Basic Principles of Immunology

 Chapter -2 

Basic Principles of Immunology 

Introduction  

Resistance to disease is based on innate mechanisms and adaptive or acquired immunity. Acquired immune mechanisms act in a specific manner and function to supplement the important nonspecific or natural resistance mechanisms such as physical barriers, granulocytes, macrophages, and chemical barriers (lysozymes, etc.). The specific immune mechanisms constitute a combination of less specific factors, including the activation of macrophages, complement, and necrosis factors; the early recognition of invading agents, by cells exhibiting a low level of specificity, (natural killer cells, cd [gamma-delta] T cells); and systems geared toward highly specific recognition (antibodies and ab [alpha-beta] T cells).

Many components of the specific immune defenses also contribute to nonspecific or natural defenses such as natural antibodies, complement, interleukins, interferons, macrophages, and natural killer cells.

In the strict sense, “immunity” defines an acquired resistance to infectious disease that is specific, i.e., resistance against a particular disease-causing pathogen. For example, a person who has had measles once will not suffer from measels a second time, and is thus called immune. However, such specific or acquired immune mechanisms do not represent the only factors which determine resistance to infection. The canine distemper virus is a close relative of the measles virus, but never causes an infection in humans. This kind of resistance is innate and nonspecific. Our immune system recognizes the pathogen as foreign based on certain surface structures, and eliminates it. Humans are thus born with resistance against many microorganisms (innate immunity) and can acquire resistance to others (adaptive or acquired immunity; Fig. 2.1). Activation of the mechanisms of innate immunity, also known as the primary immune defenses, takes place when a pathogen breaches the outer barriers of the body. Specific immune defense factors are mobilized later to fortify and regulate these primary defenses. Responses of the adaptive immune system not only engender immunity in the strict sense, but can also contribute to pathogenic processes. The terms immunopathology, autoimmunity, and allergy designate a group of immu

phenomena causing mainly pathological effects, i.e., tissue damage due to inadequate, misguided, or excessive immune responses. However, a failed immune response may also be caused by a number of other factors. For instance, certain viral infections or medications can suppress or attenuate the immune response. This condition, known as immunosuppression, can also result from rare genetic defects causing congenital immunodeficiency.

The inability to initiate an immune response to the body’s own self antigens (also termed autoantigens) is known as immunological toleran

Anergy is the term used to describe the phenomenon in which cells involved in immune defense are present but are not functi

Our understanding of the immune defense system began with studies of infectious diseases, including the antibody responses to diphtheria, dermal reactions to tuberculin, and serodiagnosis of syphilis. Characteriztion of pathological antigens proved to be enormously difficult, and instead erythrocyte antigens, artificially synthesized chemical compounds, and other more readily available proteins were used in experimental models for more than 60 years. Major breakthroughs in bacteriology, virology, parasitology, biochemistry, molecular biology, and experimental embryology in the past 30–40 years have now made a new phase of intensive and productive research possible within the field of immune defenses against infection. The aim of this chapter on immunology, in a compact guide to medical microbiology, is to present the immune system essentially as a system of defense against infections and to identify its strengths and weaknesses to further our understanding of pathogenesis and prevention of disease.

The Immunological Apparatus

The immune system is comprised of various continuously circulating cells (T and B lymphocytes, and antigen-presenting cells present in various tissues). T and B cells develop from a common stem cell type, then mature in the thymus (T cells) or the bone marrow (B cells), which are called primary (or central) lymphoid organs. An antigen-specific differentiation step then takes places within the specialized and highly organized secondary (or peripheral) lymphoid organs (lymph nodes, spleen, mucosa-associated lymphoid tissues [MALT]). The antigen-specific activation of B and/or T cells involves their staggered interaction with other cells in a contact-dependent manner and by soluble factors

B cells bear antibodies on their surfaces (cell-bound B-cell receptors). They secrete antibodies into the blood (soluble antibodies) or onto mucosal surfaces once they have fully matured into plasma cells. Antibodies recognize the three-dimensional structures of complex, folded proteins, and hydrocarbons. Chemically, B-cell receptors are globulins (“immunoglobulins”) and comprise an astounding variety of specific types. Despite the division of immunoglobulins into classes and subclasses, they all share essentially the same structure. Switching from one Ig class to another generally requires T-cell help.

