Immunology - Overview of immunology

OVERVIEW OF IMMUNITY


INTRODUCTION
The events leading to the development of immunity directed against pathogens are exceedingly complex. There are two distinct systems—innate and adaptive—that act in concert as well as separately in the development of immunity. The innate system provides a fi rst line of defense against a foreign substance. It is nonspecifi c, rapid, lacks immunologic memory, and is usually of short duration. The adaptive system has exquisite specifi city, is slower in development, exhibits immunological memory, and is long lasting. Innate and adaptive immune systems are distinct systems but interact at several levels to develop a complete defense against invading pathogens. Both systems have mechanisms for distinguishing self from nonself, therefore, under normal situations they are not directed against the host’s tissues and cells. This chapter is intended to provide an introduction to the development of immunity. The purpose is to provide an overview of the different cells involved in both systems and the interactions occurring between the cells. The vigorous signaling mechanisms and the regulatory cells and enhancer and suppressor substances will be discussed in the context of developing immunity. This chapter serves as a broad overview.

OVERVIEW OF INNATE IMMUNITY
Elements of the innate immune system have been known for many years. However, in the past few years there has been a greater focus on innate immunity and its role in protection against infection and tissue injury [1] and its role in tolerance to selfantigens. Innate immunity defi nes a collection of protective mechanisms the host uses to prevent or minimize infection. The innate immune system operates in the absence of the specifi c adaptive immune system but is tied to adaptive immunity in many ways. The innate immune system is characterized by a rapid response to an invading pathogen or foreign or effete cells. In addition to the rapid response, it is also nonspecifi c and usually of a short duration. Innate immunity lacks immunological memory and there is no clonal expansion of lymphocytes as seen in the adaptive immune response. The innate immune response is also important in directing the specifi c, long-lived adaptive immune response. The host defense mechanisms associated with innate immunity consist of a number of physical barriers (intact skin) and secretions accompanied by a number of serum factors such as complement, certain cytokines, and natural immunoglobulins [2]. The cellular components of innate immunity include a number of cell types, many of which are found at potential points of entry of pathogens [3]. Examples of these cells include natural killer (NK) cells, polymorphonuclear neutrophils (PMNs), macrophages, and dendritic cells (DCs). The intact skin and mucosal tissues provide considerable protection against invading infectious agents. However, once the agents pass through the skin a number of important events take place. This includes activation of the complement cascade that triggers the development of a number of substances to attract phagocytes to the area. A number of antimicrobial peptides are produced at epithelial cell surfaces. These antimicrobial peptides play an important role in local defense mechanisms, disrupt bacterial cell membranes, and probably play a role in preventing skin infections.
ANTIMICROBIAL PEPTIDES
Human β-defensins are produced by epithelial cells in the mucous membranes of the airways and intestinal tract [4]. Defensins are small cationic peptides that have broad antimicrobial activities against a number of microbial agents [4] including Gram-positive and Gram-negative bacteria, fungi, and enveloped viruses. Defensins are nonglycosylated peptides containing approximately 35 amino acid residues, and β-defensins have six cysteine residues that provide a distinct structure. Stimulation of the epithelium by certain cytokines can induce defensin production. The exact mode of action of defensins’ antimicrobial activity is unknown. It is likely that defensins cause membrane disruption resulting from electrostatic interaction with the polar head groups of membrane lipids [5].
There are three defensin subfamilies: α-defensins, β-defensins, and θ-defensins [5]. The α- and β-defensins are products of distinct gene families and are structurally different from the θ-defensins. The θ-defensins are not seen in humans and probably represent a mutated form of the α-defensins. The α-defensins were fi rst purifi ed from azurophilic granules of PMNs [6]. Several species, including rabbits, humans, and some rodents have α-defensins in their PMNs. Human monocytes and NK cells produce α-defensins that are similar to the α- defensins of PMNs [6]. The α-defensins play a role in the oxygen-independent killing of microorganisms after phagocytosis by PMNs [5]. The β-defensins are expressed in epithelial cells and leukocytes throughout the body. The β-defensins work in concert with a number of other components of the innate system to provide an important defense against microorganisms. There are at least four human β-defensins (HBD-1–HBD-4) [7]. Production of β-defensins may be constitutive or inducible. HBD-2, -3, and -4 are inducible. There is evidence that certain epithelial cells can be stimulated to produce HBD-2 in response to activation by bacterial products and toll-like receptors (TLRs) found on the epithelial cells [7]. Defensins also appear to have immunoregulatory properties in addition to their antimicrobial properties [8]. Immunoregulatory properties include chemoattractants for PMNs, immature DCs (iDCs), mast cells, and some memory T cells. Defensins may also stimulate iDCs to undergo maturation [8]. Several other antimicrobial peptides have been described in epithelial cells and PMNs. Lysozyme was described as an antimicrobial peptide found in human neutrophils and is known to attack the peptidoglycan cell walls of bacteria. Cathelicidin is expressed in human cells such as epithelial cells, PMNs, monocytes, and T, B, and NK cells [9]. It is usually expressed by cells lining the respiratory and gastrointestinal tracts. Cathelicidin is a well-known chemoattractant for various cells including PMNs, mast cells, monocytes, and thymus-derived lymphocytes (T lymphocytes). Cathelicidin has antimicrobial activity against most Gram-positive and Gram- negative bacteria. Histatins are a family of cationic peptides (MW = 3–4 kDa) that are present in human saliva [6]. Histatins probably play an important role in oral health by providing potent antibacterial and antifungal actions. Of the several known histatins, histatin 5 is the most potent antifungal agent and is secreted by human parotid and submandibular glands [10].
The human skin, when intact, is refractory to most pathogens. This natural resistance is reportedly due to the presence of constitutively produced and inducible antimicrobial peptides. These peptides are cathelicidins, defensins, and dermicidins [9,7,11]. These antimicrobial peptides appear to act by directly inhibiting pathogen growth and enhancing other components of the immune responses. Psoriasin is another antimicrobial peptide found in the skin, especially in areas where bacterial invasion is likely to occur [12]. Psoriasin shows bactericidal activity preferentially against Escherichia coli, and also shows activity against other organisms that may colonize the skin. Together, these antimicrobial peptides and proteins contribute signifi cantly by providing a “chemical barrier” to reenforce the physical barriers of the intact skin and mucous membranes.

