The structure of the immune system (T– and B-system). Interraction of cells in immune responses. Mechanism of antibacterial and antiviral defence .

ORIGIN OF IMMUNE CELLS

The capability of responding to immunologic stimuli rests mainly with lymphoid cells. During embryonic development, blood cell precursors originate mainly in the fetal liver and yolk sac; in postnatal life, the stem cells reside in the bone marrow. Stem cells differentiate into cells of the erythroid, myeloid, or lymphoid series. The latter evolve into two main lymphocyte populations: T cells and B cells (Figure 1 and Table 1). The ratio of T cells to B cells is approximately 3:1.

T cell precursors differentiate into immunocompetent T cells within the thymus. Stem cells lack antigen receptors and CD3, CD4, and CD8 molecules on their surface, but during passage through the thymus they differentiate into T cells that can express these glycoproteins. The stem cells, which initially express neither CD4 nor CD8 (double-negatives), first differentiate to express both CD4 and CD8 (double-positives) and then proceed to express either CD4 or CD8. A double-positive cell will differentiate into a CD4-positive cell if it contacts a cell bearing class II MHC proteins but will differentiate into a CD8-positive cell if it contacts a cell bearing class I MHC proteins. (Mutant mice that do not make class II MHC proteins will not make CD4-positive cells, indicating that this interaction is required for differentiation into single-positive cells to occur.) The double-negative cells and the double-positive cells are located in the cortex of the thymus,. whereas the single-posith cells are located in the medulla, from which they migrate outside of the thymus. Within the (hymns, two very important processes called thymic education occur.

(1) CD-4-positive, CD8-positive cells, bearing antigen receptors for "self proteins, are killed (clonal deletion) by a process of "programmed cell death" called apoptosis (Figure 2). The removal of these self-reactive cells, a process called negative selection, results in tolerance to own proteins, ie, self-tolerance, and prevents autoimmune reactions.

(2) CD4-positiva, CDS-positive cells, bearing antigen receptors, that do not react with self MHC proteins are also killed. This results in a positive selection for T cells that react well with self MHC proteins.

These two processes produce T cells that are selected for their ability to react both with foreign antigens via their antigen receptors and with self MHC proteins. Both of these features are requir for an effective immune response by T cells.

Note that MHC proteins perform two essential functions in the immune response; one is the tive selection of T cells in the thymus, as just mentioned, and the other, which is described below, is the presentation of antigens to T cells, the initial step required to activate those cells. MHC teins are also the most important antigens recognized in the graft rejection process.

Table 1. Comparison of T cells and B cells

Feature	T Cells	B Cells
Antigen receptors on surface IgM on surface	Yes No	Yes Yes
CD3 proteins on surface	Yes	No
Clonal expansion after contact with specifie antigen	Yes	Yes
Immunoglobulin synthesis	No	Yes
Regulator of antibody synthesis	Yes	No
IL-2, IL- 4, IL-5, and gamma interferon synthesis	Yes	No
Effector of cell mediated immunity	Yes	No
Maturation in thymus	Yes	No
Maturation in bursa or its equivalent	No	Yes

During their passage through the thymus, each double-positive T cell synthesizes a different, j highly specific antigen receptor called the T cell receptor (TCR). The rearrangement of the vari-i able, diversity, and joining genes that encode the receptor occurs early in T cell differentiation and accounts for the remarkable ability of T cells to recognize millions of different antigens.

Some T lymphocytes, perhaps as much as 40% of the total, do not develop in the thymus butJ rather in the "gut-associated lymphoid tissue" (GALT). These intraepithelial lymphocytes (lELs) are^! thought to provide protection against intestinal pathogens. Their antigen receptors and surface proteins are different from those of thymes-derived lymphocytes. lELs cannot substitute for thymus-derivcd lymphocytes because patients with DiGeorgc's syndrome who lack a thymus (arc profoundly immunodeficient and have multiple infections.

The thymus involutes in adults, yet T cells continue to be made. Two explanations have been offered for this apparent paradox. One is that a remnant of the thymus remains functional throughout life and the other is that an extrathymic site takes over for the involuted thymus. Individuals who have had their thymus removed still make T cells, which supports the latter explanation.

Bone marrow stem сell

B cell

Bursa equivalent(bone marrow inhumans)

B cell precursors differentiate into immunocompetent B cells in the bone marrow; they do not pass through the thymus. B cells also undergo clonal deletion of those cells bearing antigen receptors for self proteins, a process that induces tolerance and reduces the occurrence of autoimmune diseases.The site of clonal deletion of B cells is uncertain, but it is not the thymus.

Natural killer (NK) cells are large granular lymphocytes that do not pass through the thymus, do not have an antigen receptor, and do not bear CD4 or CD8 proteins. They recognize and kill target cells, such as virus-infected cells and tumor cells, without the requirement that the antigens be presented in association with class I or class II MHC proteins. Rather, NK cells target those cells to be killed by detecting that they do not display class I MHC proteins on the cell surface. For example, many cells after they have been infected by a virus lose their ability to synthesize class I MHC proteins.

In contrast to T cells, B cells, and NK cells, which differentiate from lymphoid stem cells, macrophages arise from myeloid precursors. Macrophages have two important functions, namely, phagocytosis and antigen presentation. They do not pass through the thymus and do not have an antigen receptor. On their surface, they display class II MHC proteins, which play an essential role in antigen presentation to helper T cells. The cell surface proteins that play an important role in the immune response are listed in Table 2.

T Cells. T cells perform several important functions, which can be divided into two main categories, namely, regulatory and effector. The regulatory functions are mediated primarily by helper (CD4-positive) T cells, which produce interleukins (Table 3). For example, helper T cells make (1) interleukin-4 (IL-4) and IL-5, which help B cells produce antibodies; (2) IL-2, which activates CD4 and CD8 cells; and (3) gamma interferon, which activates macrophages, the main mediators of delayed hyper-sensitivity against intracellular organisms such as Mycobacterium tuberculosis. (Suppressor T cells are postulated to down-regulate the immune response, but evidence to support the existence of these cells is lacking.) The effector functions are carried out primarily by cytotoxic (CD8-positive) T cells, which kill virus-infected cells, tumor cells, and allografts.

CD4 and CD8 Types of T Cells.

Within the thymus, perhaps within the outer cortical epithelial cells (nurse cells), T cell progenitors differentiate under the influence of thymic hormones (thymosins and thymopoietins) into T cell subpopulations. These cells are characterized by certain surface glycoproteins, eg, CD3, CD4, and CD8. All T cells have CD3 proteins on their surface in association with antigen receptors (T cell receptor [see below]). The CD3 complex of five transmembrane proteins is involved with transmitting, from the outside of the cell to the inside, the information that the antigen receptor is occupied. One of the CD3 transmembrane proteins, the zeta chain, is linked to a tyrosine kinase called fyn, which is involved with signal transduction. The signal is transmitted via several second messengers, which are described in the section on activation (see below). CD4 is a single transmembrane polypeptide whereas CD8 consists of two transmembrane polypeptides. They may signal via tyrosine kinase (the Ick kinase) also.

Table 2. Cell surface proteins that play an important role in the immune response.¹

Type of Cells	Surface Proteins
Helper T cells	CD4, TCR.^2 , CD28
Cytotoxic T cells	CD8, TCR
B cells	IgM, B7
Macrophages^3	Class II MHC
Natural killer cells	Receptors for class 1 MHC
All cells other than mature red cells	Class 1 MHC

There are many other cell surface proteins that play a role in the immune response, but the proteins listed in this table are the most important for understanding the fundamental aspects of this response; ²TCR, T cell antigen receptor; ³Macrophages and other "antigen-presenting cells."

T cells are subdivided into two major categories on the basis of whether they have CD4 or CD8 proteins on their surface. Mature T cells have either CD4 or CD8 proteins but not both.

CD4 lymphocytes perform the following helper functions: (1) they help B cells develop into antibody-producing plasma cells; (2) they help CD8 T cells to become activated cytotoxic T cells; and (3) they help macrophages effect delayed hypersensitivity (eg, limit infection by M tuberculosis). These functions are performed by 2 subpopulations of CD4 cells: Th-1 cells help activate cytotoxic T cells by producing IL-2 and help initiate the delayed hypersensitivity response by producing primarily IL-2 and gamma interferon, whereas Th-2 cells perform the B cell helper function by producing primarily IL-4 and IL-5 (Figure 3). One important regulator of the balance between Th-l cells and Th-2 cells is interleukin-12 (IL-12), which is produced by macrophages. IL-12 increases the number of Th-1 cells, thereby enhancing host defenses against organisms that are controlled by a delayed hypersensitivity response (Table 4). Another important regulator is gamma interferon which inhibits the production of Th-2 cells. CD4 cells make up about 65% of peripheral T cells and predominate in the thymic medulla, tonsils, and blood.

To mount a protective immune response against a specific microbe requires that the appropriate subpopulation, ie, either Th-1 or Th-2 cells, play a dominant role in the response. For example, if an individual is infected with M tuberculosis and Th-2 cells are the major responders, then humoral immunity will be stimulated rather than cell-mediated immunity.

Main functions of helper T cells

Table 3

Main Functions	Cytokine That Mediates That Function
Activates the antigen-specific helper T cell to produce a clone of these cells	IL-2
Activates cytotoxic T cells	IL-2
Activates B cells	IL-4 and IL-5
Activates macrophages	Gamma-interferon

Humoral immunity is not protective against M tuberculosis and the patient will suffer severe tuberculosis. Similarly, if an individual is infected with Streptococcus pneumoniae and Th-1 cells are the major responders, then humoral immunity will be not be stimulated and the patient will have severe pneumococcal disease. Precisely what component of a microbe activates either Th-1 or Th-2 cells is unknown.

Comparison of Th-1 cells and Th-2 cells

Table 4.

Property	Th-1 Cells	Th-2 Cells
Produces IL-2 and gamma interferon	Yes	No
Produces IL-4, IL-5, IL-6, and IL-10	No	Yes
Enhances cell-mediated immunity and delayed hypersensitivity primarily	Yes	No
Enhances antibody production primarily	No	Yes
Stimulated by IL-12	Yes	No
Stimulated by IL-4	No	Yes

CD8 lymphocytes perform cytotoxic functions; that is, they kill virus-infected, tumor, and allograft cells. They kill by either of two mechanisms, namely, the release of performs, which destroy cell membranes, or the induction of programmed cell death (apoptosis). CD8 cells predominate in human bone marrow and gut lymphoid tissue and constitute about 35% of peripheral T cells,

Activation of T Cells. The activation of helper T cells requires that they recognize a complex on the surface of antigen-presenting cells (APCs), eg, macrophages consisting of both the antigen and a class II MHC protein (Macrophages are the most important antigen-presenting cells, but B cells, dendritic cells in the spleen, and Langer-hans cells on the skin also present antigen, ic, have class II proteins on their surface: An essential first step for certain antigoH-prosnilinj; cells, eg. Umgcrhans cells ui the skin, is migration from the site of the skin infection to ihe local lymphoid tissue, where helper T cells are encountered). Within the cytoplasm of the macrophage, the foreign protein is cleaved into small peptides that associate with the class II MHC proteins. The complex is transported to the surface of the macrophage, where the antigen, in association with a class II MHC protein, is presented to the receptor on the CD4-positive helper cell. Similar events occur within a virus-infected cell, except that the cleaved viral peptide associates with a class I rather than a class II MHC protein. The complex is transported to the surface, where the viral antigen is presented to the receptor on a CD8-positive cytotoxic cell. Remember the rule, of eight: CD4 cells interact with class II (4 x 2 = 8), and CD8 cells interact with class I (8 x 1 = 8).

There are many different alleles within the class I and class II MHC genes; hence, there are many different MHC proteins. These various MHC proteins bind to different peptide fragnients. The polymorphism of the MHC genes and the proteins they encode are a means of presenting many different antigens to the T cell receptor. Note that class I and class IIMHC proteins can only present peptides; other types of molecules do not bind and therefore cannot be presented. MHC proteins can present peptides derived from self proteins as well as from foreign proteins; therefore whether an immune response occurs is determined by whether a T cell bearing a receptor specific for that peptide has survived the positive, and negative selection processes in the thymus.

The first step in the activation process is the interaction of the antigen with the T cell receptor specific for that antigen (Figure 4). IL-1 produced by the macrophage is also necessary for efficient helper T cell activation. Note that when the T cell receptor interacts with the antigen-MHC protein complex, the CD4 protein on the surface of the helper T cell also interacts with the class II MHC protein. In addition to the binding of the CD4 protein with the MHC class II protein, other proteins interact to help stabilize the contact between the T cell and the APC; eg, LFA-1 protein on T cells (both CD4-positive and CDS-positive) binds to ICAM-1 protein on APCs (LFA proteins belong to a family of cell surface proteins called integrins, which mediate adhesion to other cells. Inte-grin proteins are embedded in the surface membrane and have both extracellular and intracellular domains. Hence they interact with other cells externally and with the cytoplasm internallyAbbreviations: LFA, lymphocyte function-associated antigen; 1CAM, intercellular adhesion molecule).