T cells recognize peptides presented on the cell surface by major histocompatibility (gene) complex (MHC) molecules. A T-cell response can only be initiated within organized lymphoid organs. Naive Tcells circulate through the blood, spleen, and other lymphoid tissues, but cannot leave these compartments to migrate through peripheral nonlymphoid tissues and organs unless they are activated. Self antigens (autoantigens), presented in the thymus and lympoid tissues by mobile lymphohematopoietic cells, induce T-cell destruction (so-called negative selection). Antigens that are expressed only in the periphery, that is outside of the thymus and secondary lymphoid organs, are ignored by T cells; potentially autoreactive T cells are thus directed against such self antigens. T cells react to peptides that penetrate into the organized lymphoid tissues. New antigens are first localized within few lymphoid tissues before they can spread systemically. These must be present in lymphoid tissues for three to five days in order to elicit an immune response. An immune response can be induced against a previously ignored self antigen that does not normally enter lymphoid tissues if its entry is induced by circumstance, for instance, because of cell destruction resulting from chronic peripheral infection. It is important to remember that induction of a small number of T cells will not suffice to provide immune protection against a pathogen. Such protection necessitates a certain minimum sum of activated T cells.

T cells recognize peptides presented on the cell surface by major histocompatibility (gene) complex (MHC) molecules. A T-cell response can only be initiated within organized lymphoid organs. Naive Tcells circulate through the blood, spleen, and other lymphoid tissues, but cannot leave these compartments to migrate through peripheral nonlymphoid tissues and organs unless they are activated. Self antigens (autoantigens), presented in the thymus and lympoid tissues by mobile lymphohematopoietic cells, induce T-cell destruction (so-called negative selection). Antigens that are expressed only in the periphery, that is outside of the thymus and secondary lymphoid organs, are ignored by T cells; potentially autoreactive T cells are thus directed against such self antigens. T cells react to peptides that penetrate into the organized lymphoid tissues. New antigens are first localized within few lymphoid tissues before they can spread systemically. These must be present in lymphoid tissues for three to five days in order to elicit an immune response. An immune response can be induced against a previously ignored self antigen that does not normally enter lymphoid tissues if its entry is induced by circumstance, for instance, because of cell destruction resulting from chronic peripheral infection. It is important to remember that induction of a small number of T cells will not suffice to provide immune protection against a pathogen. Such protection necessitates a certain minimum sum of activated T cells.

The human immunological system can be conceived as a widely distributed organ comprising approximately 1012 individual cells, mainly lymphocytes, with a total weight of approximately 1 kg. Leukocytes arise from pluripotent stem cells in the bone marrow, then differentiate further as two distinct lineages. The myeloid lineage constitutes granulocytes and monocytes, which perform important basic defense functions as phagocytes (“scavenger cells”). The lymphoid lineage gives rise to the effector cells of the specific immune response, T and B lymphocytes. These cells are constantly being renewed (about 106 new lymphocytes are produced in every minute) and destroyed in large numbers (see Fig. 2.17, p. 88). T and B lymphocytes, while morphologically similar, undergo distinct maturation pro cesses (Table 2.1, Fig. 2.2). The antigen-independent phase of lymphocyte differentiation takes place in the so-called primary lymphoid organs: T lymphocytes mature in the thymus and B lymphocytes in the bursa fabricI (in birds). Although mammals have no bursa, the term B lymphocytes (or B cells) has been retained to distinguish these cells, with their clearly distinct functions and maturation in the bone marrow, from T lymphocytes, which mature in the thymus (Table 2.1). B cells mature in the fetal liver as well as in fetal and adult bone marrow. In addition to their divergent differentia

The B-Cell System

B lymphocytes produce antibodies in two forms; a membrane-bound form and a secreted form. Membrane-bound antibody forms the B-cell antigen receptor. Following antigen stimulation, B lymphocytes differentiate into plasma cells, which secrete antibodies exhibiting the same antigen specificity as the B-cell receptor. This system is characterized as humoral immunity, due to this release of receptors into the “humoral” system which constitutes vascular contents and mucous environments. The humoral

Immunoglobulin Structure

All immunoglobulin monomers have the same basic configuration, in that they consist of two identical light chains (L) and two identical heavy chains (H). The light chains appear as two forms; lambda (k) or kappa (j). There are five main heavy chain variants; l, d, c, a, and e. The five corresponding immunoglobulin classes are designated as IgM, IgD, IgG, IgA, or IgE, depending on which type of heavy chain they use (Fig. 2.3b). A special characteristic of the immunoglobulin classes IgA and IgM is that these comprise a basic monomeric structure that can be doubled or quintupled (i.e., these can exist in a dimeric or pentameric form). Table 2.2 shows the composition, molecular weights and serum concentrations of the various immunoglobulin classes