THE COMPLEMENT SYSTEM
The complement system is another important component of innate immunity. The system consists of 30 proteins found in serum or on the surface of certain cells [13]. Activation of the complement system results in a cascade of biochemical reactions that ultimately ends in lysis and disruption of foreign or effete cells. Without activation, the components of the complement system exist as proenzymes in body fl uids. As a by-product of the activation of the cascade, a number of biologically reactive complement fragments are generated. The complement fragments can modulate other parts of the immune system by binding directly to T lymphocytes and bone marrow–derived lymphocytes (B lymphocytes) of the adaptive immune system and also stimulate the synthesis and release of cytokines. There are three activation pathways for the complement system. Although the activation pathways are different, they all act at the microbial surface to assemble an enzyme convertase that cleaves C3 to form C3b that binds to a microbial surface where it activates C5 and the other components of the cascade. The three pathways are the classical, mannan-binding lectin (MBL), and the alternative. Each of the pathways has its own recognition mechanism and is activated through different mechanisms, but all result in the formation of a membrane attack complex (MAC) and lysis of a target cell. The classical pathway is activated by either IgM or IgG attached to a microbial surface antigen. The recognition molecule for the classical pathway is complement component C1q. A conformational change occurs in C1q, which results in activation of C1r and C1s, which, in turn, activates C4 and C2, which leads to the formation of the C4b2a complex (C3 convertase). The C3 covertase acts on C3, which ultimately leads to the formation of the MAC.
Activation of the MBL pathway begins after the recognition of mannose-binding lectin on various carbohydrate ligands [14]. MBLs and fi colins are found in serum and are structurally similar to C1q. MBLs and fi colins bind to mannose-containing carbohydrates on the surface of microbes. The MBLs and fi colins are considered to be typical pattern recognition molecules and as such attach to the MBL-associated serine proteases. On activation, the MBL-associated serine proteases cleave C4 and C2 to generate the C3 convertase C4bC2a and activate the remainder of the cascade [15]. The alternative pathway is important in innate immunity because it does not require specifi c antibodies for activation of C3. There are low levels of C3 present in body fl uids at all times. C3 undergoes hydrolysis to produce C3(H2O), which is an activated form. C3(H2O) can bind to factor B that is then cleaved by the factor D to form the fl uid-phase C3 convertase C3(H2O)Bb. Small amounts of C3b are needed to activate the alternative pathway at microbial surfaces [16]. C3b on the microbial surface binds to factor B, which is cleaved by factor D to form C3bBb, the C3 convertase. Properdin serves to stabilize the convertase whose role is to cleave C5, which activates the remainder of the cascade. There are several agents that can activate the alternative pathway: bacterial cells, tumor cells, enveloped viruses, and damaged mast cells. The complement system and its by-products serve to facilitate opsonization and may ultimately remove or destroy invading microorganisms. Tissue and circulating PMNs and macrophages are the cells that are most often involved in the ingestion of intracellular pathogens and killing of the invading microbes. Surface-bound C3b and iC3b (on the microbes) facilitate the attachment of the microbes to phagocyte complement receptors, which activates the ingestion and intracellular killing by the phagocytes [17].

In addition to enhancing opsonization in the presence or absence of antibodies, complement components have other important biological functions [17]. For example, free cleavage fragments of C3 and C5 are known to promote host infl amatory responses. C3a and C5a stimulate the bone marrow to release additional PMNs (C3b) and to serve as strong chemoattractants (C3a) for PMNs, monocytes, and eosinophils. Complement components C4a and C5a behave as anaphylotoxins. The three pathways of complement activation.


to induce histamine release, which, in turn, causes increased vascular dilatation and permeability. As mentioned earlier, the complement system helps modulate the adaptive immune response by enhancing antigen recognition and by stimulating the synthesis and release of cytokines. Together, the complement system is another important factor in defense against invading microbes and it functions to provide a rapid response.

NATURAL ANTIBODIES (OR IMMUNOGLOBULINS)
Natural antibodies have been recognized for some time but recently they were described as a component of the innate immune system [18]. Natural antibody is defi ned as an antibody that is found in normal, healthy individuals who have no evidence of exogenous antigenic stimulation. Natural antibodies are believed to develop in a highly regulated manner; they are usually found in low titer in serum and are low-affi nity antibodies [17]. A high percentage of the natural antibodies found in serum are of the IgM class. These antibodies are produced by a primitive B lymphocyte, called the B-1 lymphocytes [18]. B-1 cells are usually CD5+ and considered to be long-lived and self-replicating.

Natural antibodies play an important role as a fi rst line of defense against pathogens and other types of cells, including precancerous, cancerous, cell debris, and some self-antigens [19]. The cells of innate immunity apparently rely on an array of nonclonally expressed “pattern-recognition receptors (PRRs)” found in the target cells [20]. This response does not recognize specifi c single antigenic structures or epitopes, as in adaptive immunity, but respond to specifi c patterns, which are expressed independent of mutational events. This recognition system allows the innate immune mechanism to respond rapidly by focusing on structures most likely found in pathogens or effete cells. The B-1 cells are positioned at possible sites of entry of pathogens, along with monocytes, to mediate a rapid response to pathogens. The B-1 lymphocytes differ from the usual B lymphocyte (B-2) in terms of phenotype, anatomic location, and mechanisms of activation and signaling [21]. Thus, innate immunity uses an inherited set of receptors found on NK cells, γδ T cells, and CD5+ B cells to recognize and interact with a broad spectrum of different antigens.

TOLL-LIKE RECEPTORS
TLRs are found on phagocytic cells, including mononuclear phagocytes, circulating monocytes, tissue macrophages, and endothelial cells, and are important components of the innate immune system [22]. TLRs make up a family of cell surface protein receptors present on several cell types that function to recognize certain conserved molecular components of microorganisms and signal that microbes have breached the body’s barrier defenses [23]. TLRs serve as fi rst responders in a mammalian host to recognize the presence of an invading pathogen. They also generate an infl ammatory response to attempt to remove the invading agent. There are at least 10 TLRs in humans and they are capable of detecting a broad range of microbial ligands (see Table 1.1). The primary role of TLRs, as mentioned earlier, is to recognize and control bacterial infection. The mechanism of recognition is based on the receptor binding to a structurally conserved and unique pathogen-associated molecular pattern (PAMP) [24]. PAMPs are structural components of microbes that are important to them physiologically and are expressed on the pathogen but not on the host cells. TLRs consist of a family of “PRRs,” which are inherited molecules that exist as transmembrane proteins that detect the presence of pathogenic agents. The recognition mechanism between the TLRs and PAMPs provides an effi cient method for self/ nonself discrimination. The interaction between the TLR and the microbial PAMPs triggers host cell activation [24]. Despite intensive investigations, the molecular details of the interaction between TLRs and pathogens is still unclear. Binding sites for the different PAMPs are known to contain different extracellular leucine-rich repeat units. However, binding and recognition of many diverse bacterial ligands is not understood. After a TLR binds to a PAMP, the TLR dimerizes either with itself or, in other cases, it binds with a different TLR to induce an intracellular conformational change, resulting in the recruitment of certain other proteins (adaptor proteins) in the cytoplasm [25]. These adaptor proteins, such as MyD88, CD14, and others, transmit the message that TLR activation has occurred and initiate an intracellular cascade resulting in induction of an infl ammatory response [26]. Activation of some TLRs induces the expression of a costimulatory molecule, B7 (CD80), which is found on antigen-presenting cells (APCs) and is needed for activation of naïve T lymphocytes [27].
Table 1.1 gives a listing of the TLRs found in mammals. TLRs of humans primarily recognize microbial structures and PAMPs and go on to trigger host cell activation and infl ammation. In summary, TLRs function to curb an acute infection by activation and regulation of rapid effector responses in the innate immune system. TLR activation

