Cellular Basis of the Immune Response

Origin of Immune Cells
Cellular Basis of the Immune Response includes 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. Figure 1 describes the origin of B cells and the two types of T cells: helper T cells and cytotoxic T cells. Table 1 compares various important features of B cells and T cells. These features will be described in detail later in the chapter.

Origin of T and B cells. Stem cells in the bone marrow (or fetal liver) are the precursors of both T and B lymphocytes. Stem cells differentiate into T cells in the thymus, whereas they differentiate into B cells in the bone marrow. Within the thymus, T cells become either CD4-positive (helper) cells or CD8-positive (cytotoxic) cells. B cells can differentiate into plasma cells that produce large amounts of antibodies (immunoglobulins). Dotted lines indicate interactions mediated by interleukins.

Table 1 Comparison of T Cells and B Cells


Feature
T Cells
B Cells
Antigen receptors on surface
Yes
Yes
Antigen receptor recognizes only processed peptides in association with MHC protein
Yes
No
Antigen receptor recognizes whole, unprocessed proteins and has no requirement for presentation by MHC protein
No
Yes
IgM on surface
No
Yes
CD3 proteins on surface
Yes
No
Clonal expansion after contact with specific 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

T-cell precursors differentiate into immunocompetent T cells within the thymus. Prior to entering the thymus, stem cells lack antigen receptors and lack CD3, CD4, and CD8 proteins on their surface. During passage through the thymus they differentiate into T cells that can express both antigen receptors and the various CD proteins. 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-positive cells are located in the medulla, from which they migrate out of the thymus into the blood and extrathymic tissue.

Within the thymus, two very important processes called thymic education occur:

• CD4-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 our own proteins, i.e., self-tolerance, and prevents autoimmune reactions.

For negative selection to be efficient, the thymic epithelial cells must display a vast repertoire of "self" proteins. A transcriptional regulator called the "autoimmune regulator" (AIRE) enhances the synthesis of this array of self proteins. Mutations in the gene encoding the AIRE protein results in the development of an autoimmune disease called autoimmune polyendocrinopathy.

• CD4-positive, CD8-positive cells bearing antigen receptors that do not react with self MHC proteins (Figure 2) are also killed. This results in a positive selection for T cells that react well with self MHC proteins.

             Development of T cells. Note the positive and negative selection that occurs in the thymus.



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 required for an effective immune response by T cells.

Note that MHC proteins perform two essential functions in the immune response: one is the positive 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 proteins are also the most important antigens recognized in the graft rejection process.

During their passage through the thymus, each double-positive T cell synthesizes a different, highly specific antigen receptor called the T-cell receptor (TCR). The rearrangement of the variable, 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 but rather in the "gut-associated lymphoid tissue" (GALT). These intraepithelial lymphocytes (IELs) are thought to provide protection against intestinal pathogens. Their antigen receptors and surface proteins are different from those of thymus-derived lymphocytes. IELs cannot substitute for thymus-derived lymphocytes because patients with DiGeorge's syndrome who lack a thymus are 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.

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. This detection process is effective because many cells lose their ability to synthesize class I MHC proteins after they have been infected by a virus (see Natural Killer Cells).

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. Macrophages also display class I MHC proteins, as do all nucleated cells. The cell surface proteins that play an important role in the immune response are listed in Table 2.

Table 2 Cell Surface Proteins that Play an Important Role in the Immune Response1


Type of Cells
Surface Proteins


Helper T cells
CD4, TCR,2 CD28


Cytotoxic T cells
CD8, TCR


B cells
IgM, B7


Macrophages3
Class II MHC


Natural killer cells
Receptors for class I MHC


All cells other than mature red cells4
Class I MHC




1There 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."


4Mature red blood cells do not synthesize class I MHC proteins because they do not have a functioning nucleus.


T Cells
This Cellular Basis of the Immune Response 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 hypersensitivity against intracellular organisms such as Mycobacterium tuberculosis. (Suppressor T cells are postulated to downregulate the immune response, but the precise action of these cells is uncertain.) The effector functions are carried out primarily by cytotoxic (CD8-positive) T cells, which kill virus-infected cells, tumor cells, and allografts.

Table 3 Main Functions of Helper T Cells


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

CD4 & CD8 Types of T Cells
Cellular Basis of the Immune Response also includes 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, e.g., CD3, CD4, and CD8. All T cells have CD3 proteins on their surface in association with antigen receptors (T-cell receptor, TCR [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 lck kinase) also.

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 (e.g., limit infection by M. tuberculosis). These functions are performed by two sub-populations 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). Note also that the cytokines produced by Th-1 cells (e.g., gamma interferon) help B cells to class switch (see Antibody Production) to produce two subclasses of IgG (namely IgG 1 and IgG 3) that are very effective opsonizers of bacteria.

The origin of Th-1 and Th-2 cells. On the left side, the origin of Th-1 cells is depicted. Microorganisms are ingested by macrophages and IL-12 is produced. IL-12 induces naïve Th-0 cells to become Th-1 cells that produce gamma interferon and IL-2. These interleukins activate macrophages and cytotoxic T cells, respectively, and cell-mediated immunity occurs. On the right side, the origin of Th-2 cells is depicted. Microorganisms are ingested by an unknown type of cell (see footnote below) and IL-4 is produced. IL-4 induces naïve Th-0 cells to become Th-2 cells that produce IL-4 and IL-5. These interleukins activate B cells to become plasma cells and antibodies are produced. Not shown in the figure is an important regulatory step, namely, that IL-10 produced by Th-2 cells inhibits IL-12 production by macrophages and drives the system toward an antibody response and away from a cell-mediated response.
*The human cell that produces the IL-4, which induces naïve helper T cells to become Th-2 cells, has not been identified.

One important regulator of the balance between Th-1 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.