Immunoglobulins contain numerous domains, as illustrated by the structure of IgG. In monomeric IgG each domain consists of a protein segment which is approximately 110 amino acids in length. Both light chains possess two such domains, and each heavy chain possesses four or five domains. The domain structure was first revealed by comparison of the amino acid sequence derived from many different immunoglobulins belonging

same class. In this way a high level of sequence variability was revealed to be contained within the N-terminal domain (variable domain, V), whilst such variability was comparably absent within the other domains (constant domains, C). Each light chain consists of one variable domain (VL) and one constant domain (CL). In contrast, the heavy chains are roughly 440–550 amino acids in length, and consist of four to five domains. Again, the heavy chain variable region is made up of one domain (VH), whereas the constant region consists either of three domains (c, a, d chains), or four domains (l, e chains) (CH1, CH2, CH3, and CH4). Disulfide bonds link the light chains to the heavy chains and the heavy chains to one another. An additional disulfide bond is found within each domain

The three-dimensional form of the molecule forms a letter Y. The two short arms of this ’Y’ consist of four domains each (VL, CL, VH, and CH1), and this structure contains the antigen-binding fragments—hence its designation as Fab (fragment antigen binding). The schematic presented in Fig. 2.3 is somewhat misleading, since the two variable domains of the light and heavy chains are in reality intertwined. The binding site—a decisive structure for an epitope reaction—is formed by the combination of variable domains from both chains. Since the two light chains, and the two heavy chains, contain identical amino acid sequences (this includes the variable domains  immunoglobulin monomer has two identical antigen-binding sites (ABS), and these form the ends of the two short arms of the ’Y’. An area within the antibody consisting of 12–15 amino acids contacts the peptide region contained within the antigen and consisting of approximately 5–800 A˚ 2 (Table 2.3). The trunk of the ’Y’ is called the Fc fragment (named, “fraction crystallizable” since it crystallizes readily) and is made up of the constant domains of the heavy chains (CH2 and CH3, and sometimes CH4)

Diversity within the Variable Domains of the Immunoglobulins

The specificity of an antibody is determined by the amino acid sequence of the variable domains of the H and L chains, and this sequence is unique for each corresponding cell clone. How has nature gone about the task of producing the needed diversity of specific amino acid sequences within a biochemically economical framework? The genetic variety contained within the B-cell population is ensured by a process of continuous diversification of the genetically identical B-cell precursors. The three gene segments (variable, diversity, joining) which encode the variable domain (the VDJ region for the H chain, and the VJ region for the L chain) are capable of undergoing a process called recombination. Each of these genetic segments are found as a number of variants (Fig. 2.4, Table 2.4). B-cell maturation involves a process of genetic re

combination resulting in a rearrangement of these segments, such that one VH, one DH, and one JH segment become combined. Thus the germ line does not contain one gene governing the variable domain, but rather gene segments which each encode fragments of the necessary information. Mature B cells contain a functional gene which, as a result of the recombination process, is comprised of one VHDHJH segment. The diversity of T-cell receptors is generated in a similar manner (

The major factors governing immunoglobulin diversity include:

 Multiple V gene segments encoded in the germ lines. 

& The process of VJ, and VDJ, genetic recombination. 

& Combination of light and heavy chain protein structures. 

& Random errors occurring during the recombination process, and inclusion of additional nucleotides.

 & Somatic point mutations.

In theory, the potential number of unique immunoglobulin structures that could be generated by a combination of these processes exceeds 1012, however, the biologically viable and functional range of immunoglobulin specificities is likely to number closer to 10

The Different Classes of Immunoglobulins

Class switching. The process of genetic recombination results in the generation of a functional VDJ gene located on the chromosome upstrea

Variability types. The use of different heavy or light chain constant regions results in new immunoglobulin classes known as isotypes. Individual Ig classes can also differ, with such genetically determined variations in the constant elements of the immunoglobulins (which are transmitted according to the Mendelian laws) are known as allotypes. Variation within the variable region results in the formation of determinants, known as idiotypes. The idiotype determines an immunoglobulins antigenic specificity, and is unique for each individual B-cell clone

Functions. Each different class of antibody has a specific set of functions. IgM and IgD act as B-cell receptors in their earlier transmembrane forms, although the function of IgD is not entirely clear. The first antibodies produced in the primary immune response are IgM pentamers, the action of which is directed largely against micro-organisms. IgM pentamers are incapable of crossing the placental barrier. The immunoglobulin class which is most abundant in the serum is IgG, with particularly high titers ofthis isotype being found following secondary stimulation. IgG antibodies pass through the placenta and so provide the newborn with a passive form of protection against those pathogens for which the mother exhibits immunity.