Source: Adapted from Sigal, L.H., J. Clin. Rheumatol., 10, 353, 2004.
regulates a number of systems known to be important in innate immunity. These include the release of infl ammatory cytokines and chemokines, the oxidative burst in phagocytic cells, as well as the activation of various cationic peptides. TLRs serve to discriminate between self and nonself through the recognition and reactivity to PAMPs that are not expressed on host cells. TLRs also impact and moderate the adaptive immune response system through the induction of costimulatory molecular and cytokines that are involved in T- and B-lymphocyte reactivity.
1.2.5 PHAGOCYTOSIS
Polymorphonuclear neutrophilic leukocytes have been well-known components of the innate immune system for many years. Detailed studies of PMN phagocytosis and intracellular killing of microorganisms have led to a better understanding of important defense mechanisms against invasion by pathogenic bacteria, fungi, and enveloped viruses. PMNs are attracted to the site of microbial invasion, recognize the microbe, become activated, kill the microorganisms, resolve the infection, undergo apoptosis, and are then ingested and removed by either macrophages or neighboring endothelial cells to resolve the infl ammatory response. PMNs arise as myeloid progenitors in the bone marrow. Specifi c growth factors and cytokines mediate the differentiation of myeloid precursors into mature PMNs [28]. After entering the circulation, the PMNs have a half-life of about 8–12 h before undergoing a programmed cell death (apoptosis) and are reabsorbed through endothelial walls. The PMN turnover is about 1011 cells per day [29]. PMNs are actively recruited to the site of an infection by a complicated multistep process whereby they are mobilized both from the circulation and bone marrow. PMNs are constantly rolling along walls of the postcapillary venules where they readily detect the presence of a chemoattractant signal generated either by endothelial cells or the microbes themselves [30]. The chemokine, interleukin-8 (IL-8), is an important chemo attractant produced by multiple host cell types during an infl amatory event [31]. Bacteria also produce substances that are chemoattractants for PMNs. Once the PMNs reach the site, they become primed and develop enhancedfunctional capabilities [32]. Phagocytes are actively recruited to the site of microbial invasion as a response to a number of lectin glycoproteins called selectins [33]. Selectins are found on the surface of endothelial cells lining the blood vessels at the site of infection. P-selectin (from activated platelets) is upregulated on the surface of the endothelial cells and the selectins facilitate the “rolling and tethering” of PMNs [30]. PMNs secrete several molecules to support their migration from the circulation through the endothelium into the tissues. A number of neutrophil chemoattractants have been reported, including C5a, N-formyl bacterial oligopeptides, and leukotriene B4 [34]. As PMNs move toward the site, they become activated or primed to produce their antibacterial substances [32].
Phagocytosis is the process whereby PMNs recognize, bind, and ingest the micro organisms that stimulated the infl ammatory reaction. Phagocytosis is greatly enhanced by opsonization of the bacteria. Attachment of a specifi c IgG or complement fragment, C3, can greatly enhance the effi ciency of phagocytosis, although phagocytosis can occur without opsonization [35]. Complement receptors, CR1 and CR3, are the primary receptors for opsonization by the complement. The PMNs also express receptors for IgG fragment Fc (FcγRs) facilitates phagocytosis [36]. The two most prominently expressed Fcγ receptors on circulating PMNs are known as FcγRII (CD32) and FcγIII (CD16), and binding to these receptors triggers the oxidative burst in PMNs. The binding of antibody and complement receptors at the PMNs surface activates phagocytic process. Activation of phagocytosis causes changes in the cytoskeletal contractile elements, which leads to an invagination of the cell membrane of the PMNs. This occurs at the site of the attachment of the opsonized microorganism [37]. Pseudopods extend from the PMNs and fuse around the invagination encasing the microorganism inside the phagolysosomal vacuole [37]. The PMNs have two broad types of killing mechanisms. One is oxygendependent
and the other is oxygen-independent [38]. Phagocytosis of microbes stimulates the production of superoxide radicals and other reactive oxygen species. These are potent microbicidal agents and include, among others, hydrogen peroxide and chloramines. The enzyme NADPH oxidase is found in the cell membrane of the PMNs and generates the superoxide (called the respiratory burst) [38]. The superoxide is unstable and quickly dismutates to hydrogen peroxide and other substances that are microbicidal. These reactions take place inside the phagolysosome (also called phagosome). The PMNs contain two types of cytoplasmic granules, azurophilic and specific (also referred to as secondary and tertiary granules) [39]. Each type of granule houses a number of proteins and peptides with microbicidal properties. Lysozyme is found in both types of granules and cleaves peptidoglycans of bacterial cell walls to disrupt the microbes [40]. The azurophilic granules contain a number of small cationic proteins with microbicidal activity. Azurophilic granules also contain myeloperoxidase (MPO), which during PMN activation is directed to phagosomes where it catalyzes a reaction with chloride and hydrogen peroxide to form hypochlorous acid [37], which is extremely microbicidal. The beta2 integrin CD11b/CD18 is present in the plasma membrane and secondary granules of neutrophils, and functions as a major adhesion molecule. On PMN activation, there is translocation of intracellular pools of CD11b/ CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defi ned. α-Defensins are also found in azurophilic granules. These are small cationic peptides that can interact with negatively charged molecules at the pathogen surface to change the permeability of the bacterial cell membranes and cause death [41]. Lactoferrin is an iron-binding protein found in azurophilic granules and is capable of binding iron, which is needed for bacterial growth [42]. Iron is also needed by the PMN to form other antibacterial compounds. After death of the invading microorganisms, the PMNs also die through a process known as apoptosis. This process is affected by both proinfl ammatory host reactions and microbes and is important in the resolution of the infl ammatory process. Phagocytosis is well known and extremely important to host defenses as noted by the severe infections seen in patients with phagocytic defects.