For full activation of helper T cells, an additional "costimulatory" signal is required; that is, B7 protein on the APC must interact with CD28 protein on the helper T cell (Figure 4). If the costimulatory signal occurs, IL-2 is made by the helper T cell, and it is this step that is crucial to producing a helper T cell capable of performing its regulatory, effector, and memory functions. If, on the other hand, the T cell receptor interacts with its antigen (epitope) and the costimulatory signal does not occur, a state of unresponsiveness called anergy ensues. The anergic state is specific for that epitope, since other helper T cells specific for other epitopes are not affected. After the T cell has been activated, a different protein called CTLA-4 appears on the T cell surface and binds to B7 by displacing CD-28.

The interaction of B7 with CTLA-4 inhibits T cell activation by blocking IL-2 synthesis. This restores the activated T cell to a quiescent state and thereby plays an important role in T cell homeostasis. Mutant T cells that lack CTLA-4 and therefore cannot be deactivated participate with increased frequency in autoimmune reactions. Furthermore, administration of CTLA-4 reduced the rejection of organ transplants in experimental animals.

T cells recognize only polypeptide antigens. Furthermore, they recognize those polypeptides only when they are presented in association with MHC proteins. Helper T cells recognize antigen in association with class II MHC proteins, whereas cytotoxic T cells recognize antigen in association with class 1 MHC proteins. This is called MHC restriction; ie, the two types of T cells (CD4 helper and CD8 cytotoxic) are "restricted" because they are able to recognize antigen only when the antigen is presented with the proper class of MHC protein. This restriction is mediated by specific binding sites primarily on the T cell receptor, but also on the CD4 and CD8 proteins that bind to specific regions on the class II and class I MHC proteins, respectively.

Generally speaking, class I MHC proteins present endogenously synthesized antigens, eg, viral proteins, whereas class II MHC proteins present the antigens of extracellular microorganisms that have been phagocyti/cd, eg, bacterial proteins. One important consequence of these observations is that killed viral vaccines do not activate the cytotoxic (CD8-positive) T cells, because the virus does not replicate within cells and therefore viral epitopes are not presented in association with class I MHC proteins.

This distinction between endogenously synthesized and.extracellularly acquired proteins is achieved by processing the proteins in different compartments within the cytoplasm. The endogenously synthesized proteins, eg, viral proteins, are cleaved by a proteasome, and the peptide fragments associate with a 'TAP transporter^” that transports the fragment into the rough endoplasmic reticulum, where it associates with the class I MHC protein. The complex of peptide fragment and class I MHC protein then migrates via the Golgi apparatus to the cell surface. In contrast, the extracellularly acquired proteins are cleaved to peptide fragments within an endosome, where the fragment associates with class II MHC proteins. This complex then migrates to the cell surface.

An additional protection that prevents endogenously synthesized proteins from associating with class II MHC proteins is the presence of an "invariant chain" that is attached to the class II MHC proteins when these proteins are outside of the endosome. The invariant chain is degraded by proteases within the endosome, allowing the peptide fragment to attach to the class II MHC proteins only within that compartment.

B cells, on the other hand, can interact directly with antigens via their surface immunoglobulins (IgM and IgD). Antigens do not have to be presented to B cells in association with class II MHC proteins. unlike T cells. Note that B cells can then present the antigen, after internalization and processing, to helper T cells in association with class II MHC proteins located on the surface of the B cells (see the section on B cells, below). Unlike the antigen receptor on T cells, which recognizes only peptides, the antigen receptors on B cells (IgM and IgD) recognize many different types of molecules, such as peptides, polysaccharides, nucleic acids, and small chemicals, eg, penicillin.

These differences between T cells and B cells explain the hapten-carrier relationship described. To stimulate hapten-specific antibody, the hapten must be covalently bound to the carrier protein. A B cell specific for the hapten internalizes the hapten-carrier conjugate, processes the carrier protein, and presents a peptide to a helper T cell bearing a receptor for that peptide. The helper T cell then secretes lymphokines that activate the B cell to produce antibodies to the hapten.

When the antigen-MHC protein complex on the APC interacts with the T cell receptor, a signal is transmitted by the CD3 protein complex through several pathways that eventually lead to a large influx of calcium into the cell. (The details of the signal transduction pathway are beyond the scope of this book, but it is known that stimulation of the T cell receptor activates a series of phosphokinases which then activate phospholipase C, which cleaves phosphoinositide to produce inositol triphosphate, which opens the calcium channels.) Calcium activates calcineurin, a serine phosphatase. Calcineurin moves to the nucleus and is involved in the activation of the genes for IL-2 and the IL-2 receptor. (Calcineurin function is blocked by cyclosporine, one of the most effective drugs used to prevent rejection of organ transplants.)

The end result of this series of events is the activation of the helper T cell to produce various lymphokines, eg, IL-2, as well as the IL-2 receptor. IL-2, also known as T cell growth factor, stimulates the helper T cell to multiply into a clone of antigen-specific helper T cells. Most cells of this clone perform effector and regulatory functions, but some become "memory" cells (see below), which are capable of being rapidly activated upon exposure to antigen at a later time. (Cytotoxic T cells and B cells also form memory cells.) Note that IL-2 stimulates CD8 cytotoxic T cells as well as CD4 helper T cells. Activated CD4-positive T cells also produce another lymphokine called gamma interferon, which increases the expression of class II MHC proteins on APCs. This enhances the ability of APCs to present antigen to T cells and upregulates the immune response.

The process of activating T cells does not function as a simply "on-off'” switch.The binding

of an epitope to the T cell receptor can result in full activation, partial activation in" which only certain lymphokines are made, or no activation, depending on which of the signal transduction pathways is stimulated by that particular epitope. This important observation may have profound implications .for our understanding of how helper T cells shape our response to infectious agents.

There are three genes at the class I locus (A, B, and C) and three genes at the class II locus (DP, DQ, and DR). We inherit one set of class I and one set of class II genes from each parent. Therefore, our cells can express as many as six different class I and six different class II proteins. Furthermore, there are multiple alleles at each gene locus. Each of these MHC proteins can present peptides with a different amino acid sequence. This explains, in part, our ability to respond to many different antigens.

Memory T Cells. Memory T (and B) cells, as the name implies, endow our host defenses with the ability to respond rapidly and vigorously for many years after the initial exposure to a microbe or other foreign material. This memory response to a specific antigen is due to several features: (1) many memory cells are produced, so that the secondary response is greater than the primary response, in which very few cells respond; (2) memory cells live for many years or have the capacity to reproduce themselves; (3) memory cells are activated by smaller amounts of antigen and require less costimulation than do naive, unactivated T cells; and (4) activated memory cells produce greater amounts of interleukins than do naive T cells when they are first activated.

T Cell Receptor. The T cell receptor (TCR) for antigen consists of two polypeptides, alpha and beta ( Some TCRs have a different set of polypeptides called gamma and delta. Some of the T cells bearing these TCRs are involved in cell-mediated immunity against M tuberculosis),which are associated with CD3 proteins. TCR proteins are similar to immunoglobulin heavy chains in that (1) the genes that code for them are formed by rearrangement 6f multiple regions of DNA; (2) there are V (variable), D (diversity), J (joining), and C (constant) segments that rearrange to provide diversity, giving rise to an estimated number of more than 10⁷ different receptor proteins; and (3) the two genes (RAG-1 and RAG-2) that encode die recombinase enzymes that catalyze these gene rearrangements are similar in T cells and 8 cells.

Note that each T cell has a unique T cell receptor on its surface, which means that hundreds of millions of different T cells exist in each person. Activated T cells, like activated B cells, clonally expand to yield large numbers of cells specific for that antigen.

Although TCRs and immunoglobulins are analogous in that they both interact with antigen in a highly specific manner, the T cell receptor is different in two important ways: (1) it has two chains rather than four, and (2) it recognizes antigen only, in conjunction with MMC proteins, whereas immunoglobulins recognize free antigen.

Effect Of Superantigens on T Cells. Certain proteins, particularly staphylococcal enterotoxins and toxic shock syndrome toxin, act as "superantigens'¹ (Figure 5). In contrast to the usual antigen, which activates one (or a few) helper T cell, superantigens activate a large number of helper T cells. For example, toxic shock syndrome toxin binds directly to class II MHC proteins without internal processing of the toxin. This complex interacts with the variable portion of the beta chain (Vβ) of the T cell receptor of many T cells . This activates the T cells, causing the release of IL-2 from the T cells and IL-1 from macrophages. These interleukins account for many of the findings seen in toxin-mediated staphylococcal diseases. Certain viral proteins, eg, those of mouse mammary tumor virus (a retrovirus), also possess superantigen activity.

Features of T Cells. T cells constitute 65-80% of the recirculating poof of small lymphocytes. Within lymph nodes, they are located in the inner, subcortical region, not in the germinal centers. (B cells iruike up most of the remainder of the pool of small lymphocytes and are found primarily in the germinal centers of lymph nodes.) The life span of T cells is long: months or years. They can be stimulated to divide when exposed to certain mitogens, eg/phytohemagglutinin or concanavalin A (endotoxin, a lipopolysaccharide found on the surface of gram-negative bacteria, is a mitogen for B cells but not T cells). Most human T cells have receptors for sheep erythrocytes on their surface«and can form "rosettes" with them; this finding serves as a means of identifying T cells in a mixed population of cells.

Effector Functions of T Cells. There are two important components of host defenses mediated by T cells: delayed hypersensitivity and cytotoxicity.

A. Delayed Hypersensitivity: Delayed hypersensitivity reactions are produced particularly against antigens of intracellular microorganisms including certain fungi, eg, Histoplasma and Coccidioides, and certain intracellular bacteria, eg, mycobacteria. Delayed hypersensitivity is mediated by macrophages and CD4 cells, in particular by the Th-1 subset of CD4 cells. Important lynv phokines for Ihese reactions fnclude gamma interferon, macrophage activation factor, and macrophage" migration inhibition factor. CD4 cells, produce the interleukins, and macrophages are the ultimate effectors of delayed hypersensitivity. A deficiency of cell-mediated immunity manifests itself as a marked susceptibility to infection by such microorganisms.

B. Cytotoxicity: The cytotoxic response is concerned primarily with destroying virus-infected cells and tumor cells but also plays an important role in graft rejection. In response to virus-infected cells, the CD8 lymphocytes must recognize both viral antigens and class I molecules on the surface of infected cells. To kill the virus-infected cell, the cytotoxic T cell must also receive a cy-tokine stimulus from a helper T cell. To become activated to produce these cytokincs, helper T cells rccogni/e viial anligcns bound to class II molecules on an APC, eg, a macrophage. The activated helper T cells secrete cytokines such as IL-2, which stimulates the virus-specific cytotoxic T cell to form a clone of activated cytotoxic T cells. These cytotoxic. T cells kill the virus-infected cells by inserting "perforins" and degradative enzymes called granzymes into the infected cell. Performs form a channel through the membrane, the cell contents are lost, and the cell dies. Granzymes are proteases that degrade proteins in the cell membrane, which also leads to the loss of cell contents. They also trigger apoptosis, leading to cell death. After killing the virus-infected cell, the cytotoxic T cell itself is not damaged and can continue to kill other cells infected with the same virus. Cytotoxic T cells have no effect on free virus, only on virus-infected cells.

A third mechanism by which cytotoxic T cells kill target cells is the Fas-Fas ligand (FasL) interaction. Fas is a protein displayed on the surface of many cells. When a cytotoxic T cell receptor recognizes an epitope on the surface of a target cell, FasL is induced in the cytotoxic T cell. When Fas and FasL interact, apoptosis (death) of the target cell occurs. NK cells can also kill target cells by Fas-FasL-induced apoptosis.

In addition to direct killing by cytotoxic T cells, virus-infected cells can be destroyed by a combination of IgG and phagocytic cells. In this process, called antibody-dependent cellular cytotoxicity (ADCC), antibody bound to the surface of the infected cell is recognized by IgG receptors on the surface of phagocytic cells, eg, macrophages or NK cells, and the infected cell is killed. The ADCC process can also kill helminths (wormsjjk In this case, IgE is the antibody involved and eosinophils are the effector cells. IgE binds to surface proteins on the worm, and the surface of eosinophils displays receptors for the epsilon heavy chain. The major basic protein located in the granules of the eosinophils is released and damages the surface of the worm.

Many tumor cells develop new antigens on their surface. These antigens bound to class I proteins are recognized by cytotoxic T cells, which are stimulated to proliferate by IL-2. The resultant clone of cytotoxic T cells can kill the tumor cells, a phenomenon called immune surveillance.

In response to allografts, cytotoxic (CD8) cells recognize the class I MHC molecules on the surface of the foreign cells. Helper (CD4) cells rccognize the foreign class II molecules on certain cells in the graft, eg, macrophages and lymphocytes. The activated helper cells secrete IL-2, which stimulates the cytotoxic cell to form a clone of cells. These cytotoxic cells kill the cells in the allograft.

Regulatory Functions of T Cells. T cells play a central role in regulating both the humoral (antibody) and cell-mediated arms of the immune system.

A. Antibody Production: Antibody production by B cells usually requires the participation of helper T cells (T cell-dependent response), but antibodies to some antigens, eg, polymerized (multivalent) macromolecules such as bacterial capsular polysaccharide, are T cell-independent. These polysaccharides are long chains consisting of repeated subunits of several sugars. The repeated subunits act as a multivalent antigen that cross-links the IgM antigen receptors on the B cell and activates it in the absence of help from CD4 cells. Other macromolecules, such as DNA, RNA, and many lipids, also elicit a T cell-independent response.