Table 4 Comparison of Th-1 Cells and Th-2 Cells


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
According toCellular Basis of the Immune Response to mount a protective immune response against a specific microbe requires that the appropriate subpopulation, i.e., 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. 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.

How the appropriate response is stimulated is known for one medically important organism, namely, M. tuberculosis. A lipoprotein of that bacterium interacts with a specific "toll-like receptor" on the surface of the macrophage, which induces the production of IL-12 by the macrophage. IL-12 drives the differentiation of naïve helper T cells to form the Th-1 type of helper T cells that are required to mount a cell-mediated (delayed hypersensitivity) response against the organism.

A subset of CD4 cells called Th-17 cells play an important role in mucosal immunity, especially in the mucosa of the GI tract. These cells are characterized by producing interleukin-17 (IL-17) rather than the typical cytokines produced by Th-1 cells, namely gamma interferon, and Th-2 cells, namely IL-4. IL-17 acts to recruit neutrophils to the site of bacterial infections. One clinical finding related to Th-17 cells is that they are selectively killed by HIV. The loss of Th-17 cells results in a high rate of blood stream infections cause by colonic bacteria, such as E. coli.

CD8 lymphocytes perform cytotoxic functions, i.e., they kill virus-infected, tumor, and allograft cells. They kill by either of two mechanisms, namely, the release of perforins, 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
Cellular Basis of the Immune Response the activation of helper T cells requires that their TCR recognize a complex on the surface of antigen-presenting cells (APCs), e.g., macrophages and dendritic cells,1 consisting of both the antigen and a class II MHC protein.

What follows is a description of this activation process beginning with the ingestion of the foreign protein (or microbe) into the APC. 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 fragments. 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 II MHC proteins can only present peptides; other types of molecules do not bind and therefore cannot be presented. Note also that MHC proteins 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.

Costimulation Is Required to Activate T Cells
In this Cellular Basis of the Immune Response two signals are required to activate T cells. The first signal in the activation process is the interaction of the antigen and the MHC protein 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, e.g., LFA-1 protein2 on T cells (both CD4-positive and CD8-positive) binds to ICAM-1 protein2 on APCs.

Activation of T cells. Left: An antigen-presenting cell (APC) presents processed antigen in association with a class II MHC protein. The antigen is recognized by the T-cell receptor (TCR) specific for that antigen, and the helper T cell is activated to produce interleukin-2 (IL-2). IL-2 binds to its receptor on the helper T cell and further activates it. Note that CD4 protein on the helper T cell binds to the MHC class II protein on the APC, which stabilizes the interaction between the two cells, and that B7 on the APC must interact with CD28 on the helper T cell for full activation of helper T cells to occur. Right: A virus-infected cell presents viral antigen in association with class I MHC protein. The viral antigen is recognized by the TCR specific for that antigen and, in conjunction with IL-2 produced by the helper T cell, the cytotoxic T cell is activated to kill the virus-infected cell. The CD8 protein on the cytotoxic T cell binds to the class I protein on the virus-infected cell, which stabilizes the interaction between the two cells. Note that the class II MHC protein consists of two polypeptides, both of which are encoded by genes in the HLA locus. The class I protein, in contrast, consists of one polypeptide encoded by the HLA locus and 2-microglobulin (2 MG), which is encoded elsewhere.

A second costimulatory signal is also required, i.e., 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. Other helper T cells specific for other epitopes are not affected. Production of the costimulatory protein depends on activation of the "toll-like receptor" on the APC surface. Foreign antigens, such as bacterial proteins, induce B7 protein, whereas self antigens do not.

After the T cell has been activated, a different protein called CTLA-4 (cytotoxic T lymphocyte antigen-4) appears on the T-cell surface and binds to B7 by displacing CD28. The interaction of B7 with CTLA-4 inhibits T-cell activation by blocking IL-2 synthesis (Figure 5). 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.

Inhibition of activated helper T cells. When the activated helper T cells are no longer needed, a return to a quiescent state occurs when an inhibitory protein called CTL-4 is displayed on the surface of the helper T cell. CTL-4 binds more strongly to B7 than does CD28 and so displaces CD28 from its interaction with B7. This inhibits the synthesis of IL-2 and the T cell enters a resting state. Left: Activation of the helper T cells occurs because B7 protein is displayed on the surface of the antigen-presenting cell and interacts with CD28 on the helper T cell. (This is the same process as that depicted on the left side of Figure 4.) Right: CTL-4 protein is displayed on the surface of the helper T cell and interacts with B7 on the antigen-presenting cell. As a result, IL-2 is no longer synthesized. (Modified and reproduced with permission from Pantaleo G et al. Mechanisms of disease: The immunopathogenesis of human immunodeficiency virus infection. NEJM. 1993;328:327.)

The clinical importance of CTLA-4 is dramatically illustrated by the effectiveness of abatacept (Orencia) in rheumatoid arthritis. Abatacept is CTLA-4-IG, a fusion protein composed of CTLA-4 and a fragment of the Fc domain of human IgG. The Fc fragment provides resistance against degradation, resulting in increased plasma levels of CTLA-4 for a longer duration than CTLA-4 alone. The mechanism of action of abatacept is the binding of CTLA-4 to B7 thereby displacing CD-28 from its binding to B7. This results in a reduction of the helper T cell activity and a reduction in the inflammatory response.

T Cells Recognize Only Peptides
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 I MHC proteins. This is called MHC restriction, i.e., 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 in this Cellular Basis of the Immune Response Chapter.

Generally speaking, class I MHC proteins present endogenously synthesized antigens, e.g., viral proteins, whereas class II MHC proteins present the antigens of extracellular microorganisms that have been phagocytized, e.g., 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, e.g., 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 molecules, e.g., drugs such as penicillin.