CYTOKINES AND CHEMOKINES
Cytokines and chemokines are small, secreted polypeptides that regulate essentially all functions of the immune system. Cytokines participate in determining the nature of the immune response by regulating or controlling cell growth, differentiation, activation, immune cell traffi cking, and the location of immune cells within the lymphoid organs [43]. Cytokines are a group of “intercellular messengers” that contribute to infl ammatory
responses through activation of the host’s immune cells. Cytokines are host-derived products that enhance the recruitment of circulating leukocytes as a response to the presence of pathogens [44]. Cytokines also play important roles in leukocyte attraction by inducing the production of chemokines, which are known to be potent mediators of chemoattractant activity for infl ammatory cells. Chemokines and cytokines provide a complex network of signals that can either activate or suppress infl ammatory responses [44]. Cytokine secretions can alter the behavior or properties of that cell itself as well as the behavior of surrounding tissue cells. Cytokines are produced by many different cells. One cell type is capable of making many different cytokines and a particular cytokine may be secreted by multiple cell types. A particular cytokine may have many different effects on different cells depending on the environment. In some cases, the target cell itself may produce a cytokine that infl uences itself as well as the neighboring cells of the target cell. Some cytokines require cell-to-cell interaction to exert their effects [45]. Chemokines are another family of small structurally similar polypeptides that can regulate the traffi cking of subsets of leukocytes [46]. Chemokines differ from other cytokines because all chemokines are ligands for the G-protein-coupled receptors [44]. Chemokines are potent cell activators capable of inducing migration of immune and infl ammatory cells. Most cells in the immune system express receptors for at least one chemokine. Infl ammatory cells may release a variety of chemokines and there is some evidence that infection with certain bacteria and viruses can stimulate the host cells to produce characteristic sets of immune cells. Cytokines have multiple, broad properties. For example, cytokines may work effectively in concert with or in competition with both immune and nonimmune cells [47]. In addition to their impact on innate immunity, cytokines support B- and T-lymphocyte maturation and proliferation, differentiation of T helper cells into Th1 and Th2 subsets, maturation and polarization of DC subtypes and memory cell development [48]. These are important facets of adaptive or acquired immunity. There have been more than 30 cytokines described [49]. Most cells of the immune system and many other host cells release cytokines. In some cases, the same cell type may respond to cytokines through specifi c cytokine receptors and may release and
respond to the same cytokines it just produced. Two cytokines, IL-1 and tumor necrosis factor-α (TNF-α) play important roles in responses to bacterial infection [49,50]. They are both small polypeptides that exert a broad range of effects on multiple host reactions, including immunologic responses, infl ammation, and hematopoiesis. Experimentally, IL-1 and TNF-α if injected into mice may produce many of the features of Gram-negative sepsis in the absence of infection with the microorganisms [49]. In endotoxic shock caused by Gram-negative organism, the cytokines, IL-1 and TNF-α are secreted by mononuclear phagocytes in response to activation by TLRs by the bacterial lipopolysaccharides [49]. This reaction results in the secretion of other cytokines and chemokines, which exacerbate the reaction. Other cytokines have important roles in cell development, for example, they impact myeloid cell progenitor cell development, enhance IgG4 subclass development, regulate Th2 responses, cause mast cell proliferation in vitro, stimulate B-cell production, activate and stimulate growth of eosinophils, stimulate survival and expansion of immature precursors that are committed to T- and B-cell lineages, stimulate endothelial cells to produce adhesion molecules, enhance PMN recruitment, and many other functions.

NATURAL KILLER CELLS
NK cells were fi rst reported in the 1970s. Initially, NK cells were referred to as nonspecifi c lymphocytes because NK cells could kill certain virally infected and malignant cells without known prior sensitization. NK cells were known to resemble large lymphocytes morphologically and were referred to as large granular lymphocytes. Approximately, 10–15% of the lymphocytes circulating in peripheral blood are NK cells. NK cells are distinct from T- and B lymphocytes because they express neither immunoglobulin receptors nor T-cell antigen receptors. There are other distinctions including phenotype and function. NK cells have receptors that recognize major histocompatibility complex (MHC) class I antigens. Because NK cells have cytotoxic properties, their function is highly regulated in their interactions in both the innate and adaptive immune systems [52,53]. NK cells develop from a common lymphoid progenitor cell in the bone marrow. The NK cells diverge from other lymphocyte lineages and acquire specifi c cell surface markers to guide them through their developmental stages. After developing and maturing in the bone marrow, the NK cells migrate and circulate in the peripheral blood and may be found in various organs including the lung, liver, spleen, and uterus. On antigenic stimulation, NK cells rapidly “home” to the lymph nodes and lymphatics.
NK cells play important roles in innate immune responses and immune regulation. They communicate with other cells through a complex of both activation and inhibitory signals through cell surface receptors. There are many recognizable NK cell subsets [40–50] found in peripheral blood [54]. NK cells were fi rst defi ned by a lack of B- and T-cell surface markers but the NK cells are now identifi ed into subsets based on the expression of certain phenotypic surface markers. Most NK cells express a neural cell adhesion marker called CD56. Staining with a monoclonal antibody to CD56 permits division into two major subsets, CD56bright and CD56dim . CD56bright NK cells are characterized by having an expression of many CD56 surface molecules [55]. These cells have lower levels of some of the cytotoxic molecules such as perforin and express high levels of cytokines. CD56bright NK cells are thought to be important in infl amatory responses and probably play a role in immune regulation. NK cells are distinct from NKT cells that express CD3, and rearrange their germline DNA T-cell receptor (TCR) genes [55]. In contrast, CD56dim NK cells are the most effective killer lymphocytes. The CD56dim NK cells make up about 90–95% of NK cells in peripheral blood and they also express CD16 (Fcγ receptor) on their surface. In contrast to CD56bright NK cells, these NK cells express large amounts of perforin that mediates cytoxicity. Perforindependent cytoxicity is the major mechanism of NK cell lysis of target cells [55]. NK cells are programmed to kill target cells and they are inherently capable of killing autologous cells. They are actively inhibited from killing “self” cells by inhibitory receptors and signals. The MHC defi nes “self” and it has been suggested that “self-MHC” surface receptors engage the inhibitory receptors on the NK cell and prevent lysis of “self” cells [56]. Viral infection of a cell causes a change in the MHC class I expression, which in turn removes the normal inhibitory signal and NK cell activation occurs, resulting in cytotoxicity and death of the viral-infected cells. There are two families of inhibitory receptors affecting NK cells [57]. The bestdescribed inhibitory signals are those transduced by HLA-specifi c receptors and are members of the inhibitory killer immunoglobulin-like receptor family (KIR). There is another family of inhibitors that are lectinlike receptors identifi ed as NKG2A/3. It is believed that the net sum of activation and inhibition signals tightly regulates the function of NK cells [55]. NK cells recognize and lyse pathogen-infected cells and malignant cells [58]. They also play an important immunoregulatory role. There are several mechanisms used by NK cells to remove cells. NK cells are effective killers by releasing large number of cytolytic granules at the site of interaction with the target. A major component of the NK cell lysosomal granules is perforin, which, as mentioned earlier, is the major cytolytic substance [58]. The cytokine and chemokine secretions of NK cells are involved in the death of target cells [52]. NK cells produce IFN-γ, TNF-α, GM-CSF, IL-5, and IL-13 among other active substances. Some antiviral activity can be attributed to cytokine production by NK cells. NK cells are known to express costimulatory molecules for T- and B-lymphocytes and activate the adaptive immune system [59]. TLRs are also expressed on NK cells and these receptors participate in the early detection of an impending infection. NK cells are activated by cytokines produced by virally infected APCs, which may result in cell lysis. At the same time, NK cells may interact with DCs to participate in generating an adaptive immune response. NK cells and DCs interact to induce DCs maturation through cytokines produced by NK cells [52]. NK cell biology is very complex and appears to be directly or indirectly involved in establishing and maintaining immunity. By expression of cell surface receptors, NK cells may go through several stages of maturation and the by-products of maturation affect most components of immunity. The balance between activation and inhibition is closely regulated in the environment where NK cells and other types of immune cells exist. Patients have been described with various NK cell abnormalities. The most prevalent observation is unusual susceptibility to certain types of viral infections primarily (herpes) [60,61]. In any case, NK cell defi ciency must be rigorously documented. Most NK cell enumeration studies are performed using peripheral blood, which may not give an accurate refl ection of the numbers of functional NK cells available. Functional assays are diffi cult to perform and quantify but it is possible to determine cytolytic function and cytokine secretory properties of peripheral blood NK cells. Patients have been reported with normal numbers of peripheral blood NK cells but with a defi ciency of perforin, which would impact cytoxicity.