In the following example illustrating the T cell-dependent response, B cells are used as the APC, although macrophages commonly perform this function. In this instance, antigen binds to surface IgM or IgD, is internalized within the B cell, and is fragmented. Some of the fragments return to-the surface in association with class II MHC molecules (Figure 6) (Note that one important difference between B cells and T cells is that B cells recognise antigen itself, whereas T cell's recognize antigen onty in association with MHC proteins.)

These interact with the receptor on the helper T cell, and, if the costimulatory signal is given by the B7 protein on the B cell interacting with CD28 protein on the helper T cell, the helper T cell is then stimulated to produce lymphokines, eg, IL-2, B cell growth factor (IL-4), and B cell differentiation factor (IL-8). IL-4 and IL-5 induce "class switching" from IgM, which is the first class of immunoglobulins produced, to other classes, namely, IgG, IgA, and IgE. These factors stimulate the B cell to divide and differentiate into many antibody-producing plasma cells.

Note that interleukins alone are not sufficient to activate B cells. A membrane protein on activated helper T cells, called CD40 ligand (CD40L), must interact with a protein called CD40 on the surface of the resting B cells to stimulate the differentiation of B cells into antibody-producing plasma-cells (Figure 6B). Furthermore, other proteins on the surface of these cells serve to strengthen the interaction between the helper T cell and the antigen-presenting B cell; eg, CD28 on the T cell interacts with B7 on the B cell and LFA-1 on the T cell interacts with ICAM-1 on the B cell. (There are also ICAM proteins on the T cell that interact with LFA proteins on the B cell.)

In the T cell-dependent response, all classes of antibody are made (IgG, IgM, IgA, etc), whereas in the T cell-independent response, primarily IgM is made. This indicates that lym-phokine's produced by the helper T cell are needed for class switching. The T cell-dependent response generates memory B cells, whereas the T cell-dependent response does not; therefore, a secondary antibody response does not occur in the latter. The T cell-independent response is the main response to bacterial capsular polysaccharides, because these molecules are not effectively processed and presented by APCs and hence do not activate helper T cells. The most likely reason for this is that polysaccharides do not bind to class II MHC proteins whereas peptide antigens do.

B CELLS

B cells perform two important functions; (1) They differentiate into plasma cells and produce antibodies, and (2) they are antigen-presenting cells (APCs).

Origin. During embryogcncsis, B cell precursors are recognized first in the fetal liver. From there they migrate to the bone marrow, which is their main location during adult life. Unlike T cells, they do not require the thymus for maturation. Pre-B cells lack surface immunoglobulins and light chains but do have μ heavy chains in the cytoplasm. The maturation of B cells has two phases: the antigen-independent phase consists of stem cells, pre-B cells, and B.cells, whereas the antigen-dependent phase consists of the cells that arise subsequent to the interaction of antigen with the B cells, eg, activated B cells and plasma cells. B cells display surface IgM, which serves as a receptor for antigens. This surface IgM is a monomer, in contrast to circulating IgM, which is a pentamer. Surface IgD on some B cells may also be an antigen receptor. Pre-B cells are found in the bone marrow, whereas B cells circulate in the blood stream.

B cells constitute about 30% of the recirculating pool of small lymphocytes, and their life span is short, ie. days or weeks. Approximately 10⁹ B cells are produced each day. Within lymph nodes, they are located in germinal centers; within the spleen, they are found in the white pulp. They are also found in the gut-associated lymphoid tissue, eg, Peyer's patches.

Clonal Selection. How do antibodies arise? Does the antigen "instruct" the B cell to make an antibody, or does the antigen "select" a B cell endowed with the preexisting capacity to make the antibody?

It appears that the latter alternative, ie, clonal selection, accounts for antibody formation. Each individual has a large pool of B lymphocytes (about 10⁷). Each immunologically responsive B cell bears a surface receptor (either IgM or IgD) that can react with one antigen (or closely related group of antigens); ie, there are about l0⁷ different specificities. An antigen interacts with the B lymphocyte that shows the best "lit" with its immunoglobulin surface receptor. After the antigen binds, the B cell is stimulated to proliferate and form a clone of cells. These selected B cells soon become plasma cells and secrete antibody specific for the antigen. Plasma cells synthesize the immunoglobulins with the same antigenic specificity (ie, they have the same heavy chain and the same light chain) as those carried by the selected B cell. Antigenic specificity does not change when heavy-chain class switching occurs.

Note that clonal selection also occurs with T cells. The antigen interacts with a specific receptor located on the surface of either a CD4-positive or a CD8-positive T cell. This "selects" this cell and activates it to expand into a clone of cells with the same specificity.

Activation of B Cells. In the following example, .the B cell is the APC. Multivalent antigen binds to surface IgM (or IgD) and cross-links adjacent immunoglobulin molecules. The immunoglobulins aggregate to form "patches" and eventually migrate to one pole of the cell to form a cap. Endocytosis of the capped material follows, the antigen is processed, and epitopes appear on the surface in conjunction with class II MHC proteins. This complex is recognized by a helper T cell with a receptor for the antigen on its surface.

(Macrophages bearing antigen bound to class II MHC proteins can also present antigen to the T cell, resulting in antibody formation. In general, B cells are poor activators of "virgin" T cells, in the primary response because B cells do not make IL-1. B cells are, however, very good activators of memory T cells because little, if any, IL-I is needed). The T cell now produces various lymphokines (IL-2, IL-3, and IL-5) that stimulate the growth and differentiation of the B cell.

The activation of B cells to produce the full range of antibodies requires two other interactions in addition to recognition of the epitope by the T cell antigen receptor and the production of IL-4 and IL-5 by the helper T cell. These costimulatory interactions which occur between surface proteins on the T and B cells, are as follows: (1).CD28 on the T cell must interact with B7 on the B cell, and (2) CD40L on the T cell must interact with CD40 on the B cell. The CD28-B7 interaction is required for activation of the T cell to produce IL-2, and the CD40L-CD40 interaction is required for class switching from IgM to IgG and other immunoglobulin classes to occur.

Many plasma cells that produce large amounts of immunoglobulins specific for the epitope are the end result. Plasma cells secrete thousands of antibody molecules per second for a few days and then die. Some activated B cells form memory cells, which can remain quiescent for long pounds but are capable of being activated rapidly upon reexposure to antigen. Most memory B cells have surface IgG that serves as the antigen receptor, but some have IgM. Memory T cells secrete interleukins that enhance antibody production by the memory B cells. The presence of these cells explains the rapid appearance of antibody in the secondary response.

Lymphocytes:

• Lymphocytes are differentiated into B lymphocytes (B cells), T lymphocytes (T cells), natural killer cells (NK cells), and killer cells (K cells); all have different roles in the immune defenses of the body.

• Lymphocytes are differentiated based on the presence of specific cell surface proteins or antigens bound to their plasma membranes.

• A surface marker that identifies a specific line of cells or a stage of cell differentiation because it interacts with a group or cluster of individual antibodies is called a CD (cluster of differentiation) antigen.

• B lymphocytes (B cells), which are differentiated in bone marrow, give rise to plasma cells when stimulated by antigen.

• Plasma cells produce and release antibodies. Each clone of a plasma cell line secretes a single specific antibody.

• T lymphocytes are differentiated in the thymus.

• Cytotoxic T cells (CD8⁺ cells) lyse abnormal cells.

• Helper T cells (CD4⁺ cells) activate the immune response.

• Suppressor T cells (CD8⁺ cells) regulate the immune response.

• Natural killer or null cells are a special subset of lymphocytes that are neither T nor B cells.

• Antigen-presenting cells (APCs) include macrophages and other specialized cells (dendritic cells) in skin and lymphoid organs. APCs help activate the immune response. APCs bind antigen on their surfaces in association with a special protein called the major histocompatibility complex (MHC).

• B cells that have processed antigens become antigenpresenting cells that activate the immune response.

• T cells respond to antigens associated with MHC on antigen-presenting cells.

Antibody-mediated Immunity:

• Antibody-mediated immunity is important in preventing and eliminating microbial infections.

• In the antibody-mediated immune response, specific glycoproteins called antibodies or immunoglobulins are made when foreign antigens activate B cells.

• The key to antibody immunity is the ability of antibodies to react specifically with antigens.

• The activation of a particular B cell leads to clonal selection and expansion of B cells specific to an individual antigen.

Cell-mediated Immunity:

• The cell-mediated immune response depends on natural killer cells and various T lymphocytes.

• T cells recognize antigens by specific surface molecules called T cell receptors (TCRs).

• T cells produce cytokines when they are stimulated.

• Cytotoxic and aggressor T cells detect abnormal cells by antigens on tissue cell surface. These antigens are called major histocompatibility complex antigens. These MHC antigens are important.

Immunoglobulins. Characteristics of main Immunoglobulins classes. Cells cooperation in immune response. Mechanism of antibacterial and antiviral immunity.

Antibodies (Immunoglobulins)

Antibodies are specific substances produced in the bodies of vertebrates upon the introduction of antigens; they are capable of a specific association with them. As a result of an obvious or latent infectious disease antibodies appear in the sera of animals and man. Antibodies appear not only as a result of an infection, but also due to immunization by live (attenuated) or dead bacteria, rickettsiae, viruses, toxins and other antigenic substances.

Antibodies which occur under the influence of active immunization are named immune antibodies in contrast to normal antibodies which are often found in the sera of man and animals who have not had infectious diseases and who have not been exposed to immunization. It is believed that they form under the effect of normal microflora during the development of the organism.

Any normal serum contains a small amount of cryogglutinins (cold haemagglutinins) which react with the erythrocytes of the same person. When cryogglutinins appear in a large number, paroxysmal haemoglobinuria develops. Normal as well as immune antibodies are capable of rendering harmless the causative agents of infectious diseases in various ways.

Young poisonous snakes of some species are devoid of antibodies, as a consequence of which they are sensitive to their own poison; adult snakes have antibodies which neutralize their own poison.

Immunoglobulins are synthesized by immunocytes which form from B-lymphocytes. The sources of immunocytes are the stem cells of the haematopoietic tissue, which acquire immunologic activity due to the effect of the thymus. Antigens come into contact with the cells continuously migrating from the thymus (T-cells) and bone marrow (B-cells) into the lymph nodes and spleen where with the participation of the macrophages clonal immunocytes are formed. The immune process is therefore realized as the result of cooperation of three groups of cells: T- and B-lymphocytes and macrophages which phagocytose and digest the antigens and transmit the information concerning the antigen to the corresponding immunocom petent cells producing immunoglobulins.

IMMUNOGLOBUL1N STRUCTURE

Immunoglobulins are glycoproteins made up of light (L) and heavy (H) polypeptide chains. The terms "light" and heavy" refer to molecular weight; light chains have a molecular weight of about 25,000, whereas heavy chains have a molecular weight of 50,000-70,000. The simplest antibody molecule has a Y shape (Figure 2) and consists of four polypeptide chains: two H chains and two L chains. The four chains are linked by disulfide bonds. An individual antibody molecule always consists of identical H chains and identical L chains. This is primarily the result of two phenomena: allelic exclusion and regulation within the B cell, which ensure the synthesis of either kappa (κ) or lambda (λ) L chains but not both.

L and H chains are subdivided into variable and constant regions. The regions are composed of three-dimensionally folded, repeating segments called domains. An L chain consists of one variable (VL) and one constant (CL) domain. Most H chains consist of one variable (VH) and three constant (CH) domains. (IgG and IgA have three CH domains, whereas IgM and IgE have four.) Each domain is approximately 110 amino acids long. The variable regions are responsible for antigen-binding, whereas the constant regions are responsible for various biologic functions, eg, complement activation and binding to cell surface receptors. Carboxy terminal end.

Figure 2. Structure of IgG The Y-shaped IgG molecule consists of two tight chains-and-two heavy chains. Each light chain consists of a variable region and a constant region. Each heavy chain consists of a variable region and a constant region that is divided into three domains: CH1, CH2, and CH3. The CH2 domain contains the complement-binding site, and the CH3 domain is the site of attachment of IgG to receptors on neutrophils and macrophages. The antigen-binding site is formed by the variable regions of both the light and heavy chains. The specificity of the antigen-binding site is a function of the amino acid sequence of the hypervariable regions.

The variable regions of both L and H chains have three extremely variable ("hypervariable") amino acid sequences at the amino-terminal end that form the antigen-binding site. Only 5-10 amino acids in each hypervariable region form the antigen-binding site. Antigen-antibody binding involves electrostatic and van der Waals' forces and hydrogen and hydrophobic bonds rather than covalent bonds. The remarkable specificity of antibodies is due to these hypervariable regions.

L chains belong to one of two types, k (kappa) or λ (lambda), on the basis of amino acid differences in their constant regions. Both types occur in all classes of immunoglobulins (IgG, IgM, etc), but any one immunoglobulin molecule contains only one type of L chain. The ammo-tion of each L chain participates in the antigen-binding site. H chains are distinct for each of the five immunoglobulin classes and are designated y, a, ji, e, and 8 (Table 1). The amino-terminal portion of each H chain participates in the antigen-binding site; die carboxy terminal forms the Fc fragment, which has the biologic activities described above and in Table 1.