These differences between T cells and B cells explain the hapten-carrier relationship. To stimulate hapten-specific antibody, the hapten must be covalently bound to the carrier protein. The hapten binds to the IgM receptor on the B-cell surface. That IgM is specific for the hapten, not the carrier protein. The hapten-carrier conjugate is internalized and the carrier protein processed into small peptides that are presented in association with class II MHC proteins 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, e.g., 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. (Gamma interferon also enhances the microbicidal activity of macrophages.)

The process of activating T cells does not function as a simple "on–off" switch. The binding of an epitope to the T-cell receptor can result in either 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 (see Chapter 62). 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 according to Cellular Basis of the Immune Response chapter.

Memory T Cells
This important Cellular Basis of the Immune Response Chapter also describes about 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 naïve, unactivated T cells; and (4) activated memory cells produce greater amounts of interleukins than do naïve T cells when they are first activated.

T-Cell Receptor
The T-cell receptor (TCR) for antigen consists of two polypeptides, alpha and beta,3 which are associated with CD3 proteins.

TCR polypeptides are similar to immunoglobulin heavy chains in that (1) the genes that code for them are formed by rearrangement of 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; (3) the variable regions have hypervariable domains; and (4) the two genes (RAG-1 and RAG-2) that encode the recombinase enzymes that catalyze these gene rearrangements are similar in T cells and B 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 (antibodies) 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 MHC proteins, whereas immunoglobulins recognize free antigen. Note that the receptor on the surface of B cells (either IgM or IgG) recognizes antigen directly without the need for presentation by MHC proteins. Also TCR proteins are always anchored into the outer membrane of T cells. There is no circulating form as there is with certain antibodies (e.g., monomeric IgM is in the B-cell membrane, but pentameric IgM circulates in the plasma).

Effect of Superantigens on T Cells

Certain proteins, particularly staphylococcal enterotoxins and toxic shock syndrome toxin, act as "superantigens" (Figure 6). In contrast to the typical (nonsuper) antigen, which activates one (or a few) helper T cell, superantigens are "super" because they 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 (V) of the T-cell receptor of many T cells are very well described in this Cellular Basis of the Immune Response Chapter,
.

Activation of helper T cells by superantigen. Top: The helper T cell is activated by the presentation of processed antigen in association with class II MHC protein to the antigen-specific portion of the T-cell receptor. Note that superantigen is not involved and that only one or a small number of helper T cells specific for the antigen are activated. Bottom: The helper T cell is activated by the binding of superantigen to the V portion of the T-cell receptor outside of its antigen-specific site without being processed by the antigen-presenting cell. Because it bypasses the antigen-specific site, superantigen can activate many helper T cells. (Modified and reproduced with permission from Pantaleo G et al. Mechanisms of disease: The immunopathogenesis of human immunodeficiency virus infection. NEJM. 1993;328:327.)

This activates the T cells, causing the release of IL-2 from the T cells and IL-1 and TNF from macrophages. These interleukins account for many of the findings seen in toxin-mediated staphylococcal diseases. Certain viral proteins, e.g., those of mouse mammary tumor virus (a retrovirus), also possess superantigen activity.

Features of T Cells
T cells constitute 65% to 80% of the recirculating pool of small lymphocytes. Within lymph nodes, they are located in the inner, subcortical region, not in the germinal centers. (B cells make 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, e.g., 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.

Delayed Hypersensitivity
Delayed hypersensitivity reactions are produced particularly against antigens of intracellular microorganisms including certain fungi, e.g., Histoplasma and Coccidioides, and certain intracellular bacteria, e.g., M. tuberculosis. Delayed hypersensitivity is mediated by macrophages and CD4 cells, in particular by the Th-1 subset of CD4 cells. Important interleukins for these reactions include 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.

In the case of M. tuberculosis, a lipoprotein of the bacterium stimulates a specific "toll-like receptor" on the macrophage, which signals the cell to synthesize IL-12. IL-12 then induces naïve helper T cells to differentiate into the Th-1 type of helper T cells that participates in the cell-mediated (delayed hypersensitivity) response.

Th-1 cells produced gamma interferon, which activates macrophages, thereby enhancing their ability to kill M. tuberculosis. This "IL-12–gamma interferon axis" is very important in the ability of our host defenses to control infections by intracellular pathogens, such as M. tuberculosis and Listeria monocytogenes.

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 be activated by IL-2 produced by a helper (CD-4-positive) T cell. To become activated to produce IL-2, helper T cells recognize viral antigens bound to class II molecules on an APC, e.g., 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. Perforins 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 activate caspases that initiate apoptosis, resulting in 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, e.g., macrophages or NK cells, and the infected cell is killed. The ADCC process can also kill helminths (worms). 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 recognize the foreign class II molecules on certain cells in the graft, e.g., 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.

Antibody Production
Antibody production by B cells usually requires the participation of helper T cells (T-cell–dependent response), but antibodies to some antigens, e.g., 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 7A).5 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, e.g., IL-2, B-cell growth factor (IL-4), and B-cell differentiation factor (IL-5). 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.

A: B-cell activation by helper T cells. B0 is a resting B cell to which a multivalent antigen ( ) is attaching to monomer IgM receptors (Y). The antigen is internalized, and a fragment () is returned to the surface in conjunction with a class II molecule (). A receptor on an activated T cell recognizes the complex on the B-cell surface, and the T cell produces B-cell growth factor (BCGF, IL-4; ) and B-cell differentiation factor (BCDF, IL-5; ). These factors cause the progression of the B1 cell to form B2 and B3 cells, which differentiate into antibody-producing (e.g., pentamer IgM) plasma cells (PC). Memory B cells are also produced.