Natural Killer T Cells
Natural killer T (NKT) cells are a subset of T lymphocytes that share some properties of NK cells and conventional T cells. Classical NKT (or type 1) cells are CD1drestricted T cells that express a semi-invariant TCR Vα24-Jα18, which distinguishes them from CD1d-dependent T cells that do not express this semi-invariant TCR (type 2 NKT cells) [62]. Most NKT cells express both an invariant TCR and the NK receptor NK1.1 type 1; CD1d-restricted NKT cells are found primarily in the liver, thymus, spleen, and bone marrow [63]. Cells with type 1 TCR can be activated by a synthetic ligand α-galactosylceramide (α-GalCer) presented on CD1d [64]. Type 2 or nonclassical NKT cells fail to be activated by α-GalCer [65]. An endogenous ligand for type 1 NKT cells was identifi ed as a lysosomal glycosphingolipid [66]. There are distinct subsets of CD1d-restricted T cells. NK cell associated markers were expressed primarily within the CD4− CD8− Vα 24 NKT subset. However, both CD4+- and CD4− CD8− Vα 24 NKT cells were capable of Th1 cytokine production. This includes IFNγ and TNF-α. The Th2 cytokines, such as IL-4 and IL-13, were secreted by the CD4+ subset [67]. Both the CD4+ and the CD4− CD8− Vα 24 NKT exist wherever other T lymphocytes are found. The fact that NKT cells recognize glycolipid antigens in association with CD1d (a nonclassical antigen-presenting molecule) sets the NKT cells apart from conventional T cells. Type 1 NKT cells recognize both foreign and self-glycolipids. Recently, it has been shown that type 1 NKT cells recognize various types of glycolipids and related compounds found in a number of parasites and bacteria but the specifi c ligands have not been identifi ed. This suggests that the classical NK cells focus activity on viruses and viral infection while NKT cells, however, are primarily involved in detection of parasite and bacterial pathogens [68]. On activation, NK cells respond within a few hours with vigorous production of cytokines [69]. NKT cells release Th1-type cytokines including IFNγ and TNF-α as well as the Th2-type cytokines IL-4 and IL-13 [70]. Individual NKT cells are able to produce both Th1- and Th2-type cytokines at the same time following stimulation in vivo [71]. This is unusual because it is possible that Th1 and Th2 antagonize each other. The implications of this observation and mechanisms are unknown. NKT cells appear to be involved in immediate immune responses, tumor rejection, control of autoimmune disease, and immune surveillance [63]. NKT cells may act as effector cells as seen with their cytotoxic activity; they may also act as regulators. The important question concerns how they determine which way to go. The NKT cells may produce either pro- or anti-infl ammatory cytokines. This depends on the type of signal they receive. This is probably related to cytokine profi les produced. The cytokine profi le is dependent on the type of TCR stimulation the NKT cells receive. The study of NKT cells is an important topic of investigation today and probably will be for some time. It is clear that NKT cells are involved in a number of pathological conditions and they appear to regulate a number of others. NKT cells appear to have both protective and harmful roles in disease progression of certain allergic and autoimmune disorders and they may modulate viral infections and have a role in tumor growth and progression.

GAMMA/DELTA T LYMPHOCYTES
Gamma/delta T lymphocytes (γδ T cells) are a relatively recent discovery within the T-cell population. It is diffi cult to categorize them but it is becoming clear they are an important component of host defense and may represent a different parallel immune system component [72]. It is likely that γδ T-cell functions fall somewhere “in-between” the innate and adaptive immune systems. A major characteristic of the γδ T-cell population is that they have a TCR consisting of a γδ heterodimer rather than the more prevalent αβ TCR [73]. γδ T cells make up a small percentage of T cells (1–5%) in peripheral blood and other lymphoid organs. However, they are found in higher concentrations in the skin, gastrointestinal tract, and the genitourinary system [73]. These locations may be related to the types of antigens they encounter and the immunological responses they deliver. γδ T cells may be important in preventing infection with organisms such as Mycoplasma penetrans, an organism capable of causing urethritis and respiratory diseases in immunocompromised individuals [74]. In addition, Mycobacterium tuberculosis has been shown to elicit a γδ T-cell response [75]. γδ T cells may also recognize and express cytoxic activity against certain types of tumors, including both hematopoietic and solid tumors [73]. It has been suggested that γδ T cells recognize ligands, which are different from the short peptides that are detected by αβ T cells in the context of MHC class I or II molecules [76]. At least one subclass of γδ T cells recognizes lipidlike antigens from pathogens. Another functional subtype recognizes stress-inducible MHC-related molecules and a number of other ligands [76]. γδ T cells are active producers of cytokines, which are cytotoxic for many tumor cells. Activated γδ T cells, through cytokine production, may modulate conventional immune responses by acting on macrophages and DCs. It has been shown that γδ T cells function as both activators and inhibitors of immune reactions through surface-bound receptors [73]. Most γδ T cells possess the NKG2D receptor, which may provide a costimulatory signal that is essential for the γδ T-cell response against certain types of tumor cells. Through their interactions, both directly and indirectly, γδ T cells appear to supplement the cellular immune response by recognizing and responding to antigens that may not be detected by the more prevalent αβ T cells. These γδ T cells appear to have an infl uence on both innate and adaptive responses through patternlike recognition systems, cytoxicity against tumor cells, and providing protection against intracellular pathogens.

DENDRITIC CELLS
DCs have been known since 1973; however, the importance of DCs in both innate and adaptive immunity has been defi ned more clearly in the past few years. The DCs develop in the bone marrow and are found in the circulating blood and tissues such as the spleen, lungs, gut mucosa, and other places where they may play a role in immunosurveillance. The DCs develop in the bone marrow from hematopoietic pluripotential stem cells [77]. Precursor DCs are constantly generated in the bone marrow and are released into the peripheral blood. After leaving the bone marrow, the precursor DCs “home” to a number of different tissues where they reside as sentinels waiting to interact with antigen. The precursor DCs express low-density MHC class II antigens and after encountering a proper stimulus differentiate into highly endocytic and phagocytic iDCs [78]. DCs probably make up a heterogeneous population of cells. However, precursor DCs, iDCs, and mature DCs play different roles in the immune system. Precursor DCs circulate in the environment and on contacting a pathogen produce cytokines, that is, γ-interferon, and undergo maturation to iDCs. The iDCs acquire new properties, that is, markedly increased phagocytic and endocytic capabilities that lead to binding antigen by the iDCs and then maturation to mature DCs [78]. The mature DCs have specialized properties (receptors) to bind foreign or effete cells through lectin and Fcγ receptors. Once the antigens enter the mature DCs, they are processed for antigen presentation and the mature DCs become an APC and lose their phagocytic properties. The mature DCs are the only cells capable of activation of naïve T cells and are defi ned by this characteristic [79]. A variety of different stimuli can initiate DCs maturation including pathogens, damaged tissue-derived antigens, and ultraviolet light. The mature DCs, carrying the antigen, then migrates to the secondary lymphoid tissues [78].