If an antibody molecule is treated with a proteolytic enzyme such as papain, peptide bonds in the "hinge" region are broken, producing two identical Fab fragments, which carry the antigen-binding sites, and one Fc fragment, which is involved in placenta! transfer, complement fixation, attachment site for various cells, and other biologic activities (Figure 2).

Properties of immunoglobulins

IMMUNOGLOBULIN CLASSES

Each IgG molecule consists of two L chains and two H chains linked by disulfide bonds (molecular formula H2L2). Because it has two identical antigen-binding sites, it is said to be divalent. There are four subclasses, IgGl-IgG4, based on antigenic differences in the H chains and on the number and location of disulfide bonds. IgGl makes up most (65%) of the total IgG. IgG2 antibody is directed against polysaccharide antigens and is an important host defense against encapsulated bacteria.

IgG is the predominant antibody in the secondary-response and constitutes an important defense against bacteria and viruses (Table 2). IgG is the only antibody to cross the placenta only its Fc portion binds to receptors on the surface of placenta! cells. It is therefore the most abundant un-munoglobulin in newborn. IgG is one of the two immunoglobulins that can activate complement; IgM is the other.

IgG is the immunoglobulin that opsonizes. It can opsonize, ie, enhance phagocytosis, because there are receptors for the y H chain on the surface of phagocytes. IgM does not opsonize directly, because 4here are no receptors on the phagocyte surface for the ji H chain. However, IgM activates complement, and the resulting C3b can opsonize because there are binding sites for C3b on the surface of phagocytes.

IgA. IgA is the main immunoglobulin in secretions such as colostrum, saliva, tears, and respiratory, intestinal, and genital tract secretions. It prevents attachment of microorganiimi, eg, bacteria and viruses, to mucous membranes. Each secretory IgA molecule consists of two H2L2 units plus one molecule each of J (joining) chain and secretory component (Figure 3). The secretory component is a polypeptide synthesized by epithelial cells that provides for IgA passage to the mucosal surface. It also prelects IgA from being degraded in the intestinal tract. In serum, some IgA exists as monomeric H2L2.

IgM IgM is the main immunoglobulin produced early in the primary response. It is present as a monomer on the surface of virtually all B cells, where it functions as an antigen-binding receptor In serum, it is a pentamer composed of 5 H2L2 units plus one molecule of J (joining) chain (Figure 3). Because the pentamer has 10 antigen-binding sites, it is the most efficient immunoglobulin in agglutination, complement fixation (activation), and other antibody reactions and is important in defense against bacteria and viruses. It can be produced by the fetus in certain infections. It has the highest avidity of the immunoglobulins; its interaction with antigen can involve all 10 of its binding sites.

IgD This immunoglpbulin has no known antibody function but may function as an antigen receptor; it is present on the surface of many B lymphocytes. It is present in small amounts in serum.

IgE. IgE is medically important for two reasons: (1) it mediates immediate (anaphylactic) hypersensitivity, and (2) it participates in host defenses against certain parasites, eg, helminths (worms). The Fc region of IgE binds to the surface of mast cells and basophils. Bound IgE serves as a receptor for antigen (allergen), and this antigen-antibody complex triggers allergic responses of the immediate (anaphylactic) type through the release of mediators. Although IgE is present in trace amounts in normal serum (approximately 0.004%), persons with allergic reactivity have greatly increased amounts, and IgE may appear in external secretions. IgE does not fix complement and does not cross the placenta.

IgE is the main host defense against certain important helminth (worm)infections, such as Strongyloides, Trichinella, Ascaris, and the hookworms. The serum IgE level is usually increased in these infections. Because worms are too large to be ingested by phagocytes, they are killed by eosinophils that release worm-destroying enzymes. IgE specific for worm proteins binds to receptors on eosinophils, triggering the antibody-dependent cellular cytotoxicity (ADCC) response.

Immunoglobulin	Major Functions
IgG	Main antibody in the secondary response. Opsonizes bacteria, making them easier to phagocytize. Fixes complement, which enhances bacterial killing. Neutralizes bacterial toxins and viruses. Crosses the placenta.
IgA	Secretory IgA prevents attachment of bacteria and viruses to mucous membranes Does not fix complement.
IgM	Produced in the primary response to an antigen. Fixes complement. Does not cross the placenta. Antigen receptor on the surface of B cells.
IgD	Uncertain. Found on the surface of many B cells as well as in serum.
IgE	Mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to antigen (allergen) Defends against worm infections by causing release of enzymes. frorn eosinophils. Does not fix complement. Main host defense against helminth infections.

The immune response and tolerance

The generation and control of immune responses are a direct consequence of a complex series of antigen-mediated interactions between various cell types (Fig.1).

FIGURE 1. Induction and regulation of immune responses. Lymphocytes recognize antigens with antigen-specific receptors on their plasma membranes. Antigen recognition activates the lymphocytes, resulting in proliferation and expression of effector functions. Helper T (Th) lymphocytes produce a variety of cytokines that function as growth and differentiation factors for themselves, T-cytotoxic (Tc) cells, and B cells. In this scheme, the Th cells are given the role of immunoregulatory cells, which both augment and suppress T- and B-cell responses; Tc cells and B cells are likely to regulate Th cell function by secreting immunomodulatory cytokines as well. APC, antigen-presenting cell.

In the positive sense, these responses follow the interaction between an effector cell (eg, T-cytotoxic |Tc| cells or B cells) and a positive regulatory cell, the helper T (Th) cell. This cell-to-cell intercommunication results in either antibody production (Th—B interaction), delayed-type hypersensitivity (Th-macrophage interaction), or cell-mediated lympholysis (Th-Tc interaction). This cell-cell interaction results in the proliferation and differentiation of the effector cell and is mediated by a variety of soluble factors (monokines or lymphokines) secreted primarily by T cells and macrophages (Table).

TABLE . Monokines and Lymphokines Involved in Regulation of Immune Responses

Factor	Other Name(s)	Source	Properties	Effect on Human Cells
IL-1	LAF	Macrophages T cells B cells Epithelial cells	15-17 kd	Promotes early B-cell differentiation
IL-2	TCGF	Activated T cells	15 kd	Promotes proliferation of B and T cells
IL-3	Multi-CSF	Activated T cells	34 kd	Induces growth of hemopoietic cells
IL-4	BCGF-1 BCDF e BCDF-y BSF-I	Activated T cells	20 kd	Promotes IgE production, induces increased FceR on B cells and mast cells
IL-5	BCGF-II	Activated T cells	30-60 kd	Promotes IgM and IgA production, promotes eosinophil differentiation
IL-6	BSF-II HGF	Monocytes Fibroblasts T cells	20 kd	Promotes growth of plasma cells, promotes Ig secretion
IFN-γ		T cells NK cells	20-25 kd	Induces class II expression on cells, promotes antimicrobial activity of macrophages, promotes NK cell activity, induces FcyR expression on monocytic cells, with IL-4 promotes IgG production
IL-1O	CSIF	T cells B cells Macrophages Keratinocytes	17-21 kd	Inhibits cytokine production by Thl cells, NK cells, and macrophages; stimulates B-cell proliferation and differentiation
BCDF, B-ce1l differentiation factor; BCGF, B-cell growth factor; CSF, colony-stimulating factor; CSIF, cytokine synthesis inhibitory factor; HGF, hepatocyte growth factor; IFN-γ, interferon-gamma; 1L, interleukin; LAF, leukocyte activating factor; NK, natural killer; TCGF, T-cell growth factor.

Negative regulation of immune responses still is not well understood. However, it is likely that cell—cell interactions and cell— cytokine interactions are able to prevent the induction of a primary or a secondary response or reduce the magnitude of an ongoing response by inhibiting further activation.

Induction of Antibody Synthesis

In 1966, Henry Claman provided the first evidence of a synergistic interaction between cells in an immune response. He reported that the injection of a mixture of bone marrow cells, thymus cells, and antigen into irradiated mice produce a much higher antibody response than does the injection of antigen with either cell type alone. Other investigators soon showed that the bone marrow contains the precursors of the antibody-producing cell and that the thymus-derived cell provides some auxiliary or helper function. Soon thereafter, Donald Mosier and colleagues demonstrated that a glass-adherent, nonspecific cell also is essential for optimal antibody responses; this cell proved to be the antigen-presenting macrophage. The need for two specific lymphocytes to interact in the production of antibody explained to a great extent previous observations that a secondary immune response to hapten requires that the hapten be coupled to the same carrier for the primary and secondary immunizations. Subsequent experiments showed that the cell that recognizes the carrier is thymus-derived and the hapten-specific, antibody-producing cell is bone marrow-derived. Moreover, it became clear that for optimal responses to occur, the antigenic determinant recognized by the antibody-producing cell and that recognized by the thymus-derived cell must be on the same antigen molecule. This thymus-derived cell (ie, T cell) is called a Thelper (Th) cell, because it does not produce antibody, but it is required for most types of antibody production.

An overall scheme for the induction of the immune response and its negative control is shown in Figure 1. Each cell type shown is preprogrammed for its specificity and its function. The only role for antigen is to select that cell capable of binding antigen and, under the appropriate conditions, to activate it to carry out its preprogrammed function. The overall scheme, as is shown later in this chapter, is deceptively simple. Effector cells (B cells and Tc cells) are activated by antigen in the presence of a positive regulatory cell, the Th cell. Each activated effector cell then performs its preprogrammed function, for example, antibody synthesis or killing of virally infected cells. Down-regulation of these immune responses is provided by a negative regulatory effect brought about by cytokines such as interleukin (IL)-10, produced by the Th subset called Th₂. The lymphocytes are thought to exert positive regulatory actions mediated by IL-2 and interferon gamma (IFN-γ).

The induction and control of antibody responses to foreign and self-antigens are the topics of the remainder here. The induction and control of cell-mediated immune responses (eg, those mediated by Tc cells and Th cells) are discussed later.

SIGNAL TRANSDUCTION AND SECOND MESSENGER PATHWAYS IN LYMPHOCYTES

The literature dealing with signal transduction and second messenger pathways is replete with details regarding the events that take place after receptor—ligand interaction at the surface of a cell. Lymphocytes have served as models for the study of transmembrane signaling and the investigation of second messenger pathways in part because of the convenience in obtaining large numbers of these cells in relatively pure form and because they can be maintained in tissue culture in synchronous growth using a variety of growth factors and mitogens. From the overview of signal transduction and second-messenger regulation of lymphocyte activation, it is apparent that considerable similarity exists in the transduction and messenger pathways in T and B lymphocytes.

To begin with, B-lymphocyte membrane immunoglobulin, the T-cell antigen receptor/CD3 complex, the CD4 and CD8 coreceptor molecules, and the IL-2 receptor (IL-2R) function in their respective lymphocyte subtypes to transduce signals from the cell membrane to the interior of the cell. It is well known that protein tyrosine kinases (PTKs) are activated upon cell membrane interaction of receptors with ligands in both T and B lymphocytes. There is still much to learn regarding how these common pathways in lymphocytes from distinctly different lineages, which have different functions, can selectively activate and repress different transcriptional events in these separate cell types.

T CELLS. The a/β and β/δ chains of the T-cell receptor (TCR) complex designated Ti are involved in recognition of antigen presented by antigen-presenting cells (APCs). The TCR chains have short cytoplasmic tails and do not function as signal transduction systems for intracellular activation events. Other molecules of the TCR—the CD3 complex known as the γ, δ, and ε chains—actually serve to couple the α and β chains of the TCR to the intracellular signal transduction pathways. In addition to the γ, δ, and ε chains, a fourth chain exists known as ζ which is noncovalently associated with the CD3 complex of the TCR. The ζ chain has a short extracellular domain and a long intracellular domain. A homologue of the ζ chain on T-cell membranes is on the IgG-Fc receptor (IgG-FcR) of natural killer cells. The molecules of the CD3 complex—γ, δ, ε, and ζ—couple the TCR to the intracellular compartment, and the IgG-FcRs perform a similar function on natural killer cells. They activate protein tyro-sine phosphorylation of tyrosine residues after interaction of the TCR with antigen on an APC. At least 10 repeating cytoplasmic components, or motifs, allow one TCR to interact with, and activate, many copies of the same signal transduction system. This amplification increases the sensitivity of the TCR interaction with antigen. After transduction of the signal from the plasma membrane to the intracellular compartment, several members of the Src family of PTKs are involved in further regulation of cellular activation. Thus, the FynPTK, LckPTK, LynPTK, and ZAP-70 SrcPTKs have been shown to be involved in T cell activation. One of the major consequences of TCR-mediated PTK activation is activation of the phosphatidyl inositol second messenger pathway within the cell. The activation of the kinases ultimately results in transduction of signals into the nucleus of the cell where gene activation, DNA synthesis, transcription, and cell division are initiated (Fig. 2). As indicated earlier, considerable redundancy exists in the pathways used in transmembrane signaling and second messenger signal transfer to the nucleus in T lymphocytes and B lymphocytes (compare Fig. 2 and Fig. 3). Thus, it is not clear how common pathways in different cell types lead to expression of different cellular functions.