 B: Inducible protein B7 () on the B cell must interact with CD28 protein on the helper T cell in order for the helper T cell to be fully activated, and CD40L (CD40 ligand) on the helper T cell must interact with CD40 on the B cell for the B cell to be activated and synthesize the full range of antibodies. (Modified and reproduced with permission from Stites DP, Terr A, eds. Basic & Clinical Immunology. 7th ed. Originally published by Appleton & Lange. Copyright 1991 McGraw-Hill.)

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 7B). 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; e.g., 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 lymphokines produced by the helper T cell are needed for class switching. The T-cell–dependent response generates memory B cells, whereas the T-cell–independent response does not; therefore, a secondary antibody response (see Chapter 60) 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.

Cell-Mediated Immunity
 Cell Mediated Immunity is a part of Cellular Basis of the Immune Response in which the cell-mediated response, the initial events are similar to those described above for antibody production. The antigen is processed by macrophages, is fragmented, and is presented in conjunction with class II MHC molecules on the surface. These interact with the receptor on the helper T cell, which is then stimulated to produce lymphokines such as IL-2 (T-cell growth factor), which stimulates the specific helper and cytotoxic T cells to grow.

Suppression of Certain Immune Responses

A subset of T cells called regulatory T cells (TR) can suppress (inhibit) the effector functions of CD4 (helper) and CD8 (cytotoxic) T cells. (These cells are also called suppressor T cells.) TR cells are 5% to 10% of the CD4-positive cells and are characterized by possessing the CD25 marker. These cells also produce FoxP3, a regulator of transcription of various genes. A hallmark of TR cells that are expressing FoxP3 is the synthesis of the inhibitory surface protein, CTLA-4. Individuals whose TR cells lack the ability to make FoxP3 are predisposed to autoimmune diseases such as systemic lupus erythematosus and a rare X-linked disease characterized by polyendocrinopathy and enteropathy (IPEX).

When there is an imbalance in numbers or activity between CD4 and CD8 cells, cellular immune mechanisms are greatly impaired. For example, in lepromatous leprosy there is unrestrained multiplication of Mycobacterium leprae, a lack of delayed hypersensitivity to M. leprae antigens, a lack of cellular immunity to that organism, and an excess of CD8 cells in lesions. Removal of some CD8 cells can restore cellular immunity in such patients and limit M. leprae multiplication. In acquired immunodeficiency syndrome (AIDS), the normal ratio of CD4:CD8 cells (>1.5) is greatly reduced. Many CD4 cells are destroyed by the human immunodeficiency virus (HIV), and the number of CD8 cells increases. This imbalance, i.e., a loss of helper activity and an increase in suppressor activity, results in a susceptibility to opportunistic infections and certain tumors.

One important part of the host response to infection is the increased expression of class I and class II MHC proteins induced by various cytokines, especially interferons such as gamma interferon. The increased amount of MHC proteins leads to increased antigen presentation and a more vigorous immune response. However, certain viruses can suppress the increase in MHC protein expression, thereby enhancing their survival. For example, hepatitis B virus, adenovirus, and cytomegalovirus can prevent an increase in class I MHC protein expression, thereby reducing the cytotoxic T-cell response against cells infected by these viruses.

1Macrophages and dendritic cells are the most important antigen-presenting cells, but B cells and Langerhans' cells on the skin also present antigen, i.e., have class II proteins on their surface. An essential first step for certain antigen-presenting cells, e.g., Langerhans' cells in the skin, is migration from the site of the skin infection to the local lymphoid tissue, where helper T cells are encountered.


2LFA proteins belong to a family of cell surface proteins called integrins, which mediate adhesion to other cells. Integrin 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 internally. Abbreviations: LFA, lymphocyte function-associated antigen; ICAM, intercellular adhesion molecule.


3Some TCRs have a different set of polypeptides called gamma and delta. These TCRs are unusual because they do not require that antigen be presented in association with MHC proteins. Gamma/delta T cells constitute approximately 10% of all T cells. Some of the T cells bearing these TCRs are involved in cell-mediated immunity against M. tuberculosis.


4Each superantigen, e.g., the different staphylococcal enterotoxins, interacts with different V chains. This explains why many but not all helper T cells are activated by the various superantigens.


5Note that one important difference between B cells and T cells is that B cells recognize antigen itself, whereas T cells recognize antigen only in association with MHC proteins.

B Cells
B cells perform two important functions: (1) they differentiate into plasma cells and produce antibodies and (2) they can present antigen to helper T cells.

Origin
During embryogenesis, 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 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, e.g., activated B cells and plasma cells (Figure 8). 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. The monomeric IgM on the surface has an extra transmembrane domain that anchors the protein in the cell membrane that is not present in the circulating pentameric form of IgM. 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 bloodstream.

Maturation of B cells. B cells arise from stem cells and differentiate into pre–B cells expressing heavy chains in the cytoplasm and then into B cells expressing monomer IgM on the surface. This occurs independent of antigen. Activation of B cells and differentiation into plasma cells is dependent on antigen. Cells to the left of the vertical dotted line do not have IgM on their surface, whereas B cells, to the right of the vertical line, do have IgM. , mu heavy chains in cytoplasm; Y, IgM. (Modified and reproduced with permission from Stites DP, Terr A, eds. Basic & Clinical Immunology. 7th ed. Originally published by Appleton & Lange. Copyright 1991 McGraw-Hill.)

B cells constitute about 30% of the recirculating pool of small lymphocytes, and their life span is short, i.e., 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, e.g., 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 is very well covered in this Chapter.

It appears that the latter alternative, i.e., 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); i.e., there are about 107 different specificities. An antigen interacts with the B lymphocyte that shows the best "fit" 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 (i.e., 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.6 The T cell now produces various interleukins (IL-2, IL-4, 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.

Effector Functions of B Cells/Plasma Cells
Cellular Basis of the Immune Response describes the end result of the activation process is the production of many plasma cells that produce large amounts of immunoglobulins specific for the epitope. 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 periods 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.

6Macrophages 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-1 is needed.