Immature Dendritic Cells
iDCs are activated upon exposure to the so called danger signals, including PAMPs and become actively phagocytic for the infectious agent. As a part of their maturation to mature DCs, the iDCs become less phagocytic but more mobile once the DC has taken up the antigen and begun processing it. Activation and maturation of DCs occur through the NF-κB signaling pathway [80]. The iDC neither provides T-cell stimulation nor cosignaling.

Mature Dendritic Cells
After maturation from iDC, the mature DC migrates to the secondary lymphoid tissues where it begins processing the antigenic material. This migration occurs because of the expression of specifi c chemokine receptors, especially CCR-7. Mature DCs have highly developed antigen-processing cell capabilities and are capable of loading endocytosed antigenic peptides on both MHC class I and II molecules thereby permitting presentation to both CD8+ and CD4+ T lymphocytes [81]. Mature DCs have high-density costimulatory molecules for presenting processed antigen to T cells [79]. Maturation of DCs is associated with the up-regulation of the costimulatory molecules. Costimulatory molecules CD40, CD80, and CD86 enhance the stability of DCs interactions with naïve antigen-specifi c CD4+ and CD8+ T lymphocytes and the secretion of cytokines such as INFα, IL6, IL-10, and IL-12. The mature DCs are potent activators of T-cell responses. The DCs transmit information about the danger signals to the T cell and help defi ne the T-cell responses [82]. There are two subsets of DCs in the blood based on the expression of CD11c (β2 integrin). The subsets named, myeloid DCs (M-DCs) and lymphoid DCs or plasmacytoid DCs (P-DCs), differ in morphology and expression of markers and function
[83]. They do, however, share some surface markers for adhesion, activation, costimulation, and coinhibition. M-DCs express myeloid surface markers, that is, CD13, CD33, and CD11c. The M-DCs also express large numbers of mannose receptors and rapidly take up polysaccharide antigens [84]. The M-DCs readily capture antigen in peripheral tissues by phagocytosis and migrate as immature M-DCs to lymph nodes where stimulation with CD40L (CD40 ligand) induces maturation to mature M-DCs. The M-DCs are potent inducers of both Th1 and Th2 cytokines in naïve CD4+ T lymphocytes. The local microenvironments bias the development of either Th1 or Th2 types of reactions [84]. The P-DCs have morphology similar to plasma cells and are derived from a lymphoid lineage. Instead of myeloid markers, the P-DCs express high levels of CD123 and MHC molecules. CD40L (ligand) causes stimulation of P-DCs and results in DCs maturation. These DCs support Th2 cytokines (primarily). The P-DCs populate T-cell areas of lymph nodes and may have a special ability to recognize self-antigens or viruses [85].

The cell surface receptor CD40 plays an important role in humoral and cellmediated immune responses. CD40 is expressed on many cell types including B cells, epithelial and endothelial cells, and all APC. The ligand for CD40 is CD40L and is a trimeric TNF-α-like molecule. CD40L is expressed primarily on T helper cells [86]. Ligation of CD40 on the surface of the DCs induces maturation that is detected by markedly enhanced T-cell stimulatory capacity. This maturation of the DCs causes it to activate naïve CD8+ cytotoxic T-cells (CTL) that are important in developing immunity against certain pathogens and tumors [86]. The types of T-cell subsets induced by DCs is dependent on several factors including the DCs subset involved, the nature and dose of the antigen, and the types of cytokines present in the microenvironment where the interaction between DCs and pathogen occurs. DCs play an important role in how a host responds to foreign antigens, effete, or other cells that have initiated its maturation. The DCs help direct the T cell to respond—how it should respond, and where to go to respond. The DC provides a number of sequential signals to the responding T cells. The fi rst signal consists of the interaction of the TCR with the specifi c antigen in the context of the MHC protein on the surface of the DC. This determines the antigenic specifi city of the response and triggers the differentiation of naïve CD4+ and CD8+ T cells into T helper and CTL, respectively. This antigen-specifi c T-cell activation requires the engagement of the TCR/CD3 complex. The antigenic peptide is presented by the MHC and the engagement of appropriate costimulatory reception by costimulatory ligands on the DCs [87]. The DCs also provide the costimulatory signaling that T cells require to respond to antigen. The cosignaling can be either positive or negative. Cosignaling may be provided by a number of different molecules including CD80, CD86, and CD28. The activation and maturation signals may be diverse, but all involve activation of the NF-κB signaling pathway [88]. In the presence of a negative cosignal (coinhibitory) or in the absence of a positive costimulatory signal, T cells will fail to respond and may not be capable of reacting to that specifi c antigen in the future. Some pathogens have evolved mechanisms to help evade this component of the innate immune response. These pathogens possess compounds capable of arresting DCs in their immature state where they cannot produce costimulating molecules. Without costimulatory activity, the DCs are unable to completely stimulate T cells to respond [89] and the pathogens to avoid an immune response directed against them. DCs also direct the functional polarization of CD4+ T cells into Th1, Th2, and Treg cells [90]. The nature of the “danger signal” or PAMP defi nes the DCs response as Th1-, Th2-, or Treg-type responses, which result in the DCs producing certain cytokines to induce T-cell differentiation into Th1, Th2, or Treg CD4+
T lymphocytes [90]. It is unclear whether or not the DCs are restricted to one polarization
type or if they are fl exible depending on the nature of the stimulus and this microenvironment.