B CELLS. As previously indicated, B-cell membrane immunoglobulin receptors interact with antigens and signals are transduced across the plasma membrane, activating intracellular PTKs in much the same way as occurs in T lymphocytes. It is known that the membrane immunoglobulin protein complex of B cells is homologous to the CD3 complex, which is associated with the TCR on T lymphocytes. On B cells adjacent to the immunoglobulin receptor, there is a disulfide-bridged heterodimeric complex referred to as Ig a/β which contains amino acid sequences that bind to cytoplasmic effector systems, including members of the Src family of tyrosine kinases, such as are activated after T-cell recognition of antigen. Activation of a number of PTKs within the cell occurs shortly after cross-linking of B-cell immunoglobulin receptors by antigen. These kinases include Fyn, Lyn, Blk, and others. Whether these tyrosine kinases are activated in parallel or as a cascade is not clear. Nevertheless, these and other kinases serve as a transduction system ultimately resulting in nuclear activation of specific genes in the B lymphocytes. The biochemical cascade that occurs intracellularly after tyrosine kinase activation involves protein tyrosine phosphorylation. These activation events include the phosphorylation of phosphatidylinositol 4,5-diphosphate-specific phospholipase C, PLC-γ. This, in turn, results in the hydrolytic cleavage of phospholipids producing inositol 1,4,5-triphosphate (InsP₃) and diacylglycerol (DAG). InsP₃ mediates Ca²⁺ changes, which activate calcium calmodulin kinase II (CaM kinase II), which phos-phorylates the Ets-1 DNA binding protein. The regulatory action of Ets-1 on gene transcription is altered by phosphorylation, thus completing the cascade from the cell membrane to the gene.

A second biochemical pathway activated intracellularly in B cells after antigen binding involves a G protein known as p21 ras. Ras is activated within minutes of immunoglobulin-antigen interaction and regulates the sequential activation of serine/threonine phosphorylation of the microtubule-associated protein kinases, ERK2 kinase, MEK, and subsequently microtubule-associated protein kinase 2 (MAPk). MAPk may indirectly regulate gene expression; this kinase is known to catalyze the phosphorylation of c-Jun, which regulates gene activity in B lymphocytes.

A third event occurring intracellularly in B cells after stimulation by antigen involves the activation of phospha-tidylinositol 3 kinase. This enzyme phosphorylates inositol phospholipids, and these phospholipids are important in stimulating proliferation, possibly by activating protein kinase C (PKC) f, which activates NFkB, a mediator of gene transcription and cell division.

Ultimately, signal transduction from the B-lymphocyte membrane immunoglobulin receptor involves the activation of PTKs, which diverge intracellularly, activating several enzymatic processes that stimulate serine/ threonine kinases. These kinases may, in turn, regulate gene transcription in the B cells, resulting in a differentiated cellular response (see Fig. 3). As indicated previously, such signal transduction events and second messenger pathways are recognized as a common pattern in receptor-mediated signaling from the cell surface to the interior of the cell. Much is yet to be learned regarding the specificity of these various second-messenger pathways, and it is still undetermined how genes are differentially activated and inactivated in T cells and B cells so as to permit these cells to maintain their unique functional properties.

HELPER T-CELL ACTIVATION

An essential step in the induction of immune responses to most antigens is the activation of the Th cell. (The cell-surface phenotype of the Th cell in humans is CD3⁺4⁺8^-.) This activation occurs in several steps:

1. Antigen is taken up and processed by the antigen presenting cell (APC) (eg, a macrophage, a dendritic cell, or a B lymphocyte). This involves antigen interaction with the APC" cell surface, endocytosis of the antigen, partial or incomplete digestion of the antigen, and the expression of the antigen fragment in the groove or cleft of a major histocompatibility complex (MHC) molecule on the APC cell surface (Fig. 4).

⁄⁄ Class II Molecule; ● & ○ Fully Degraded Antigen; ■ & □ Antigen-Derived Peptides

FIGURE 4. Antigen processing by the antigen-presenting cell (APC). The activation of helper T (Th) cells depends on the uptake and processing of soluble foreign antigen molecules by nonspecific APCs. The antigen undergoes endocytosis by the cell and is degraded in acidic intracellular compartments (thought to be the light endosomes). Fragments of the antigen are taken up by class II molecules and presented as peptide—class II complexes on the surface of the APC The association between the peptide antigen and the class II molecule is thought to first occur in the intracellular compartment before appearance of the complex on the cell surface. Class II-restricted T cells specifically recognize and interact with this complex. A number of cell types can perform this antigen-presentation function.

Processing of antigen within the cell occurs in acidic compartments such as endosomes or the phagolysosomes. Drugs, such as chloroquine, that increase the pH in these compartments inhibit antigen presentation, although they do not prevent endocytosis of the antigen by the cell. Similarly, processing of protein antigens is blocked by the addition of protease inhibitors (eg, leupeptin). Indeed, pre-processed protein antigen (ie, peptides derived from an in vitro digestion of the antigen) can substitute for whole antigen, even if fixed APCs are used that are unable to internalize and process antigen. Thus, in most situations, processing of most protein antigens is a prerequisite for T-cell recognition of antigen on the APCs cell surface.

2. It is well established that Th cells recognize antigen on the surface of APCs in the context of class II MHC molecules. This dual recognition of an antigen and a class II molecule is responsible for the MHC restriction discussed in Chapters 9 and 10. Specific binding of peptides, derived from protein antigen, to isolated class II molecules has been directly demonstrated. Therefore, the Th cell recognizes a complex of peptide antigen and a class II molecule, and only cells that express class II molecules can function as APCs in the activation of Th cells. The class II molecule exhibits a relatively broad specificity (compared with the highly specific B-cell receptors and TCRs) in that a given class II molecule can bind peptides derived from a broad range of proteins that have no apparent structural similarity with one another. Because of the importance of this binding of peptides derived from protein antigens by class II molecules (and, is seen later, by class I molecules as well) to the initiation of immune responses and to the development of synthetic vaccines, the nature and specificity of this binding are areas of intense investigation.

3. The APC subsequently releases a soluble factor called IL-1 (see Table 1). Incidentally, macrophage-de-rived factors are collectively referred to as monokines. The recognition of the class II-antigen peptide complex by the Th on the APC surface, combined with the binding of IL-1 to IL-1 receptors on the T-cell membrane, provides the first signal which drives the Th from the resting G_o state into G₁of the cell cycle (Fig. 5).

FIGURE 5. Antigen specific activation of helper I (Th) cells. Th cells are activated after the recognition of a peptide antigen class II major histocompatibilitv complex (MHC) by the I -cell receptor. Interleukin I (II,-I } is involved in this initial event, which causes the G₀ to G₁ transition in the cell cycle and the acquisition of receptors for the growth factors IL-2 and IL-4. Interaction of IL.-2 and IL-4 with these newly acquired receptors causes the T cells to continue through the remainder of the cell cycle. In addition, expression of other genes is induced, leading to the secretion of a number of lymphokines by the Th cells. Th 1 cells secrete IL-1, IL-3, interferon gamma (INF-γ), and various colony stimulating factors (CSF). Th2 cells secrete IL-4, IL-5, and IL-10, as well as CSFs. The types of T- and B cell responses regulated by each of the helper T cell subsets (Th1 and Th2) depend on the cytokine that each secretes.

4. As the Th cell progresses into the cell cycle, it begins to express cell-surface receptors for another soluble factor, IL-2 (or T-cell growth factor; see Table 1). Subsequent interaction of these IL-2R-positivc Th cells with IL-2 drives the cells through the remainder of the cell cycle (see Fig. 11-5). This interaction is the second signal required for Th activation. The rate and extent of proliferation induced are directly proportional to the amount of IL-1 released, the density of cell-surface IL-2R, and the concentration of IL-2.

5. The overall result of this APC-antigen-Th cell interaction is the clonal proliferation of the Th cell and the production, by the Th cell, of a variety of soluble factors collectively referred to as lymphokines. These lymphokines include R-cell and T-cell growth and differentiation factors including IL-2 (see Table 1). IL-2 is a product of the Th cell as well as a growth factor for Th cells and is a good example of an autocrine growth factor (ie, it is required by the cell that produces it).

Interaction of antigen (with or without other signals) with its membrane receptor activates membrane phospholipase C, releasing inositol triphosphate and DAG by the hydrolysis of membrane phosphatidyl inositol biphosphate. Activation of PKC by DAG indirectly affects ion pumps and intracellular ion concentrations. Ion concentrations, in turn, affect the activity of certain protein kinases. Inositol triphosphate induces the release of Ca²⁺ from intracellular stores, activating other protein kinases and thus protein phosphorylation, which precedes DNA synthesis.

The proliferation of the Th cell yields an expanded pool of antigen-specific Th cells. Subsequent activation of this enlarged pool of Th cells by second contact with antigen produces a more rapid and heightened response, that is, a secondary response.

Co-Stimulatory Signals Required for T-Cell Activation

The interaction of the TCR complex with a peptide antigen in the groove of a self-MHC molecule is the primary signal that a T lymphocyte must receive to become activated and participate in an immune response. The TCR, in conjunction with either the CD4 or CD8 molecules on helper and cytotoxic T cells, respectively, provides the primary stimulus to T cells to enter into an antigen-specific immune response.

Several years ago, Kevin Lafferty and others recognized that antigen-specific T-lymphocyte responses require more than antigen recognition by the T cell. It soon became clear that second or co-stimulatory signals are required for lymphocyte activation and entry into specific immune responses. Because it is known that antigen presentation to T lymphocytes is performed by so-called professional APCs, it is clear that, in addition to the recognition of antigen on self-MHC molecules, the T cells have to form a second or co-stimulatory receptor-ligand relationship with the APC. Many investigators have contributed to the discovery and molecular characterization of various receptors and ligands on APCs and T cells. For example, the molecular characterization of the CD28 molecule on T cells and the B7 molecule on APCs preceded the functional characterization of these molecules.

Within the last few years, Peter Linsley and coworkers and Jeffrey Bluestone and colleagues elucidated the function of the CD28 receptor and its B7 ligand in T-cell co-stimulation. These investigators showed that after the interaction of the TCR with a self-MHC-peptide complex, the CD28 and B7 interaction must take place for the T cell to undergo activation, functional differentiation, and participation in an antigen-specific immune response (Fig. 7). There are other examples of T-cell APC co-stimulatory interactions; the CD28-B7 interaction will be used here as the prototype to define the functional significance of the co-stimulatory signaling in T-cell activation.

FIGURE 7. T-lymphocyte co-stimulatory signaling. The primary signal that a T lymphocyte receives is through the binding of the T-cell receptor (TCR) to the antigen presented on a self-MHC antigen molecule. An example of a second or co-stimulatory signal, which T lymphocytes must receive, is the interaction of the CD28 molecule on the T lymphocyte with the B7 ligand on an antigen-presenting cell. This second, or co-stimulatory, interaction must take place for the T cell to respond to the antigen.

CD28 is a 44-kilodalton glycoprotein that is present on the surface of all CD4⁺ and approximately 50% of CD8* human T lymphocytes; all mouse T cells express this glycoprotein. Perturbation of the CD28 molecule with anti-CD28 antibodies mimics the co-stimulatory CD28-B7 signal to the T cell, causing the T cell to produce lymphokines and enter the cell cycle. Both similarities and differences exist between the signal transduction events that take pace after the TCR interacts with the self-MHC-peptide complex and CD28 interacts with the B7 molecule. The TCR transduction pathway is sensitive to the immunosuppressive drug, cyclosporin A, and inhibitors of PKC, whereas CD28-B7 transduction is not affected by these mediators. Interestingly, both CD28-B7 and TCR signal transduction are sensitive to tyrosine kinase inhibitors. Thus, there are some common elements and some differences in both receptor-ligand interactions.

Gordon Freeman and Richard Hodes have discovered a variant of the B7 molecule on APCs. Thus, the original B7 molecule is one of a family of at least two B7 subtypes designated B7-1 and B7-2. In the latest modeling of the expression of B7 molecules by APCs, it seems that the B7-2 molecule may be constitutively expressed; mRNA transcripts for the B7-2 molecule are present in APCs before they are actively involved in antigen presentation. The B7-1 molecule, on the other hand, is expressed by APCs after some delay. B7-1 appears on the APC] membrane 24 to 48 hours after the APC has provided the antigenic stimulus to T cells through the interaction of self-MHC-peptide complex with the TCR.

The significance of the observations regarding the necessity of co-stimulatory signals for antigen-specific T-cell activation lies in the potential to manipulate this interaction to modulate and even prevent antigen-specific immune responses both experimentally and clinically. Evidence suggests that if a T cell receives an antigenic stimulus by the TCR interacting with antigen on self-MHC but fails to receive the co-stimulatory signal provided by the CD28-B7 interaction, the T cells are rendered impotent or anergic. Some investigators have speculated that blocking the co-stimulatory interaction may be one route to the induction of antigen-specific T-cell tolerance. The results of several studies reveal that if the CD28-B7 interaction is blocked, an antigen-specific T-cell response is prevented from developing.

The discovery of a CD28 homologue on activated T cells called CTLA4, which has a higher affinity for B7 than CD28, has been used to block T-lymphocyte co-stimulation (Fig. 8).

