viremia stage (viruses free in the blood as they spread from infected to uninfected cells), such as poliomyelitis or yellow fever, can be neutralized by circulating antibody. Pathogenic bacteria that multiply outside of cells (nearly all bacteria) at sites accessible to antibody can  can be stopped by the forces of AMI. Diseases caused by circulating bacterial toxins (e.g. diphtheria and tetanus) are controlled by circulating antibodies that neutralize toxins. Circulating antibodies (and perhaps secretory IgA, as well) present in immune animals can prevent reinfection by pathogens.

In other types of infections CMI is of supreme importance in recovery. These tend to be infections where the microbe grows or multiplies intracellularly. Bacterial infections of this nature include tuberculosis, brucellosis and syphilis. Recovery is associated with development of a pronounced CMI response, even though it is CMI that contributes to the pathology of the disease.

The clearest picture of the importance of CMI in recovery from disease is seen in certain viral infections (e.g. herpes, pox viruses and measles). Viruses are always intracellular parasites and may only rarely expose themselves to the extracellular forces of AMI. Antibodies could neutralize free virus particles liberated from cells but often have little influence on infected cells. The best strategic defense against virus-infected cells seems to be to kill the infected cell when the virus may be in a replicative (noninfectious) form. Many viruses, as they mature, cause foreign (viral) antigens to appear on the infected cell surface. These cells are recognized by the host's CMI defenses and they become target cells for cytolysis. The infected cell can be destroyed before virus is liberated.

The CMI response also plays a role in destruction of tumor cells and in rejection of tissue transplants in animals. A major problem in transplantation of tissues from one individual to another is rejection which is often based on CMI response to "foreign" cells (not a perfect match antigenically). Since tumor cells contain specific antigens not seen on normal cells they also may be recognized as foreign and destroyed by the forces of CMI. If tumor cells develop on a regular basis in animals, it may be the forces o CMI that eliminate them or hold them in check The increase in the incidence of many types of cancer (tumors) in humans with advancement of age may be correlated with a decline in the peak efficiency of the immune system that begins about 25 years of age.

In summary, antibody-mediated immunity (AMI) is probably most useful as an immune defense because of its ability to neutralize or destroy extracellular pathogens and to prevent occurrence of reinfection. Cell-mediated immunity (CMI) plays the major role in immune defense against infections caused by intracellular parasites, infections caused by viruses (either virulent or oncogenic), rejection of transplanted tissues or cells, and in the destruction of tumor cells. The contrasting roles of AMI and CMI as specific immunological responses are presented in the following table.
 
 

Table 2. Relative Importance of AMI and CMI in Various Types of Infections

Type of Infectious Agent	Immune Defense	Mechanisms	Examples
MULTIPLIES INSIDE TISSUE CELLS	Prevent entry  Kill infected cell	AMI: IgG, IgA, IgM  CMI: Tc, NK, ADCC	viruses, Rickettsia
MULTIPLIES INSIDE PHAGOCYTES	Activate phagocytes	CMI: lymphokines	viruses, Mycobacterium tuberculosis
	Kill infected phagocytes	CMI: Tc, NK, ADCC
MULTIPLIES OUTSIDE CELLS	Kill microbe extracellularly	AMI: Complement- mediated lysis	most bacteria
	Opsonized phagocytosis and lysis	AMI: IgG, IgM
	Neutralize toxins	AMI: IgG, IgM
MULTIPLIES OUTSIDE CELLS BUT ATTACHMENT TO BODY SURFACES REQUIRED FOR INVASION	Prevent attachment	AMI: IgA	streptococci E. coli Neisseria

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Written and edited by Kenneth Todar  University of Wisconsin-Madison  Department of Bacteriology  All rights reserved