Antigen-Presenting Cells

Macrophages

Macrophages have three main functions: phagocytosis, antigen presentation, and cytokine production (Table 58–5).

1. Phagocytosis. Macrophages ingest bacteria, viruses, and other foreign particles. They have surface Fc receptors that interact with the Fc portion of IgG, thereby enhancing the uptake of opsonized organisms. Macrophages also have receptors for C3b, another important opsonin. After ingestion, the phagosome containing the microbe fuses with a lysosome. The microbe is killed within this phagolysosome by reactive oxygen and reactive nitrogen compounds and by lysosomal enzymes.
2. Antigen presentation. Foreign material is ingested and degraded, and fragments of antigen are presented on the macrophage cell surface (in conjunction with class II MHC proteins) for interaction with the TCR of CD4-positive helper T cells. Degradation of the foreign protein stops when the fragment associates with the class II MHC protein in the cytoplasm. The complex is then transported to the cell surface by specialized "transporter" proteins.
3. Cytokine production. Macrophages produce several cytokines (macrokines, monokines), the most important of which are IL-1 and TNF. IL-1 (endogenous pyrogen) plays a role in the activation of helper T cells, and TNF is an important inflammatory mediator (see Cytokines Affecting Macrophages & Monocytes). In addition, macrophages produce interleukin-8 (IL-8), an important chemokine that attracts neutrophils and T cells to the site of infection.

Table 5 Important Features of Macrophages


Functions
Mechanisms


Phagocytosis
Ingestion and killing of microbes in phagolysosomes. Killing caused by reactive oxygen intermediates such as superoxides, reactive nitrogen intermediates such as nitric oxide, and lysosomal enzymes such as proteases, nucleases, and lysozyme.


Antigen presentation
Presentation of antigen in association with class II MHC proteins to CD4-positive helper T cells. Also displays B7 protein, which acts as a costimulator of helper T cells.


Cytokine production
Synthesis and release of cytokines such as IL-1 and TNF, and chemokines such as IL-8.

These three functions are greatly enhanced when a process called macrophage activation occurs. Macrophages are activated initially by substances such as bacterial lipopolysaccharide (LPS, endotoxin), by bacterial peptidoglycan, and by bacterial DNA. (Human DNA is methylated, whereas bacterial DNA is unmethylated and therefore is perceived as foreign.) These substances interact with "toll-like receptors" on the macrophage surface and signal the cell to produce certain cytokines. Macrophages are also activated by gamma interferon produced by helper T cells. Gamma interferon increases the synthesis of class II MHC proteins, which enhances antigen presentation and increases the microbicidal activity of macrophages.

Macrophages are derived from bone marrow histiocytes and exist both free, e.g., monocytes, and fixed in tissues, e.g., Kupffer cells of the liver. Macrophages migrate to the site of inflammation, attracted by certain mediators, especially C5a, an anaphylatoxin released in the complement cascade.

Dendritic Cells
Dendritic cells are a third type of cell that function as "professional" antigen-presenting cells (macrophages and B cells are the other two), i.e., they express class II MHC proteins and present antigen to CD4-positive T cells. They are particularly important because they are the main inducers of the primary antibody response. The name "dendritic" describes their many long, narrow processes (that resemble neuronal dendrites), which make them very efficient at making contact with foreign material. Dendritic cells are primarily located under the skin and the mucosa, e.g., Langerhans' cells in the skin. Dendritic cells migrate from their peripheral location under the skin and mucosa to local lymph nodes, where they present antigen to helper T cells.

Summary of the Interaction of Antigen-Presenting Cells, T Cells, & B Cells
The interactive process is initiated by the ingestion of a microbe by an antigen-presenting cell, for example, the ingestion of a bacterium by a dendritic cell in the skin. The dendritic cell migrates to the lymph node via lymph vessels, attracted there by chemokines. In the lymph node, the dendritic cell presents antigen to the T cell bearing a receptor specific for that antigen. While this process is occurring, fragments of the microbe circulate to the lymph node and bind directly to the B cell antigen receptor (membrane IgM). The antigen is internalized, processed, and presented to helper T cells with the correct receptor. Various chemokines and chemokine receptors (such as CCR7) facilitate the migration of these cells to a junctional area in the lymph node where they have a high probability of interacting with each other. The proximity of the B cell to the helper T cell allows interleukins produced by the helper T cell to efficiently activate antibody synthesis by the B cell.

Follicular Dendritic Cells
These cells have a similar appearance to the dendritic cells mentioned above but are quite different from them in their location and function. Follicular dendritic cells (FDCs) are located in the B-cell–containing germinal centers of the follicles in the spleen and lymph nodes. They do not present antigen to helper T cells because they do not produce class II MHC proteins. Rather, they capture antigen–antibody complexes via Fc receptors located on their surface. The antigen–antibody complexes are then detected by activated B cells. The antibody produced by these B cells undergoes affinity maturation. (Affinity maturation is the improvement in the affinity of an antibody for the antigen that occurs upon repeated exposure to the antigen.) Affinity maturation is described in Chapter 60. In addition, FDCs produce chemokines that attract B cells to the follicles in the spleen and lymph nodes.

Natural Killer Cells
NK cells play two important roles in our innate host defenses: (1) they kill virus-infected cells and (2) they produce gamma interferon that activates macrophages to kill bacteria ingested by the macrophage (Table 6).