FIGURE 8. Blocking T-lymphocyte co-stimulation. The production of a high-affinity soluble ligand termed CTLA4-Ig has been used to prevent the co-stimulatory signaling of T lymphocytes upon antigen recognition on an antigen-presenting cell (APC). CTLA4-Ig binds to the B7 coreceptor molecule on the APC and prevents the T cell from receiving the second signal through the CD28 receptor. The failure to receive the second signal renders the T lymphocyte unresponsive or anergic.

The creation of a soluble version of the CTLA4 molecule called CTLA4-Ig has been used in the laboratories of Linsley and Ledbetter and of Jeffrey Bluestone to modulate antigen-specific T-cell responses in mice. Remarkably, it was shown that human pancreatic acinar cells survived for 100 days or more under the kidney capsule of mouse hosts. The survival of human cells in mice for this period of time suggests that useful clinical opportunities lie ahead; it should be possible to engineer molecules such as CTLA4-Ig, which can be used to safely and effectively suppress unwanted T-lymphocyte responses.

B-CELL ACTIVATION

Antigens can be divided into two categories based on their apparent need for Th cells for the induction of antibody synthesis: (1) those that require Th cells, referred to as T-dependent antigens (TD-antigens); and (2) those that do not require Th cells, called T-independent antigens (TI-antigens).

The first step in the induction of antibody responses to TD-antigens is the activation of Th cells. This has been discussed earlier. B cells can take up antigen either specifically, via their immunoglobulin receptors, or by nonspecific endocytosis. Specific uptake is a far more efficient process, requiring at least 1000-fold less antigen for stimulation than does nonspecific endocytosis. Therefore, in normal situations where antigen is in low concentrations (eg, early in infection), the immunoglobulin receptor serves to focus the antigen on the important B cells. This focusing of antigen results in a specific antibody response rather than in generalized immunoglobulin biosynthesis.

This immunoglobulin receptor-mediated endocytosis results in the internalization and processing of the antigen by B cells in a manner essentially identical to that described earlier for the APC in Th activation (see Fig. 4). Processed antigen (ie, peptide) then is repositioned on the B-cell surface in association with the B cell's class II molecules (Fig. 9).

FIGURE 9. Induction of an antibody response to a T-dependent antigen. Antigen is recognized and bound by the B-cell immunoglobulin antigen-specific receptor. Receptor-mediated endocytosis of the antigen (and the immunoglobulin receptor itself) leads to processing of the antigen, as described for APCs in Figure 2. A fragment of the antigen is bound by a class II molecule and is presented on the B-cell surface as a peptide-class II complex. A helper T (Th) cell, which has been preactivated by interaction with an identical peptide-class II complex on an APC, interacts with this complex on the B cell. This Th cell is then activated and delivers both proliferative and differentiative signals (many in the form of secreted lymphokines) to the B cell, causing the formation of memory B cells and plasma cells, which synthesize and secrete antibody. On subsequent contact with antigen and Th cells, the memory B cell gives rise to more memory B cells and to more plasma cells secreting antibody.

The B cell, therefore, is also an APC. The role of IL-1 in the interaction between B cells and Th cells is uncertain. Apparently, B cells do not secrete this monokine, but several reports suggest that IL-1 is present in a membrane-bound form on B cells. In addition, although B cells can function as APCs for activating Th cells during induction of antibody responses, it is not clear that they can present antigen to virgin Th cells.

The activated Th cell then interacts with this peptide-class II complex, resulting in the release of lymphokines by the Th cell at the B-cell surface. These factors provide signals of growth and differentiation that, together with signals derived from its interaction with antigen, drive the B cell to proliferate and to differentiate into antibody-producing plasma cells and memory B cells.

The peptide bound by the class II molecule for presentation to the Th cell can be any part of the protein that makes up a complex antigen recognized by the B-cell receptor. The induction of antibody responses to viruses is an important example. Virus are complex structures composed of several proteins, DNA or RNA and, in certain cases, lipids from the host cell in which the virus replicated. A given B cell will recognize only a single epitope on one of the viral surface proteins. Uptake of the viral particle via immunoglobulin receptor-mediated endocytosis and the subsequent degradation of the viral proteins may result in many antigenic peptides, one or more from each viral protein. Thus, B cells that recognize epitopes on viral surfaces can be helped by Th cells, which are specific for peptides derived from any protein of that virus, whether on the surface (viral glycoprotein) or not (eg, nucleocapsid or matrix proteins). Therefore, the epitope recognized by the Th cell need not be on the same molecule as the epitope recognized by the B cell, but it must be on the same antigenic complex that was recognized and processed by that B cell. The chief requirement is that the antigenic epitope recognized by the Th cell must be part of whatever the B cell specifically recognizes, internalizes, and processes, whether it be a complex, multiprotein antigen such as a virus, or a simple soluble protein antigen such as tetanus toxin.

This Th cell-B cell interaction can cause the B cell to switch from producing IgM to producing antibody of another isotype. As discussed in Chapter 8, this switch involves a translocation of the heavy-chain VD J gene from a position adjacent to the /u-chain constant-region gene to another constant-region gene. Because of this switch, some plasma cells will be producing antibody of one isotype, and the memory B cells will begin to express receptors of a different isotype.

This Th cell-driven switch in heavy-chain constant-region expression is also accompanied by a dramatic increase in somatic mutation in the VDJ gene, presumably giving rise to some of the amino acid sequence diversity and the maturation in affinity of the antibody.

HELPER T-CELL FACTORS (LYMPHOKINES)

As discussed earlier, the Th cell is responsible for a number of the growth and differentiation signals given to the B cell during immune responses. Evidence suggests that the Th cell controls B-cell proliferation and differentiation by secreting soluble factors, generally referred to as cytokines or lymphokines which interact with the B cell (and, as is seen later, other T cells).

The first step in B-cell activation requires cell-cell contact with the Th cell, resulting in progression of the resting B cell from G_o to Gi of the cell cycle. During this progression, the B cell begins to express receptors for IL-2 and a number of other Th-derived lymphokines. Subsequent interaction of the B cell with IL-2 drives the B cell through the remainder of the cell cycle (see Fig.10), resulting in clonal expansion of the antigen-specific B cell.

Further differentiation into memory B cells and antibody-producing plasma cells appears to require other Th-derived lymphokines. A number of these lymphokines have been isolated, and their effect on B-cell growth and differentiation is the subject of intense study (see Table 11-1). The possibility that the roleofIL-1 in B-cell activation is the same as it is in Th activation (ie, induction of expression of receptors for IL-2) has already been discussed. During or after clonal expansion, interaction of other lymphokines (IL-4, IL-5, IL-6, and IFN-γ) with their specific receptors on the B cell will induce a variety of effects, such as differentiation into plasma cells and memory cells (Fig. 11), increased expression of class II molecules, increased antibody secretion, expression of Fc receptors (FcRs), and preferential expression of antibody of a given isotype (see Fig. 11).

FIGURE 11. Role of lymphokines in B-cell proliferation and differentiation. A number of factors are released by macrophages and other antigen-presenting cells (monokines) and by T cells (lymphokines). These factors act at specific points in the proliferative and differentiative pathway leading to memory B cells (A) and antibody-secreting plasma cells (B). Some factors act early, some act later, and others (eg, IL-4) exert effects at several points in this pathway.

HETEROGENEITY OF HELPER T CELLS

Multiple examples exist in humans and in experimental animals of antibody responses in which a single immunoglobulin class, allotype, or idiotype is dominant. There is evidence that selective secretion of certain lymphokines is responsible for this phenomenon (Fig. 12).

FIGURE 12. Role of helper T (Th) cells in the preferential expression of immunoglobulin isotypes. Certain Th cell clones can have a preferential effect on the expression of one or the other antibody isotypes. This effect is probably mediated by the type and concentration of lymphokine secreted by the Th cell clone. For example, it has been shown that secretion of IL-4 results in elevated production of IgE antibody. Secretion of interferon gamma and IL-4 increases IgG antibody production, and it is thought that secretion of IL-5 promotes IgM or IgA production. It is not known whether these factors influence production of a given isotype by promoting expansion of B cells already producing that isotype or by inducing an isotype switch in antibody-producing cells.

For example, it has been shown that two classes of Th cells, Thl and Th2, exist. Thl cells secrete IL-2 and IFN-y, whereas Th2 cells secrete IL-4 and IL-5. Both subsets of Th cells activate B lymphocytes; Thl cells induce B cells to switch from IgM to IgG, and Th2 cells induce B-cell switching from IgM to IgG or IgE. The subsets promote IgG synthesis, although it is unclear whether both promote synthesis of all four IgG subclasses. The conditions that result in preferential activation of one type of Th or preferential secretion of certain lymphokines by a single Th cell are not clear. Furthermore, evidence suggests that preferential expression of certain allotypes or idiotypes by B cells occurs, but this property has not yet been linked to any known lymphokine. It is not known whether these lymphokines can directly induce the isotype switch in B cells or whether they selectively expand B cells that have already undergone the switch as the result of other signals. Either way, the control of isotype expression permits the immune system to be focused on the synthesis of the antibody with the required biologic activity (eg, IgE and IgG in response to parasitic infections, IgA in response to infections on mucosal surfaces, and IgM and IgG to infections by organisms sensitive to complement).

T-INDEPENDENT ANTIGENS

As mentioned earlier, certain antigens do not require Th to induce antibody responses. These are called T independent or TI-antigens. They are normally large molecules that are poorly catabolized, possess repeating antigenic determinants, and often possess properties that render them mitogenic for lymphocytes when present at high concentrations. TI-antigens are present on the surface of infectious organisms. An example is the lipopolysaccharide of gram-negative organisms such as Escherichia coli or Salmonella typhi or the pneumococcal polysaccharide of the various species of S pneumoniae. Induction of antibody responses by these antigens may involve binding to both immunoglobulin receptors and mitogen receptors on B cells. Binding of a Tl-antigen to a B cell results in extensive cross-linking of the immunoglobulin receptors. This cross-linking combined with the mitogenic activity of other structures on the antigen suffices to provide proliferative signals and certain signals of differentiation to the B cell. It is not known why these antigens are T-cell independent. Perhaps it is because they are poorly catabolized, or perhaps class II antigens do not bind these antigens or smaller oligomers derived from them. Either way, Th cells, which require corecognition of antigen and class II, are not stimulated.

KINETICS OF ANTIBODY FORMATION

As one might expect, the rate and extent of antibody formation are influenced by several different factors. These include the nature and concentration of the antigen, the frequency of Th and B cells specific for the antigen, and the efficiency of antigen processing by the APC. An adjuvant is a substance that is used to enhance the immunogenicity of soluble antigens. The exact mechanisms by which adjuvants exert their effect on immunogenicity is not clear, but it is interesting that many adjuvants contain substances that are mitogenic for lymphocytes. Examples of adjuvants are alum-precipitated toxoids, which have been used extensively in human immunizations, and water and oil emulsions of antigen containing killed tubercle bacilli, which have been used widely in animal immunizations. This latter mixture, called Freund's complete adjuvant, causes a fairly intense inflammatory response at the site of injection.

Primary Response

The appearance of antibodies in the blood after an initial exposure to antigen is known as the primary antibody response. The rate and the extent of the primary response are dependent on the nature of the antigen, the size of the dose administered, the route of administration, and the frequency of the antigen-specific Th and B cells. Figure 13 shows the kinetics of an antibody response after an initial (primary) and subsequent (secondary) exposure to a theoretical antigen.

FIGURE 13. Kinetics of the antibody response. The kinetics of the appearance of antibody in blood plasma arc shown after first and subsequent contacts with antigen. After a primary response (1°), there is a latent period before appearance of antibody. IgM antibody is the first to appear, followed by IgG and other isotypes. After a short period, the concentration of circulating antibody declines. On subsequent contact (2°) with antigen, memory B cells are stimulated and immediately respond by differentiating to plasma cells secreting antibody. The shortened latent period in the secondary response is primarily because of the expanded pool of B cells after primary stimulation (ig, there arc more B cells and, thus, antibody is detected earlier). The secondary response is also of a much greater magnitude, again reflecting the greatly expanded pool of B cells after primary stimulation. In addition, the relative amount of IgG antibody in the secondary response is much greater than that of IgM (which is approximately the same as in the 1° response). This enhanced IgG response reflects the fact that many of the memory B cells have already undergone an isotype switch from µ- to -µ-heavy chain expression. The affinity of antibody for its antigen also increases with time after antigen contact. This increasing affinity for antigen reflects an in vivo selection for B cells with high-affinity receptors while the antigen concentration decreases.

Measurable antibody begins to appear about 5 days after the initial exposure to an antigen and reaches a peak in 2 to 3 weeks. The duration of antibody in the scrum is dependent on continued stimulation of antibody production and on the normal catabolic turnover rate of immunoglobulin, which is different for each class of immunoglobulin. (The half-life of human IgG is about 23 days.)

Notice that IgM is the first antibody produced in the primary response and that the affinity of the antibody for the antigen increases with time. The fact that the first antibody produced is IgM should not be surprising, because after DNA rearrangement during differentiation from a stem cell to a B cell, the VDJ gene is directly upstream from the µ-chain constant-region gene. Thus, B cells with the VDJ µ-chain constant-region arrangement are present in a high frequency.