The T-Cell System

T-Cell Receptors (TCR) and Accessory Molecules

Like B cells, T cells have receptors that bind specifically to their steric counterparts on antigen epitopes. The diversity of T-cell receptors is also achieved by means of genetic rearrangement of V, D, and J segments (Fig. 2.4b). However, the T-cell receptor is never secreted, and instead remains membrane-bound.

Each T-cell receptor consists of two transmembrane chains, of either the a and b forms, or the c and d forms (not to be confused with the heavy chains of Ig bearing the same designations). Both chains have two extracellular domains, a transmembrane anchor element and a short intracellular extension. As for Ig, the terminal domains are variable in nature (i.e., Va and Vb), and together they form the antigen binding site (see Fig. 2.9, p. 65). T-cell receptors are associated with their so-called co-receptors—other membrane-enclosed proteins expressed on the T cell surface—which include the multiple-chain CD3 complex, and CD4 or CD8 molecules (depending on the specific differentiation of the T cell). CD stands for “cluster of differentiation” or “cluster determinant” and represents differentiation antigens defined by clusters of monoclonal antibodies. (Table 2.13, p. 135f., provides a summary of the most important CD antigens.)

T-Cell Specificity and the Major Histocompatibility Complex (MHC)

T-cell receptors are unable to recognize free antigens. Instead the T-cell receptor can only recognize its specific epitope once the antigen has been cleaved into shorter peptide fragments by the presenting cell. These fragments must then be embedded within a specific molecular groove and presented to the T-cell receptor (a process known as MHC-restricted T-cell recognition or MHC restriction). This “binding groove” is located on the MHC molecule. The MHC encodes for the powerful histocompatibility or transplantation antigens (also known in humans as HLA, human leukocyte antigen molecules, immunological rejection of cell transfusions or tissue and organ transplants. Its true function as a peptide-presenting molecule was not discovered until the seventies, when its role became apparent whilst testing the specificity of virus-specific cytotoxic Tcells. During these experiments it was observed that immune Tcells were only able to destroy infected target cells if both cell types were derived from the same patient or from mice with identical MHC molecules. The resulting conclusion was that a T-cell receptor not only recognizes the corresponding amino acid structure of the presented peptide, but additionally recognizes certain parts of the MHC structure. It is now known that this contact between MHC on the APC and the T-cell receptor is stabilized by the co-receptors CD4 and CD8.

MHC classes. Molecules encoded by the MHC can be classified into three groups according to their distribution on somatic cells, and the types of cells by which they are recognized:

MHC class I molecules. These molecules consist of a heavy a chain with three Ig-like polymorphic domains (these are encoded by 100–1000 alleles, with the a1 and a2 domains being much more polymorphic than the a3 domain) and a nonmembrane-bound (soluble) single-domain b2 microglobulin (b2M, which is encoded by a relatively small number of alleles). The a chain forms a groove that functions to present antigenic peptides (Fig. 2.7). Human HLA-A, HLA-B, and HLA-C molecules are expressed in varying densities on all somatic cells (the relative HLA densities for fibroblasts and hepatic cells, lymphocytes, or neurons are 1x, 100x and 0.1!, respectively). Additional, nonclassical, class I antigens which exhibit a low degree of polymorphism are also present on lymphohematopoietic cells and play a role in cellular differentiation

MHC class II molecules. These are made up by two different polymorphic transmembrane chains that consist of two domains each (a1 is highly polymorphic, whilst b1 is moderately polymorphic, and b2 is fairly constant). These chains combine to form the antigen-presenting groove. Class II molecules are largely restricted to lymphohematopoetic cells, antigen-presenting cells (APC), macrophages, and so on. (see Fig. 2.9a, p. 65) In humans, but notin mice, they are also found on some epithelial cells, neuroendocrine cells, and T cells. The products of the three human gene regions HLA-DP, HLA-DQ and HLA-DR can additionally form molecules representing combinations of two loci—thus providing additional diversity for peptide presentation