Table 6 Important Features of Natural Killer (NK) Cells


I. Nature of NK Cells
Large granular lymphocytes
Lack T-cell receptor, CD3 proteins, and surface IgM and IgD
Thymus not required for development
Normal numbers in severe combined immunodeficiency disease (SCID) patients
Activity not enhanced by prior exposure
II. Function of NK Cells
Kill virus-infected cells and cancer cells
Killing is nonspecific and is not dependent on foreign antigen presentation by class I or II MHC proteins
Produce gamma interferon that activates macrophages to kill ingested bacteria



NK cells specialize in killing virus-infected cells and tumor cells by secreting cytotoxins (perforins and granzymes) similar to those of cytotoxic T lymphocytes and by participating in Fas-Fas ligand-mediated apoptosis. They are called "natural" killer cells because they are active without prior exposure to the virus, are not enhanced by exposure, and are not specific for any virus. They can kill without antibody, but antibody (IgG) enhances their effectiveness, a process called antibody-dependent cellular cytotoxicity (ADCC) (see the section on effector functions of T cells [above]). IL-12 produced by macrophages and interferons alpha and beta produced by virus-infected cells are potent activators of NK cells. Approximately 5% to 10% of peripheral lymphocytes are NK cells.

NK cells are lymphocytes with some T-cell markers, but they do not have to pass through the thymus in order to mature. They have no immunologic memory and, unlike cytotoxic T cells, have no T-cell receptor; also, killing does not require recognition of MHC proteins. In fact, NK cells have receptors that detect the presence of class I MHC proteins on the cell surface. If a cell displays sufficient class I MHC proteins, that cell is not killed by the NK cell. Many virus-infected cells and tumor cells display a significantly reduced amount of class I MHC proteins, and it is those cells that are recognized and killed by the NK cells. Humans who lack NK cells are predisposed to life-threatening infections with varicella-zoster virus and cytomegalovirus.

NK cells detect the presence of cancer cells by recognizing a protein called MICA that is found on the surface of many cancer cells but not normal cells. Interaction of MICA with a receptor on NK cells triggers the production of cytotoxins by the NK cell and death of the tumor cell.

Polymorphonuclear Neutrophils
Neutrophils are a very important component of our innate host defenses, and severe bacterial infections occur if they are too few in number (neutropenia) or are deficient in function, as in chronic granulomatous disease. They have cytoplasmic granules that stain a pale pink (neutral) color with blood stains such as Wright stain, in contrast to eosinophils and basophils whose granules stain red and blue, respectively. These granules are lysosomes, which contain a variety of degradative enzymes that are important in the bactericidal action of these cells in this Cellular Basis of the Immune Response  Chapter.

Neutrophils have receptors for IgG on their surface so IgG is the only immunoglobulin that opsonizes, i.e., makes bacteria more easily phagocytosed. Note that neutrophils do not display class II MHC proteins on their surface and therefore do not present antigen to helper T cells. This is in contrast to macrophages that are also phagocytes but do present antigen to helper T cells.

Neutrophils can be thought of as a "two-edged" sword. The positive edge of the sword is their powerful microbicidal activity, but the negative edge is the tissue damage caused by the release of degradative enzymes. An excellent example of the latter is the damage to the glomeruli in acute poststreptococcal glomerulonephritis. The damage is caused by enzymes released by neutrophils attracted to the glomeruli by C5a activated by the antigen–antibody complexes deposited on the glomerular membrane.

Eosinophils
Eosinophils are white blood cells with cytoplasmic granules that appear red when stained with Wright stain. The red color is caused by the negatively charged eosin dye binding to the positively charged major basic protein in the granules. The eosinophil count is elevated in two medically important types of diseases: parasitic diseases, especially those caused by nematodes, and hypersensitivity diseases, such as asthma and serum sickness. Diseases caused by protozoa are typically not characterized by eosinophilia.

The function of eosinophils has not been clearly established. It seems likely that their main function is to defend against the migratory larvae of nematodes, such as Strongyloides and Trichinella. They attach to the surface of the larvae and discharge the contents of their granules, which in turn damages the cuticle of the larvae. Attachment to the larvae is mediated by receptors on the eosinophil surface for the Fc portion of the heavy chain of IgG and IgE.

Another function of eosinophils may be to mitigate the effects of immediate hypersensitivity reactions because the granules of eosinophils contain histaminase, an enzyme that degrades histamine, which is an important mediator of immediate reactions. However, the granules of the eosinophils also contain leukotrienes and peroxidases, which can damage tissue and cause inflammation. The granules also contain major basic protein that damages respiratory epithelium and contributes to the pathogenesis of asthma.

Eosinophils can phagocytose bacteria but they do so weakly and are not sufficient to protect against pyogenic bacterial infections in neutropenic patients. Although they can phagocytose, they do not present antigen to helper T cells. The growth and differentiation of eosinophils is stimulated by interleukin-5.

Basophils & Mast Cells
Basophils are white blood cells with cytoplasmic granules that appear blue when stained with Wright stain. The blue color is caused by the positively charged methylene blue dye binding to several negatively charged molecules in the granules. Basophils circulate in the bloodstream, whereas mast cells, which are similar to basophils in many ways, are fixed in tissue, especially under the skin and in the mucosa of the respiratory and gastrointestinal tracts.

Basophils and mast cells have receptors on their surface for the Fc portion of the heavy chain of IgE. When adjacent IgE molecules are cross-linked by antigen, immunologically active mediators, such as histamine, and enzymes, such as peroxidases and hydrolases, are released. These cause inflammation and, when produced in large amounts, cause severe immediate hypersensitivity reactions such as systemic anaphylaxis.

Mast cells also play an important role in the innate response to bacteria and viruses. The surface of mast cells contain Toll-like receptors that recognize bacteria and viruses. The mast cells respond by releasing cytokines and enzymes from their granules that mediate inflammation and attract neutrophils and dendritic cells to the site of infection. Dendritic cells are important antigen-presenting cells that initiate the adaptive response. The role of mast cells in inflammation has been demonstrated in rheumatoid arthritis. These cells produce both inflammatory cytokines and the enzymes that degrade the cartilage in the joints.