The increase in affinity during the primary response probably results from two factors. First, the heavy-chain switch is accompanied by somatic mutation in the VDJ genes. These somatic mutations may yield antibody molecules with a greater affinity for antigen. Second, as antibody is produced, the concentration of antigen capable of stimulating B cells decreases. Residual antigen binds preferentially to B cells bearing immunoglobulin receptors with high affinity. Thus, as antigen concentration decreases, stimulation of B cells with high-affinity receptors occurs.

As B cells undergo the heavy-chain switch, antibody of the IgG and other classes begins to appear. The nature of the antibody produced depends on a number of factors, not the least of which is the nature of the immunoglobulin-specific Th cell that is activated (see earlier). In most cases in which one examines antibody in the blood, the IgG isotype is predominant. However, IgA is the predominant isotype in the secretions (eg, tears and colostrum).

As antigen is removed, fewer and fewer cells are stimulated. Serum antibody concentrations begin to decline as the terminally differentiated plasma cells cease to produce antibody and die.

Secondary Response

As is shown in Figure 13, a second exposure to antigen, months or even years later, produces an almost immediate appearance of antibodies (within 1 to 3 days), reaching concentrations that may be 10 to 15 times greater than that which occurred during the primary response. This may happen even if there was no measurable antibody at the time of the second exposure to antigen. This heightened response is caused almost entirely by the activation of relatively large numbers of memory Th and memory B cells that resulted from the clonal proliferation after primary stimulation. Because most of these memory cells have already undergone the switch, IgG antibody is synthesized almost immediately.

The affinity of the antibody produced during a secondary response begins at about the level that was present at the end of the primary response. Again, this should not be surprising, because the majority of the memory cells activated are those that were generated near the end of the primary response.

The response to Tl-antigens is different. First, the isotype of the antibody produced is largely IgM, regardless of the number of times antigen is encountered. Second, because memory Th cells, to a large extent, are responsible for the heightened response after second exposure to antigen and because Tl-antigens do not activate Th cells, the response to most Tl-antigens will be of about the same magnitude on second and subsequent contacts as it is on first contact.

MHC Restriction and Dual Recognition

As mentioned earlier, activation of the Th cell requires that the Th cell interact with both antigen and a class II molecule on the APC surface. This phenomenon is referred to as dual recognition. Moreover, such dual recognition is restricted in that the Th cell can respond only to an antigen that is complexed with class II molecules of its own genotype. This is referred to as MHC restriction. Dual recognition and MHC restriction are general phenomena that are common to Th cells and cytotoxic T cells. Th cells are restricted to recognizing antigen plus class II molecules, whereas cytotoxic T cells are restricted to recognizing antigen plus class I molecules.

The T-Cell Recognition Complex

The minimal T-cell recognition complex includes the heterodimeric antigen-specific TCR (termed Ti), the CD3 molecule, and either the CD4 or CDS molecule, depending on whether the T cell is CD4⁺8^– or CD4^–8⁺, respectively. A diagrammatic representation of this recognition complex is shown in Figure 14.

The CD3, CD4, and CD8 molecules from different individuals and different T-cell clones are invariant in structure. Antibody to the CD4 or CD8 molecules blocks activation of T cells, regardless of their antigen specificity. The CD3 and TcR molecules are physically associated with each other on the T-cell surface, and antibody to cither can activate the T cell. However, antibody to CD3 activates T cells, regardless of the antigen specificity of the T cell, whereas antibody to TcR activates only the T-cell clone used to produce the anti-TcR antibody (ie, anti-TcR antibody is clonally specific).

The CD4 molecule appears to react with a nonvariable (monomorphic) portion of the class II molecule, whereas the CD8 molecule appears to react with a monomorphic portion of the class I molecule. Thus, the CD4 molecule on a CD4⁺ T cell may serve to direct that cell to a target cell bearing the class II MHC products (eg CD4⁺ Th cells would be directed to class II⁺ macrophages and B cells). Similarly, the CDS molecule on a CD8⁺ T cell may serve to direct that cell to a target bearing the class I MHC products. For example, a CD8⁺ Tc cell would be directed to class I⁺ virally infected target cells.

The model shown in Figure 14 is a minimal model and, at first glance, one might question the need for such complexity. However, the complexity is explicable if the recognition complex is viewed as having an antigen-specific receptor (the TCR molecule) linked to a signal transducer (the CD3 molecule) whose interaction with specific cell types is directed and stabilized by an anchor (the CD4 or CDS molecule).

Figure 14 shows the interaction of a CD4⁺8^– Th cell with an APC that has processed the antigen for which the Th cell is specific. The complementarity-determining regions (CDRs) of the TCR polypeptide chains have formed a binding site that interacts with the peptide-class II complex. (The structure of class I and class II molecules is discussed in Chap. 10.) Some polymorphic residues of the class II molecule are involved in binding the peptide and others in binding to the TCR. The CD4 molecule binds to monomorphic (invariant) residues on the Class II molecule. This specific interaction leads to signal transduction via the CD3 molecule.

Other invariant, cell-surface adhesions are involved in this cell-cell interaction: for example, lymphocyte function antigen-1 on the T-cell surface and intracellular adhesion molecule-1 on the APC or target-cell surface. However, their role is to nonspecifically stabilize the complex formed by the more specific interactions described earlier.

Because the binding pocket of each class II (and class I) molecule has a specific composition and stereochemical configuration, it will bind only certain peptide fragments of antigen. The differences in peptide specificity of different class II molecules are determined by polymorphic residues in this pocket. Thus, during processing in the cell, individual allelic class II products can bind only certain of the peptides produced from a given protein. Some class II molecules may not bind to any peptide derived from a given protein. If this occurs for all class II molecules in an individual, no immune response against that protein will be made. Thus, an individual who has a limited number of class II alleles (eg, individuals homozygous at the class II loci) may be more susceptible to infection by a particular organism. Conversely, heterozygous individuals (with different alleles at the class II loci) will have more different class II products and thus a greater chance of binding and presenting antigens from a variety of infectious organisms.

That this may be true is suggested by the following observations:

· Administration of IL-2 prevents induction of tolerance in adult animals.

· Neonatal T cells produce little IL-2 on stimulation with agents known to induce it.

· Treatments known to reduce the numbers of IL-2-producing T cells facilitate the induction of tolerance in adult animals (eg, total lymphoid irradiation and
injections of anti-T-cell antibodies such as antilymphocyte serum [ALS], anti-CD3, and anti-CD4).

· Drugs (eg, cyclosporin A) that inhibit IL-2 production prevent tolerance induction in adult animals.

Cell-mediated immunity

The immune system of vertebrate animals can be divided into two parts based on the function of each: the humoral immune system, which involves antibody-mediated functions; and the cellular immune system, which involves T-lymphocyte-mediated functions. The two parts or systems are separate entities, but they are not mutually exclusive. Thus, the same antigen frequently induces both antibody synthesis and a cellular immune response.

Antigen-specific cell-mediated immunity is mediated by T lymphocytes. A second, smaller population of cells (which have the morphologic features of lymphocytes but lack T- or B-cell markers, mediate cellular cytotoxicity, and are not antigen specific) includes natural killer (NK) and killer (K) cells. The T-cell group is composed of (I) helper T (Th) cells, which, on recognition of foreign antigens presented on the surface of antigen-presenting cells (APCs), perform their function by secreting biologically active factors that stimulate other cell types that are involved in cellular immune reactions; and (2) T cytotoxic cells (Tc cells), which kill target cells (tumor cells, virus-infected cells, or allograft cells) by release of lysins after recognition of foreign antigens on the target cell membrane. The NK group kills target cells by mechanisms similar to those that the Tc cell uses. The NK cells kill some tumor cells but lack antigen-specific receptors. K cells recognize antibody-coated target cells by means of a cell-surface Fc receptor on the K cell.

It was soon noticed that lymphocytes from normal, nonimmune individuals could nonspecifically kill a variety of tumor cells under certain conditions. These cells are referred to as natural killer (NK) cells. It was also observed that a population of lymphocyte-like cells could kill antibody-coated target cells. This antibody-dependent cellular cytotoxicity (ADCC) is mediated by cytolytic cells called killer (K) cells. Both these cells belong to a subpopu-lation of lymphocytes called large granular lymphocytes (LGLs) because of their size and the presence of large azurophilic granules in their cytoplasm.

Tc cells with TCRs composed of the γ/δ heterodimer have been demonstrated in the intestine. These cells have a CD3⁺4^–8⁺ cell-surface phenotype but are not restricted by MHC molecules in their killing of target cells, and their antigen specificity is unknown.

A summary of the properties of these four cytolytic cells is given in Table. A more detailed description of each cell type and the mechanisms by which they kill their target cells is given below.

TABLE. Properties of Cytotoxic Cells

Cytotoxic Cell	Receptor	MHC Restriction	Cell Surface CD Phenotype	Target
Tc-I*	α/β	Class I	CD3^+4^–8^+	Class I plus peptide
Tc II	α/β	Class II	CD3^+4^+8^–	Class II plus peptide
Tc-III	γ/δ	Unknown'	CD3^+4^–8^– CD3^+4^–8^+	Bacteria(?)
NK	Unknown	No	CD16^+3^–4^–8^–	Various tumor cells
K	FcγR	No	CD16^+3^–4^–8^–	Antibody-coated cells
Tc, cytotoxic T lymphocyte; K, killer; MHC, major histocompatibility complex; NK, natural killer. * – The I, II, and Ill categories are not an official designation but are used merely to distinguish between the three types of Tc cells. ' – There have been some reports that these Tc cells may be restricted by class I-like molecules.

CYTOTOXIC T LYMPHOCYTES (Tc CELLS)

Most cytotoxic T lymphocytes (Tc cells) bear the α/β heterodimeric TCR (the α/β TCR). They are involved in elimination of virus-infected cells and tumor cells and in the rejection of allogeneic and xenogeneic transplants.

Tc Cells With the α/β T-Cell Receptors. All of these Tc cells recognize antigen in association with either a class I or a class II MHC molecule (ie, they are MHC restricted). Those α/β TCR⁺ Tc cells that are class I restricted have the CD3⁺4^–8⁺ phenotype, whereas those that are restricted by class II molecules are CD3⁺4⁺8^– (see Table “Properties of Cytotoxic Cells”). The Class I-restricted Tc cells are by far the most numerous cytolytic cells in the peripheral lymphoid organs.

Tc Cells With the γ/δ T-Cell Receptors. Most γ/δ TCR-expressing cells are of the CD3⁺4~8~ phenotype and are found in small numbers in the thymus and peripheral lymphoid organs. T lymphocytes have been demonstrated residing between the columnar epithelial cells of the villi of the small intestine. These intraepithelial lymphocytes (IEL) express the γ/δ TCR but are CD4^–8⁺ or CD4^–8^–. The cytotoxic activity of γ/δ TCR-expressing cells appears not to be MHC restricted, and until recently there were few clues as to which antigens these cells recognize. However, Lefrancois and Goodman found that IELs taken from mice born and raised in a germ-free environment showed little or no cytolytic activity, whereas IELs taken from mice raised in a conventional (or natural) environment were constitutively cytolytically active (as measured by indirect methods). This result suggests that IELs in normal mice have been activated by environmental pathogens in the intestine. Lefrancois and Goodman also reasoned, as have others, that the γ/δ TCR may have evolved before the α/β TCR, because the primordial immune system would develop a protective mechanism against infection of the digestive tract before requiring one monitoring the internal milieu.

Tc-Cell Activation. The following discussion of Tc-cell activation and the mechanisms by which Tc cells recognize and kill target cells has been shown to be true for the α/β TCR-expressing Tc cells. The mechanisms by which the γ/δ TCR-expressing Tc cells recognize antigen, are activated, and kill target cells are unknown. However, because both cell types have similar antigen-specific receptors, one might assume that except for the difference in MHC restriction during antigen recognition, they operate by similar methods.

The kinetics of a primary and secondary Tc-cell response are much the same as those seen in an antibody response. A measurable Tc-cell response to initial contact with antigen (a primary response) can be detected in about 10 days to 2 weeks. On subsequent contact with the same antigen (eg, 1 or 2 months later, during which time cytolytic activity has waned), memory Tc cells undergo rapid clonal proliferation and activation into cytolytically active Tc cells. The lag period between contact with the antigen and the detectable appearance of Tc cells, in both the primary and the secondary response, depends on the nature of the antigen (viral antigen, or minor or major transplantation antigen) and the number of precursor Tc cells with TCRs specific for the antigen in question.

The activation of Tc cells occurs in several steps, as shown in Figure 2. The first step is the activation of Th cells, as described in Chapter 11. Antigen is ingested, processed, and presented in association with a class II molecule to the Th cell by an APC. This activated Th cell synthesizes and secretes IL-2. The antigen may be a viral glycoprotein or an alloantigen released from an allograft, that is, a major or minor histocompatibility (transplantation) antigen.

At the same time, the Tc cell interacts with antigen, either on the surface of an APC, a virus-infected cell, a tumor cell, or cells expressing foreign major or minor histocompatibility antigens. This interaction induces the Tc cell to express a receptor for IL-2. Other poorly understood differentiative events occur as well. The IL-2 secreted by the Th cell reacts with the newly acquired IL-2 receptor on the Tc cell and induces proliferation and further differentiation. In contrast to what occurs in induction of antibody responses, direct cell-cell contact between the Th cells and Tc cells is not required, and the Th cell requirement in Tc cell activation can be replaced, at least in vitro, by IL-2. Therefore, the primary role of the Th cell in Tc-cell activation may be the synthesis and secretion of this lymphokine.