The nature of the binding of the stimulating agent (type of PAMP) to certain types of TLRs induces the DC maturation, which results in the development of Th1 T cells [91]. For example, binding of microbial double strand RNA (ds RNA) to TLR3 triggers the formation of the Th1 T cells. Other TLRs associated binding signals produce either Th1 or Th2 types of responses depending on the TLR type and the danger signal. It appears that the nature of the PAMP is important in defi ning the T-cell response. In general, Th1-type responses are directed to cell-mediated types of responses and the nature of the cytokines produced drives these responses. A Th2 T-cell response may result from antigens of a parasites-inducing type 2 DCs, which go on to produce a Th2 type of response. Th2 responses are usually antibody or humoral immune responses [89,92]. Treg-type T cells are CD4+ T cells that can suppress responses of other T cells and probably play an important role in regulating self-tolerance. Multiple subtypes of Treg cells have been identifi ed and each has its own specifi c phenotype, cytokine profi le, and mechanism of activation for suppressing immune responses. The most frequently described phenotype includes CD25 as a surface marker [93,94]. The Treg cells have been shown to interfere with tumor immunity and a number of parasites are known to induce regulatory DCs that induce Treg responses. For example, a hemagglutin from Bordetella pertusis serves as a ligand for TLR2 and this induces the development of regulatory DCs and ultimately Treg T cells. It is important that immune responses are initiated against foreign substances but immune responses should not be directed at self-antigens. DCs are responsible for the induction of peripheral and central tolerance. A major role of the DCs is to recognize “danger signals” and activate the appropriate response by passing information to T cells through cytokines and chemokines to direct the T-cell response. As expected, cytokines production by DCs is tightly regulated. The DCs possess PRRs that can detect concerned motifs on invading pathogens and distinguish them from self-antigens. There is constant communication between DCs and T cells with information being shared in both directions. The exact mechanisms involved in how DCs and T cells combine to distinguish self from non-self are not understood completely. However, iDCs are involved in the maintenance of tolerance to self-antigens by constantly defi ning self in the periphery. The iDCs constantly sample different self-antigens and present them to T cells under noninfl ammatory conditions permitting the detection of autoreactive T cells. The iDCs may also induce tolerance to self by stimulating naïve CD4+ and CD8+ T cells to differentiate into Treg cells that produce IL-10, which in turn causes these cells to inhibit Th1 T-cell differentiation and suppress CD8+ memory cell responses. Figure 1.2 summarizes the pivotal role of DCs in immunity.

OVERVIEW OF ADAPTIVE IMMUNITY
In contrast to innate immunity, adaptive immunity is fl exible, specifi c, and has immunological memory, that is, it can respond more rapidly and vigorously on a second exposure to an antigen. Immunologic memory provides a more powerful response to a repeated exposure to the same foreign substance or antigen. Adaptive immunity is more complex because it provides the ability to respond very specifi cally. Innate and adaptive immunity responses interact effectively to enhance the body’s defense mechanisms against foreign or damaged host cells. Inherent in both innate and adaptive immune responses are the mechanisms to distinguish self from nonself. The primary blood cell elements of the adaptive immune system are T lymphocytes and B lymphocytes. These T- and B-cells provide the unique specifi city for their target antigens by virtue of the antigen-specifi c receptors expressed on their surfaces. The B- and T-lymphocyte antigen-specifi c receptors develop by somatic rearrangement of germline gene elements to form the TCR genes and the immunoglobulin receptor genes. This recombination mechanism provides unique antigen receptors capable of recognizing almost any antigen encountered, and provides the specific immunological memory for a rapid, vigorous, and specifi c response to a later exposure to the same antigen. It is estimated that millions of different antigen receptors may be formed from a collection of a few hundred germline-encoded gene elements. For many years, innate and adaptive immune responses were studied as separate systems because of their different mechanisms of action. However, it is now understood that synergy between the two systems is required to provide adequate immune reactivity against invading pathogens. Innate immune responses, through their barrier and relatively broad types of actions, represent the fi rst line of defense against pathogens. At the time the innate system is getting activated, the adaptive system becomes activated also. The adaptive response becomes evident a few days later because it requires time for suffi cient antigen-specifi c receptors to be generated through clonal expansion/proliferation. There are multiple interactions occurring between the two systems, which results in the coamplifi cation of each respective response and leads to the ultimate destruction and elimination of the invading pathogen.
B LYMPHOCYTES
The primary function of B lymphocytes is the production of antibodies that are specifi c for a given antigenic component of an invading pathogen. Antibodies are encoded by the heavy (H)- and light (L)-chain immunoglobulin genes. Antibodies may be secreted or cell surface–bound on B lymphocytes. There are fi ve classes of immunoglobulins: IgM, IgG, IgA, IgD, and IgE; and the classifi cation is based on the isotypes of the H chain. B lymphocytes represent roughly 10–15% of the



peripheral blood lymphocyte population and free immunoglobulins make up a considerable proportion of serum proteins. After an encounter with a specifi c pathogen and an antibody response is generated, the level of specifi c antibodies to that antigen decreases in serum over a relatively short period of time. However, immunological memory persists in the B-cell population, which is capable of rapid clonal expansion upon reexposure to that same antigen [95]. Protective immunity for a specifi c antigen requires cooperation between B and T lymphocytes. In many instances, helper T-cell interaction is required for the development of high-affi nity antibodies and complete protection. For naïve B lymphocytes to undergo proliferation and differentiation in response to most antigens, they require stimulation by a CD4+ T helper cell with the same antigenic specifi city. B lymphocytes are capable of antigen presentation to T cells through their surface MHC class II proteins [96]. Antibodies recognize the tertiary structure of proteins and react with specific epitopes in the antigen structure [97]. During an immune response, immunoglobulin formation may undergo switching from one isotype of immunoglobulin to another, usually from IgM to IgG and IgA or IgE. This switching requires additional genetic recombination between the same variable region genes and new H-chain isotope genes. As the humoral immune response continues, antibodies with higher affinity for the antigen develops. Competition for antigen provides a selective advantage to B lymphocytes with the highest affi nity for antigen.