MHC class III molecules. These molecules are not MHC antigens in the classical sense, but are encoded within the MHC locus. These include complement (C) components C4 and C2, cytokines (IL, TNF), heat shock protein 70 (hsp70), and other products important for peptide presentation

Functions of MHC molecules. MHC class I and II molecules function mainly as molecules capable of presenting peptides (Figs. 2.7–2.9). These two classes of MHC molecules present two different types of antigens:

Intracellular antigens; these are cleaved into peptides within the proteasome and are usually associated with MHC class I molecules via the endogenous antigen processing pathway

Antigens taken up from exogenous sources; these are processed into peptides within phagolysosomes, and in most cases are then presented on MHC class II molecules on the cell surface (Fig. 2.8, right side). Within the phagolysosome, a fragment called the invariant chain (CLIP, class IIinhibiting protein) is replaced by an antigen fragment. This CLIP fragment normally blocks the antigen-binding site of the MHC class II dimer, thus preventing its occupation by other intracellular peptides

The presentation groove of MHC class I molecules is closed at both ends, and only accommodates peptides of roughly 8–10 (usually 9) amino acids in length. The groove of MHC class II molecules is open-ended, and can contain peptides of 9–15 (usually 10–12) amino acids in length.     

T cells can only recognize antigenic peptides in combination with either MHC class I (which presents endogenous linear peptides, such as those derived from viruses) or MHC class II (which present exogenous linear peptides, such as those derived from bacterial toxins) (Table 2.3). In contrast to antibodies that recognize soluble, complex, nonlinear, three-dimensional structures—T-cell recognition is restricted to changes on the surfaces of cells signaled via MHC plus peptide

T-cell specificity. T-cell recognition therefore involves two levels of specificity: first, MHC presentation molecules bind peptides with a certain degree of specificity as determined by the shape of the groove and the peptide anchoring loci. Second, the MHC-peptide complex will only be recognized by specific T-cell receptors (TCR) once a minimum degree of binding strength has been obtained. For this reason diseases associated with the HLA complex are determined largely by the quality of peptide presentation, but can also be influenced by the available TCR repertoire.

The structure of the MHC groove therefore determines which, of all the potentially recognizable, peptides will actually be presented as T-cell epitopes. Thus, the same peptides cannot function as T-cell epitopes in all individuals. Nonetheless, certain combinations of peptides and MHC are frequently observed. For example, approximately 50% of Caucasians carry the HLA-A2 antigen, although this is sometimes found in a variant form

Antigen-presenting cells (APC). APCs belong to the lymphohematopoietic system. They attach peptides to MHC class II molecules for presentation to T cells, and induce T-cell responses. The complex mechanisms involved in this process have not yet been fully delineated. Stromal cells present in the thymus and bone marrow (i.e., connective tissue cells, dendritic cells and nurse cells in both thymus and bone marrow, plus epithelial cells in the thymus) can also function as APCs. The following cell types function as APCs in peripheral secondary lymphoid organs:

Circulating monocytes.

Sessile macrophages in tissues, microglia in the central nervous system.

Bone marrow derived dendritic cells with migratory potential—these occur as cutaneous Langerhans cells, as veiled cells during antigen transfer into the afferent lymph vessels, as interdigitating cells in the spleen and lymph nodes, and as interstitial dendritic cells or as M cells within MALT.

Follicular dendritic cells (FDC)—these are found within the germinal centers of the secondary lymph organs, do not originate in the bone marrow, and do not process antigens but rather bind antigen-antibody complexes via Fc receptors and complement (C3) receptors.

B lymphocytes—these serve as a type of APC for T helper cells during T-B collaborations.

The consequences of MHC variation. Because every individual differs with regard to the set of polymorphic MHC molecules and self antigens expressed (with the exception of monozygotic twins and inbred mice of the same strain), the differences between two given individuals are considerable. The high degree of variability in MHC molecules—essential for the presentation of a large proportion of possible antigenic peptides for T-cell recognition—results in these molecules becoming targets for T cell recognition following cellular or organ transplantation resulting in transplant rejection.