Important Cytokines
The important functions of the main cytokines are described in Table 7. Note that the three important proinflammatory cytokines are IL-1, IL-6, and tumor necrosis factor (TNF). The term "proinflammatory" means to stimulate or enhance inflammation. The main anti-inflammatory cytokines are IL-10 and transforming growth factor .

Table 7 Important Functions of the Main Cytokines


Major Source
Cytokine
Important Functions


Macrophages
Interleukin-1
Proinflammatory cytokine. Induces fever. Induces liver to produce acute phase proteins.




Interleukin-6
Proinflammatory cytokine. Induces fever. Induces liver to produce acute phase proteins.




Tumor necrosis factor
Proinflammatory cytokine. Low concentration: activates neutrophils and increases their adhesion to endothelial cells. High concentration: mediates septic shock, acts as cachectin, causes necrosis of tumors.




Interleukin-12
Drives development of Th-1 subset of T cells.


Th-1 subset of helper T cells
Interleukin-2
T-cell growth factor. Stimulates growth of both helper (CD4) and cytotoxic (CD8) T cells.




Gamma interferon
Stimulates phagocytosis and killing by macrophages. Increases class I and II MHC protein expression. Inhibits growth of Th-2 cells.


Th-2 subset of helper T cells
Interleukin-4
Drives development of Th-2 subset of T cells. Stimulates B-cell growth. Increases isotype class switching to IgE.




Interleukin-5
Increases number of eosinophils. Increases isotype class switching to IgA.




Interleukin-10
Antiinflammatory cytokine. Inhibits development of Th1 subset of T cells.


Th-17 subset of T cells
Interleukin-17
Recruits neutrophils to site of infection. Important in gut mucosal immunity.


Many cells including macrophages, T cells, and B cells.
Transforming Growth Factor-
Antiinflammatory cytokine. Inhibits activation of T cells. Increases isotype switching to IgA.



Cytokines Affecting Lymphocytes

cytokines Affecting Lymphocytes is an important part of Cellular Basis of the Immune Response it according to which IL-1 is produced mainly by macrophages It is a proinflammatory cytokine, i.e., plays an important role, along with tumor necrosis factor (TNF), in inducing inflammation. In addition, IL-1 is endogenous pyrogen, which acts on the hypothalamus to cause the fever associated with infections and other inflammatory reactions. (Exogenous pyrogen is endotoxin, a lipopolysaccharide found in the cell wall of gram-negative bacteria.)

IL-2 is produced mainly by helper T cells. It stimulates both helper and cytotoxic T cells to grow. IL-2 is T-cell growth factor. Resting T cells are stimulated by antigen (or other stimulators) both to produce IL-2 and to display IL-2 receptors on their surface, thereby acquiring the capacity to respond to IL-2. Interaction of IL-2 with its receptor stimulates DNA synthesis allowing cell division to occur.

IL-4 is produced by the Th-2 class of helper T cells. IL-4 stimulates the development of Th-2 cells from T cells that have been activated by exposure to antigen. It also induces class switching to IgE. IL-4 is the "signature" (most characteristic) cytokine produced by Th-2 cells (Figure 3).

IL-5 is produced by the Th-2 class of helper T cells. It induces class switching to IgA thereby increasing mucosal immunity. It also increases the number and activity of eosinophils. Eosinophils are an important host defense against many helminths (worms), e.g., Strongyloides and are increased in immediate hypersensitivity (allergic) reactions.

IL-6 is produced mainly by macrophages. It is a proinflammatory cytokine that induces fever by affecting the hypothalamus and induces the production of acute-phase proteins by the liver. Acute-phase proteins are described in Innate Immunity.

IL-10 and IL-12 regulate the production of Th-1 cells, the cells that mediate delayed hypersensitivity (Figure 58–3). IL-12 is produced by macrophages and promotes the development of Th-1 cells, whereas IL-10 is produced by Th-2 cells and inhibits the development of Th-1 cells. The relative amounts of IL-4, IL-10, and IL-12 drive the differentiation of Th-1 and Th-2 cells and therefore enhance either cell-mediated or humoral immunity, respectively. This is likely to have important medical consequences because the main host defense against certain infections is either cell-mediated or humoral immunity. For example, Leishmania infections in mice are lethal if a humoral response predominates but are controlled if a vigorous cell-mediated response occurs.

The IL-12–gamma interferon axis is very important in the ability of our host defenses to control infections by intracellular pathogens, such as M. tuberculosis and L. monocytogenes. IL-12 increases the number of Th-1 cells, and Th-1 cells produce the gamma interferon that activates the macrophages that phagocytose and kill the intracellular bacterial pathogens mentioned above.

IL-13 is implicated as the mediator of allergic airway disease (asthma). IL-13 is made by Th-2 cells and binds to a receptor that shares a chain with the IL-4 receptor. In animals, IL-13 was shown to be necessary and sufficient to cause asthma. IL-13 is involved in producing the airway hyperresponsiveness seen in asthma but not in increasing the amount of IgE.

The main function of transforming growth factor- (TGF-) is to inhibit the growth and activation of T cells. It is an "anti- inflammatory" cytokine. Although it is a "negative regulator" of the immune response, it stimulates wound healing by enhancing the synthesis of collagen. It is produced by many types of cells, including T cells, B cells, and macrophages. In summary, the role of TGF- is to dampen or suppress the immune response when it is no longer needed after an infection and to promote the healing process.

Cytokines Affecting Macrophages & Monocytes
Chemokines are a group of cytokines that can attract either macrophages or neutrophils to the site of infection. The term "chemokine" is a contraction of chemotactic and cytokine. Chemokines are produced by various cells in the infected area, such as endothelial cells and resident macrophages. The circulating neutrophils and macrophages (monocytes) are attracted to the site by an increasing gradient of chemokines, then bind to selectins on the endothelial cell surface. Chemokines also activate integrins on the surface of the neutrophils and macrophages that bind to ICAM proteins on the endothelial cell surface. The interaction between integrin and ICAM facilitates the movement of the white cells into the tissue to reach the infected area.