The activated, or effector, Tc cells recognize and bind to the antigen on the surface of the target cell. A series of subsequent events, which are described later, leads to lysis of the target cell.

These interactions between the Th cell and the APC, and between the Tc cell and the stimulator cell bearing the antigen, result in the generation of functional Tc cells as well as memory Tc cells and memory Th cells. It is not known how memory Tc cells and effector Tc cells differ; they may be the same cell in different stages of the life cycle or in different stages of activation.

Dual Recognition and MHC Restriction by Tc Cells. Although Tc cells were discovered after studies of allograft rejection in dogs, these cells did not evolve thousands of years ago on the chance that surgeons would one day appear on the evolutionary scene with the desire to transfer tissue from one individual to another. Tc cells more

likely evolved as a mechanism of defense against a class of pathogens that are capable of infecting almost any cell type. Viruses are the main example of this type of pathogen.

α/β TCR-expressing Tc cells recognize antigen in association with class I or class II MHC-encoded molecules. It was thought that Tc cells recognized two distinct entities on the surface of target cells. For example, it was believed that after virus infection, a viral antigen is synthesized and inserted into the membrane, where it is recognized as an intact viral protein along with the MHC molecule. However, clearly the Tc cell recognizes antigen-derived peptides bound to the MHC molecule (Fig. 1). Townsend and colleagues demonstrated the existence of influenza virus nucleoprotein (NP)-specific Tc cells. Since NP is a DNA-binding protein and is not expressed on the surface of infected cells as an intact protein, the Tc cells must recognize another form of the NP. Using peptides derived from NPs and a series of mutant NP molecules with deletions in selected regions of the protein, Townsend was able to demonstrate that the NP-specific Tc cell indeed recognizes peptides from selected regions of the NP molecules. Thus, both Th cells and Tc cells apparently recognize processed antigen that is bound by class II or class I MHC molecules respectively.

Another series of experiments demonstrated that two distinct pathways exist for processing and presentation of antigen to α/β TCR⁺ Tc cells. One pathway is sensitive to treatment with chloroquine or protease inhibitors and does not require protein synthesis. This pathway is responsible for presentation of antigen to class II-restricted CD4⁺8^– Tc cells. This pathway is presumably the same as that required for processing and presentation of soluble antigens to CD4⁺8^– Th cells. Thus, a common pathway involving endocytosis and processing of exogenous antigen in an acidic, intracellular compartment is responsible for presentation of antigen-derived peptides to both class II-restricted Th cells and to class II-restricted Tc cells (Fig. 13-B). The second pathway is not sensitive to chloroquine and protease inhibitors but does require protein synthesis. This pathway is responsible for presentation of peptides derived from endogenous antigen to class 1-restricted Tc cells (see Fig. 13-3A).

FIGURE 3. Class I and class II MHC-rcstricted presentation of antigen. Antigen is processed differently for presentation by class I or class II molecules to T cells. Class I-restricted T cells (including cytotoxic T [Tc] lymphocytes) recognize antigen that has been produced within the cell on which it is presented. For example, A shows the production of class I MHC molecules on membrane-bound polyribosomes and the production of a viral protein on soluble polyribosomes. Some of this endogenously produced viral antigen is then processed into peptides and combines with the class I MHC molecule. The complex is transferred to the cell membrane where it is recognized by class I-restricted, CD8⁺ T cells. Most Tc cells recognize antigens processed by this pathway, leading to lysis of cells actively producing the foreign antigen, be it viral, tumoral, or otherwise. This pathway does not require endocytosis, nor does it occur in acidic intracellular compartments. B shows the uptake of exogenous antigen (by either nonspecific or receptor-mediated endocytosis), degradation, and presentation of antigen fragments complexed to class II MHC molecules. This complex is recognized by class II-restricted, CD4⁺ T cells. The pathway shown in B requires endocytosis and occurs in acidic intracellular compartments such as light endosomes. In addition, a large portion of the class II molecules used in this pathway seem to recycle from the cell surface to the light endosomes and back to the cell surface. Most Th cells recognize antigen processed by this pathway and thus promote responses to circulating foreign antigens such as bacteria, toxins, certain viruses, and so forth. A small proportion of Tc cells seem to recognize antigen processed by this second pathway and, therefore, presumably, can kill cells that have engulfed antigen through endocytosis (eg, viruses), perhaps preventing initiation of viral replication in cells that have ingested intact virus.

ALLOREACTIVITY. In the case of most allografts, the foreign antigen is a class I molecule on the surface of cells of the allograft. Tc cell clones specific for human HLA class I molecules have been isolated that recognize the class I molecule when expressed on human cells but not when expressed on murine cells after transfection of the murine cells with the gene encoding the class I molecule. The human class I molecule expressed by the murine cell appears to be normal in all other aspects. Thus, these allo-specific Tc cell clones seem to be recognizing the alloantigen plus some other entity that is present on human cells a peptide derived from proteins endogenous to cells of the allograft. Therefore, Tc-cell responses to allografts probably are most specific for polymorphic residues on the class I molecule of the grafted cells plus a peptide derived from molecules endogenous to the allograft cell itself. Tc cells of this type would not have been eliminated during selection of the T-cell repertoire in the thymus of the graft recipient, because the class I molecule involved in thymic selection is different from that expressed on the grafted cells.

Tc cells are specific for a complex composed of two components: the MHC molecule and an antigen-derived peptide. If either component is missing, recognition and activation of that T cell will not occur. In the allograft example given above, the antigen from which the peptide is derived may be identical to the same molecule made by the recipient's own cells. One might wonder, therefore, why Tc cells induced by the allograft do not react with the recipient's own tissues. The answer is that the second component of the complex (ie, the MHC molecule) is different, and because recognition of both components must occur, the Tc cells that are specific for the allograft cannot react with self-tissue even if the peptide is present.

Heterozygosity at the MHC Loci. Different clones of Tc cells are specific for different antigenic determinants on a viral antigen in conjunction with each of the HLA-A, -B, and -C gene products. For example, for an individual who is homozygous at each of the class I loci, three types of virus-specific Tc cells may exist: one for virus plus HLA-A, one for virus plus HLA-B, and one for virus plus HLA-C. In an individual heterozygous at each allele, six types of Tc cells would theoretically be present, one for virus plus each of the two alleles of each of the three loci. Being heterozygous at each of the MHC loci, therefore, confers a selective advantage. For example, it Tc cells recognizing virus plus one allelic product are somehow lost, Tc cells recognizing the same virus in association with another allelic product confer protection. Similar arguments Apply to class II-restricted Tc cells or Th cells.

Mechanism of Lysis. The events occurring during Tc cell-mediated lysis have been divided into several steps, as shown in Figure 4 and as discussed later.

FIGURE 4. The cytotoxic T (Tc) lymphocyte lytic process. The process leading to lysis of target cells by Tc cells begins by specific recognition of the antigen-MHC complex on the target cell by the T-cell receptor. Other molecules on the T-cell surface then promote adhesion of the T cell to the target cell. Soon thereafter, cytoplasmic granules containing a number of active molecules, including perform, begin to move from the distal portion of the Tc cell and accumulate at the interface between the Tc cell and the target cell. Degranulation at the interface results in release of perform and other active components. Although the process is not completely understood, perform clearly aggregates on the surface of the target cell, forming complexes similar to those seen with antibody and complement. Assembly of this polyperforin then presumably leads to destabilization of the membrane, rapid ion exchange, and lysis. The Tc cell is not affected by this process and survives to kill other target cells.

1. There are three specific defense mechanisms:

a. Those mediated by antibody, such as the inactivation of toxins, opsonization of bacteria, neutralization of viral infectivity, antibody-mediated complement-dependent lysis, and ADCC;

b. Those mediated by Tc cells, such as the elimination of virus-infected cells or tumor cells; and

c. Those mediated by Th cells, such as the elimination of intracellular parasites.

2. There are four requirements for the induction of each of these mechanisms:

a. Antigen;

b. APCs (macrophages, dendritic cells);

c. Th cells; and

d. A resting B cell, Tc cell, or Th cell.

3.In all cases, the B or T cell is precommitted to antigen specificity and function.

4.MHC-restricted activation of Th cells enables these cells to deliver proliferative and differentiative signals to the effector cells or memory cells.

5. This same dual recognition involving antigen and MHC-encoded class II molecules occurs in the activation of virgin Th cells and of memory Th cells.

6.In each case, activation of the resting B cell, Tc cell, or Th cell (or memory cell) requires recognition of antigen (and for T cells, recognition of MHC-encoded products) in addition to the signals received from the Th cells.

7.The MHC-gene product (class I or class II) seen by the virgin, memory, or effector T cell in association with antigen depends on the cell involved (ie, class I or class II for Tc cells and class II for Th cells).

8.Differentiation of the virgin T cell results in the generation of effector cells and memory cells.

9.Activation of memory cells results in the generation of effector cells and more memory cells.

10. In the case of antibody formation, the Th cell also delivers a differentiative signal to the B cell, which results in a switch in the immunoglobulin isotype produced. This can result in expression of all of the biologic functions of antibody in the response to a given antigen.

11.In antibody responses, a Th cell also secretes lymphokines that enhance expression of a given isotype, allotype, or idiotype of immunoglobulin.

12.The negative control by the Th cell is mediated by regulatory cytokines. This prevents further induction and the amplification of immune responses.

13. In antibody formation, Th cells can specifically modify expression of a given isotype, allotype, or idiotype.

A Closer Look

The phrase cell-mediated immunity is generally synonymous with antigen-specific immunity mediated by T lymphocytes. Subcategories of cell-mediated immune reactions include the delayed-type hypersensitivity reaction, cell-mediated cytotoxicity mediated by cytotoxic T lymphocytes, and certain other forms of T-lymphocyte-mediated immune regulation.

Delayed-type hypersensitivity, discovered nearly a century ago, has become the prototypic measure of cellular immune function in humans and is widely used to determine if a patient has been infected by certain microorganisms such as Mycobacterium tuberculosis. Beyond its usefulness as a measure of an individual's exposure to an infectious agent when measured in the skin, the delayed-type hypersensitivity reaction is an essential cellular defense mechanism that enables the body to resist and suppress the spread of certain infectious agents.

Cellular cytotoxicity mediated by antigen-specific lymphocytes is a key factor in eliminating virus-infected cells expressing cell membrane antigens dictated by the virus. The role of T-cytotoxic lymphocytes in the destruction of syngeneic tumors is less clear. Evidently, all forms of antigen-specific cell-mediated immune reactions are dependent on the expression of antigenspecific receptors on T-cell membrane and the interaction of these receptors with a self-major histocompatibility complex molecule presenting a foreign peptide on the membrane of an antigen-presenting cell.

There are some forms of non-antigen-specific cellular immunity, which include natural killer (NK) and killer (K) cells. NK cells and K cells have the morphologic features of lymphocytes but do not express typical T- or B-lymphocyte surface membrane markers. These cells, which kill certain kinds of tumor cells and other target cells, are not antigen-specific, do not proliferate in response to antigen, and are not known to be involved in antigen-specific recognition, such as occurs with T and B cells. NK cells may be an important arm of a primordial immune surveillance system that evolved to eliminate effete cells, tumor cells, and certain parasites. K cells acquire specificity by virtue of their cell membrane Fc receptors for immunoglobulin.

Patients who have defects in cellular immune function may have congenital thymic deficiencies. Regardless of the origin of the T-lymphocyte deficiency, such patients are highly susceptible to viral and fungal infections. Therapies designed to reverse such deficiencies have not met with much success, although cellular and gene therapy may offer some hope.

REFERENCES:

1. Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 37-47.

2. Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P. 382-190, 404-410.

3. Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P.115-120, 131-132.

4. Wesley A.Volk et al. Essentials of Medical Microbiology. Lippincott – Raven Publishers, Inc., Philadelphia–New York.–1995.–725 p.

REFERENCES:

1. Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P.59-80, 353-362

2. Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P. 109-114, 144-175.

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http://www.mansfield.ohio-state.edu/~sabedon/biol2035.htm

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http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/Chap12.html#NSD_mechanisms

http://www2.hawaii.edu/~johnb/micro/m130/m130lect12.html

http://www.merck.com/mmpe/sec14/ch167/ch167b.html

http://en.wikipedia.org/wiki/Phagocytosis

http://en.wikipedia.org/wiki/Complementary

http://www-micro.msb.le.ac.uk/MBChB/Merralls/Merralls.html

http://en.wikipedia.org/wiki/Immune_system

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http://www.answers.com/topic/lipopolysaccharide-1

http://www.answers.com/topic/flagellar-antigen

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http://www.callutheran.edu/Academic_Programs/Departments/BioDev/omm/viral_antigens/molmast.htm

http://en.wikipedia.org/wiki/Major_histocompatibility_complex

http://www.cryst.bbk.ac.uk/pps97/assignments/projects/coadwell/MHCSTFU1.HTM

http://en.wikipedia.org/wiki/Antibody

http://www.fleshandbones.com/readingroom/pdf/291.pdf