T-Cell Maturation: Positive and Negative Selection

Maturation of T cells occurs largely within the thymus. Fig. 2.2 (p. 47) shows a schematic presentation of this process. Because the MHC-encoded presentation molecules are highly polymorphic, and are also subject to mutation, the repertoire of TCRs is not genetically pre-determined. One prerequisite for an optimal repertoire of T-cells is therefore the positive selection of T cells such that these preferentially recognize peptides associated only with self transplantation (MHC) antigens. A second prerequisite is negative selection, which involves the deletion of T cells that react too strongly against self MHC plus self peptide. The random processes governing the genetic generation of an array of T-cell receptors results ab or cd receptor chain combinations which are in the majority of cases are non-functional. Those T cells preserved through to maturity represent cells carrying receptors capable of effectively recognizing self-MHC molecules (positive selection).However, the T cells within this group which express too high an affinity for self-MHC plus self-peptides are deleted (negative selection).

The process of positive selection was demonstrated in experimental mice expressing MHC class I molecules of type b (MHC classIb) from which the thymus had been removed (and which therefore had no T cells). Implantation of a new thymus with MHC class I molecules of type a (MHC class I a) into the MHC class I b mice resulted in the maturation of T cells which only recognized peptides presented by MHC class I a molecules, and not peptides presented by MHC class I b molecules. However, recent experiments have shown that this is probable an experimental artefact and that it is not (or not solely) the thymic epithelial cells that determine the selection process, but that this process is driven by cells formed in the bone marrow.

T-Cell Subpopulations

In order to recognize the presented antigen, T cells require the specific T-cell receptor and a molecule which functions to recognize the appropriate MHC molecules (i.e. CD4 or CD8 which recognize MHC class II and MHC class I, respectively). Thus T cells are classified into different subpopulations based on the CD4 or CD8 surface molecules:

CD4+ T cells. These T cells recognize only MHC class II-associated antigens. They are also called T helper cells due to their important role in T-B cell collaboration (Fig. 2.9a), although they exhibit many other additional functions. CD4+ cells can produce, or induce, the production of cytokines by which means they can activate macrophages and exercise a regulatory effect on other lymphocytes (see p. 75f.). Although these cells sometimes demonstrate an ability to cause cytotoxic destruction in vitro, this does not hold true in vivo.

CD8+ T cells. Only MHC class I-associated antigens are recognized by the CD8+ molecule. These cells are also known as cytotoxic T cells due to their ability to destroy histocompatible virus-infected, or otherwise altered, target cells as well as allogeneic cells. This effect can be observed both in vitro and in vivo (Fig. 2.9b). Costimulatory molecules are not required for this lytic effector function. However, cytotoxicity is only one of several important functions expressed by CD8+ T cells. They also have many other non-lytic functions which they execute via the production, or induction of, cytokine release. The designation (CD8+) T suppressor cell is misleading and should not be used. It was originally coined to distinguish these cells from the function of T helper cells, mentioned above. However, plausible documentation of a suppressor effect by CD8+ T cells has only been obtained in a very small number of cases. In most cases, this suppressive effect can in fact be explained.

cd T cells. As for the homologous ab heterodimer, the cd T-cell receptor is associated with the CD3 complex within the cell membrane. The genetic sequence for the c and d chains resembles that of the a and b chains, however, there are a few notable differences. The gene complex encoding the d chain is located entirely within the V and J segments of the a chain complex. As a result, any rearrangement of the a chain genes deletes the d chain genes. There are also far fewer V segments for the c and d genes than for the a and b chains. It is possible that the increased binding variability of the d chains makes up for the small number of V segments, as a result nearly the entire variability potential of the cd receptor is concentrated within the binding region (Table 2.4, p. 53). The amino acids coded within this region are presumed to form the center of the binding site.

cd T cells. As for the homologous ab heterodimer, the cd T-cell receptor is associated with the CD3 complex within the cell membrane. The genetic sequence for the c and d chains resembles that of the a and b chains, however, there are a few notable differences. The gene complex encoding the d chain is located entirely within the V and J segments of the a chain complex. As a result, any rearrangement of the a chain genes deletes the d chain genes. There are also far fewer V segments for the c and d genes than for the a and b chains. It is possible that the increased binding variability of the d chains makes up for the small number of V segments, as a result nearly the entire variability potential of the cd receptor is concentrated within the binding region (Table 2.4, p. 53). The amino acids coded within this region are presumed to form the center of the binding site.

cd T cells can be negative for CD4+ and CD8+, or express two a chains (but no b chain) of the CD8+ molecule. Although it is assumed that cd T cells may be responsible for early, low-specificity, immune defense at the skin and mucosa, their specificities and effector functions are still largely unknown