Approximately 50 chemokines have been identified; they are small polypeptides ranging in size from 68 to 120 amino acids. The alpha-chemokines have two adjacent cysteines separated by another amino acid (Cys-X-Cys), whereas the beta-chemokines have two adjacent cysteines (Cys-Cys) (Table 8). The alpha-chemokines attract neutrophils and are produced by activated mononuclear cells. IL-8 is a very important member of this group. The beta-chemokines attract macrophages and monocytes and are produced by activated T cells. RANTES and MCAF are important beta-chemokines.

Table 8 Chemokines of Medical Importance


Class
Chemistry
Attracts
Produced by
Examples


Alpha
C-X-C
Neutrophils
Activated mononuclear cells
Interleukin-8


Beta
C-C
Monocytes
Activated T cells
RANTES,1 MCAF2

1RANTES is an abbreviation for regulated upon activation, normal T expressed and secreted.


2MCAF is an abbreviation for macrophage chemoattractant and activating factor.

There are specific receptors for chemokines on the surface of cells, such as neutrophils and monocytes. Interaction of the chemokine with its receptor results in changes in cell surface proteins that allow the cell to adhere to and migrate through the endothelium to the site of infection.

Cytokines Affecting Polymorphonuclear Leukocytes
TNF activates the phagocytic and killing activities of neutrophils and increases the synthesis of adhesion molecules by endothelial cells. The adhesion molecules mediate the attachment of neutrophils at the site of infection.

Chemotactic factors for neutrophils, basophils, and eosinophils selectively attract each cell type. Interleukin-8 and complement component C5a are important attractants for neutrophils.

Leukocyte-inhibitory factor inhibits migration of neutrophils, analogous to migration-inhibitory factor (below). Its function is to retain the cells at the site of infection.

IL-17 produced by Th-17 T cells recruits neutrophils to the site of infection. IL-17 plays an important role in mucosal immunity, especially in the GI tract.

Cytokines Affecting Stem Cells
IL-3 is made by activated helper T cells and supports the growth and differentiation of bone marrow stem cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim) is made by T lymphocytes and macrophages. It stimulates the growth of granulocytes and macrophages and enhances the antimicrobial activity of macrophages. It is used clinically to improve regeneration of these cells after bone marrow transplantation. Granulocyte colony-stimulating factor (G-CSF, filgrastim) is made by various cells, e.g., macrophages, fibroblasts, and endothelial cells. It enhances the development of neutrophils from stem cells and is used clinically to prevent infections in patients who have received cancer chemotherapy. The stimulation of neutrophil production by G-CSF and GM-CSF results in the increased number of these cells in the peripheral blood after infection.

Cytokines Produced by Macrophages that Affect Other Cells

1. TNF- is a proinflammatory cytokine produced primarily by macrophages. It has many important effects that differ depending on the concentration. At low concentrations, it increases the synthesis of adhesion molecules by endothelial cells, which allows neutrophils to adhere to blood vessel walls at the site of infection. It also activates the respiratory burst within neutrophils, thereby enhancing the killing power of these phagocytes. It also causes fever.At high concentrations, it is an important mediator of endotoxin-induced septic shock; antibody to TNF- prevents the action of endotoxin. (The action of endotoxin is described in Chapter 7.) TNF mediates septic shock by inducing fever and causing hypotension through vasodilation and an increase in capillary permeability.TNF- is also known as cachectin because it inhibits lipoprotein lipase in adipose tissue, thereby reducing the utilization of fatty acids. This results in cachexia. TNF-, as its name implies, causes the death and necrosis of certain tumors in experimental animals. It may do this by promoting intravascular coagulation that causes infarction of the tumor tissue. Note the similarity of this intravascular coagulation with the DIC of septic shock, both of which are caused by TNF-.
2. Nitric oxide (NO) is an important mediator made by macrophages in response to the presence of endotoxin, a lipopolysaccharide found in the cell wall of gram-negative bacteria. NO causes vasodilation, which contributes to the hypotension seen in septic shock. Inhibitors of NO synthase, the enzyme that catalyzes the synthesis of NO from arginine, can prevent the hypotension associated with septic shock.
3. Macrophage migration inhibitory factor (MIF) is another important mediator made by macrophages in response to endotoxin. The function of MIF is to retain the macrophages at the site of infection. Recent studies have shown that MIF plays a major role in the induction of septic shock. Antibody against MIF can prevent septic shock in animals genetically incapable of producing TNF. The mechanism of action of MIF in septic shock is unclear at this time.

Cytokines with Other Effects
Interferons are glycoproteins that block virus replication and exert many immunomodulating functions. Alpha interferon (from leukocytes) and beta interferon (from fibroblasts) are induced by viruses (or double-stranded RNA). These interferons exert a powerful antiviral activity by inducing the synthesis of a ribonuclease that degrades viral mRNA thereby inhibiting viral replication. They also activate NK cells causing those cells to kill virus-infected cells more effectively.

Gamma interferon is a lymphokine produced primarily by the Th-1 subset of helper T cells and is the "signature" cytokine involved in the inflammation mediated by those cells. It is one of the most potent activators of the phagocytic activity of macrophages, NK cells, and neutrophils, thereby enhancing their ability to kill microorganisms and tumor cells. For example, it greatly increases the killing of intracellular bacteria, such as M. tuberculosis, by macrophages. It also increases the synthesis of class I and II MHC proteins in a variety of cell types. This enhances antigen presentation by these cells.

Lymphotoxin (also known as TNF-) is made by activated T lymphocytes and causes effects similar to those of TNF-. It binds to the same receptor as TNF- and hence has the same effects as TNF. All the above topic and sub topic are very well described in this Chapter.