T LYMPHOCYTES
Whereas B lymphocyte products recognize extracellular pathogens, T lymphocytes are adept at identifying and destroying cells that have been infected by intracellular pathogens. For T cells to recognize antigenic peptides, the peptide must be presented in the context of cell surface MHC class I or class II proteins [98]. In other words, T cells can only recognize molecular complexes consisting of the antigenic peptide and a self-structure, that is, the MHC. Depending on whether the antigenic peptide has been synthesized within the host cell or ingested by the cell and modifi ed by proteolytic digestion, either MHC class I or class II proteins are required [99]. Proteins of the MHC are intimately tied to T-lymphocyte responses and recognition of antigenic peptides. The MHC class I proteins consist of three HLA classes: HLA-A, HLA-B, and HLA-C with hundreds of allelic variants of each. Structural studies have shown that class I molecules exist as cell surface heterodimers with a polytransmembrane α-chain associated (noncovalently) with a nonpolymorphic β2 microglobulin protein [99]. The protein chains are folded in such a way as to form a physical groove capable of binding up to an 11 amino acid long peptide. Antigenic proteins are degraded by proteolytic enzymes to about this size for binding to the MHC class I proteins for antigenic presentation. Antigenic peptides are bound in the groove of the HLA molecule and expressed to the cell surface for presentation to initiate a T-cell response [99]. The large number of HLA class I alleles refl ects structural polymorphism, which adds to the number of antigenic peptides that can be recognized. Most humans are heterozygous for HLA, further increasing the numbers of antigenic polypeptides bound and capable of activating T cells [100]. There are three major groups of MHC class II proteins, HLA-DR, HLA-DQ, and HLA-DP, each with large numbers of alleles. Class II proteins are also folded in such a way as to form a groove for binding peptides for presentation as a complex consisting of the protein fragment—HLA structure. Class II proteins consist of two polypeptide chains, which are MHC-encoded transmembrane proteins [101]. As mentioned earlier, the antigenic peptides bound to class II proteins are derived from exogenous antigens taken up by APCs and are degraded into peptides of a size appropriate for loading in the class II protein groove. The phagocytosed antigens were degraded by proteolytic enzymes found in the lysosomes of the APC and further processed by specialized proteins in the cytoplasm to generate peptides of the correct length for binding [101]. As with the MHC class I proteins, the large repertoire of the peptide-binding class II molecule is generated through polymorphisms of the class II proteins where there are multiple alleles of each HLA type. Once again, class I and class II HLA proteins differ in the types of peptides recognized and where and how the antigenic peptides are generated. The thymus is an organ dedicated to T-cell development. T cells originate in the bone marrow as progenitors and circulate in the blood stream before eventually homing to the thymus. The particular environment in the thymus provides the optimal milieu for thymic lineage development [102]. It is likely that interaction with the thymic stroma provides the signals required for the thymic progenitor cells to undergo proliferation and differentiation into mature naïve T cells [102]. This process is tightly regulated and is mediated by various transcription factors, cytokines,
chemokines, and one or more selectins. T lymphocytes make up the majority of lymphocytes in peripheral blood and are most readily defi ned by the expression of TCR molecules on their surface. The αβ TCR molecules recognize specifi c peptide antigens that are presented in the context of the MHC class I or class II proteins (in the form of a complex). T cells expressing αβ molecules differentiate into different subsets including the CD4+ T cells and the CD8+ T cells. The CD4+ T-cell subsets are active as regulators of cellular and humoral immune responses. The other subset, CD8+ T cells, is cytotoxic for cells infected with intracellular pathogens. T-lymphocyte receptors are different from the B-lymphocyte immunoglobulin receptors in that they are never secreted and are able to recognize peptides produced after proteolytic breakdown of antigens as opposed to B-cell receptors that react to native proteins [103]. TCRs recognize the primary amino acid structure of the protein antigen. TCRs recognize antigenic peptides only when they are presented as cell surface– bound complexes with MHC class I or II proteins. Each T cell bears a TCR with a single antigenic specifi city and these cells provide recognition for a very large number of possible pathogens. T-lymphocyte activation through the TCR is an important step in the initiation of an adaptive immune response. TCR activation requires two interactions [99]. One is the interaction between the TCR and the peptide MHC complex, which gives a partial signal for T-cell activation. Full activation requires both the TCR-peptide-MHC binding and the interaction with a costimulatory molecule, CD28, on the T cell and CD80 or CD86 present on the APC [99]. This second signal stimulates proliferation and differentiation of the T cell. If T cells do not receive the second signal they may become unresponsive and possibly anergic. There is considerable cross talk between T- and B-cells in an adaptive immune response. For proliferation and differentiation to occur, naïve B cells must be stimulated by a particular type of helper T cell, the CD4+ T cell. Moreover, the B and the CD4+ helper T cells must have receptors that are specifi c for epitopes present on the same antigenic molecule. The complex interaction between T helper cells and B cells (with matching specifi cities for a pathogen-specifi c antigen) and a second signal to the T cell (possibly provided by the B cell) results in the proliferation and differentiation of both B- and T-cells and ultimately in an adaptive immune response. T cells are divided into a number of subsets based on their migration patterns and functional abilities. The naïve T lymphocytes tend to circulate between the blood and lymph nodes in response to the homing receptor l-selectin [104]. Recirculation of naïve T cells allow them to move in and out of areas of the body where they have the best chance of detecting a pathogen bearing their specifi c antigen receptor [104]. At least two types of memory T cells are active in adaptive immunity. One subtype, the effector memory cell, is short-lived and aggressively migrates to the site of the target tissue to destroy the pathogen. The second subset, called central memory cells, serves a role in immunologic memory and has migratory patterns that place them in environments where pathogens may enter [104]. Antigenic stimulation of these memory cells leads to a rapid proliferative response leading to the production of both effector and central memory T cells. There appears to be a tendency for memory T cells to migrate back to the tissue where they fi rst encountered a specifi c antigen. This appears to be controlled by specifi c homing receptors on the surface of memory T cells. As mentioned earlier, αβ T cells, while in the thymus, differentiate into T-cell subpopulations expressing CD4 or CD8 cell surface markers. These cell populations were described fi rst as phenotypic markers and their functions were described later. Originally, these T cells were considered to be helper cells (CD4) and suppressor cells (CD8) but later the CD8 cells were identifi ed as cytotoxic T cells [105]. These associations are not absolute, since some CD4+ T cells may also have cytotoxic properties. CD4+ molecules and CD8+ molecules serve as coreceptors in the interaction between the T cells and APC. The CD4 molecules expressed on the surface of T cells bind to class II molecules expressed on the surface of APCs and serve to stabilize the interaction between that particular T cell and the APC. CD8 molecules, however, bind to class I molecules expressed on the surface of APCs and serve to stabilize the interaction between CD8 T cells and APCs. When naïve CD4+ or CD8+ T cells are activated by APC, they undergo differentiation into distinct subsets with different functions. The effector functions of the T-cell subsets are determined by the nature of the costimulatory signals given and the cytokines secreted [106]. There are two main subsets of CD4+ T cells generated or determined by the cytokines secreted by the APC, Th1, and Th2. The Th1 subsets are generated by APCs secreting IL-2. The T cells differentiate into effector cells producing high levels of IFN-γ and IL-2. This subset, Th1, generally supports cell-mediated immunity by producing cytokines INF-γ and TNF-β that are effi cient in activating macrophages and cytotoxic T cells [107]. In contrast, naïve CD4+ T cells activated by IL-4 stimulated DCs differentiate into effector T cells designated Th2. The Th2 subset actively supports the development of humoral or antibody responses [107]. Th2 lymphocytes secrete IL-4, IL-5, IL-9, and IL-13, which in turn are effi cient at stimulating B lymphocytes to differentiate into antibody- forming cells (particularly IgE) and actively secrete antibody. Generally, most immune responses show a combination of both features of Th1 and Th2 pathways and a prolonged immunization process may lead to one pathway becoming the most dominant. To a certain extent, Th1 and Th2 subsets secrete cytokines that can suppress one another, that is, Th1 secrete cytokines that can suppress Th2 responses and Th2 cells produce cytokines that can suppress Th1 responses. Another family of CD4+ T cells called Treg was recently shown to suppress the responses of other T cells [93]. It is likely the Treg cells play a role in regulating self-tolerance but may have a harmful effect on tumor immunity. Several subsets of Treg cells have been reported and each has a distinct surface phenotype, cytokine profi le, and mechanism of action for suppressing immune responses. Initially, high CD25 expression was used to identify the majority of human Treg cells. The CD4+ cells with the greatest regulatory activity had high levels of CD25 (i.e., CD4+ CD25 high T cells). A considerable number of other surface markers have been shown on Treg cells but expression is not as great or as consistent as CD25 [108]. More recently, the intracellular protein FOXP3 has been identifi ed as a key molecule involved in driving the activity of Treg and now serves as a marker to enumerate these cells.