Immune Responses and Effector Mechanisms

The effector functions of the immune system comprise antibodies and complement-dependent mechanisms within body fluids and the mucosa, as well as tissue-bound effector mechanisms executed by T cells and monocytes/macrophages. B cells are characterized by antigen specificity. Following antigen stimulation, specific B cells proliferate and differentiate into plasma cells that secrete antibodies into the surroundings. The type of B-cell response induced is determined by the amount and type of bound antigen recognized. Induction of an IgM response in response to antigens which are lipopolysaccharides—or which exhibit an highly organized, crystal-like

Some forms of T-cell responses involve the release of soluble mediators (cytokines), which effectively expands the field of T cell function beyond individual cell-to-cell contacts to an ability to regulate the function of large numbers of surrounding cells. Other T-cell effector mechanisms are mediated in a more precise manner through cell-to-cell contacts. Examples of this include perforin-dependent cytolysis and induction of the signaling pathways involved in B-cell differentiation or Ig class switching

B Cells B-Cell Epitopes and B-Cell Proliferation

Burnet’s clonal selection theory, formulated in 1957, states that every B-cell clone is characterized by an unique antigen specificity, i.e., it bears a specific antigen receptor. Accordingly, once rearrangement of the Ig genes has taken place, the corresponding protein will be expressed as a surface receptor. At the same time further rearrangement is stopped. Thus, only one ABS, or one specificity (one VH plus VL [either j or k]), derived from a single allele can be expressed on a single cell. This phenomenon is called allelic exclusion. The body faces a large number of different antigens in its lifetime, necessitating that a correspondingly large number of different receptor specificities, and therefore different B cells, must continuously be produced. When a given antigen enters an organism, it binds to the B cell which exhibits the correct receptor specificity for that antigen. One way to describe this process is to say that the antigen selects the corresponding B-cell type to which it most efficiently binds. However, as long as the responding B cells do not proliferate, the specificity of the response is restricted to a very small number of cells. For an effective response, clonal proliferation of the responsive B cells must be induced. After several cell divisions B cells differentiate into plasma cells which release the specific receptors into the surroundings in the form of soluble antibodies. B-cell stimulation proceeds with, or without, T cell help depending on the structure and amount of bound antigen.

Antigens. Antigens can be divided into two categories; those which stimulate B cells to secrete antibodies without any T-cell help, and those which require additional T-cell signals for this purpose

Type 1 T-independent antigens (TI1). These include paracrystalline, identical epitopes arranged at approximately 5–10 nm intervals in a repetitive two-dimensional pattern (e.g., proteins found on the surface of viruses, bacteria, and parasites); and antigens associated with lipopolysaccharides (LPS). Thus TI1 antigens represent structures with a repetitive arrangement, which allows the engagement of several antigen receptors at one time and results in optimal Ig receptor cross-linking; or structures which result in sub-optimal cross-linking, but which are complemented by an LPS-mediated activation signal. Either type of antigen can induce B cell activation in the absence of T cell help

Type 2 T-independent antigens (TI2). These antigens are less stringently arranged, and are usually flexible or mobile on cell surfaces. They can crosslink Ig receptors, but to a lesser extent than TI1 antigens. TI2 antigens require a small amount of indirectly associated T help in order to elicit a B-cell response (e.g., hapten-Ficoll antigens or viral glycoproteins on infected cell surfaces).

T help-dependent antigens. These are monomeric or oligomeric (usually soluble) antigens that do not cause Ig cross-linking, and are unable to induce B-cell proliferation on their own. In this case an additional signal, provided by contact with T cells, is required for B-cell activation (see also B-cell tolerance, p. 93ff.).

Proliferation of B cells. As mentioned above, contact between one, or a few, B-cell receptors and the correlating antigenic epitope does not in itself suffice for the induction of B-cell proliferation. Instead proliferation requires either a high degree of B cell receptor cross-linking by antigen, or additional T cellmediated signals

Monoclonal Antibodies

A normal immune response usually involves the response and proliferation of numerous B cell clones, bearing ABS with varying degrees of specificity for the different epitopes contained within the antigen. Thus the immune response is normally polyclonal. It is possible to isolate a single cell from such a polyclonal immune response in an experimental setting. Fusing this cell with an “immortal” proliferating myeloma cell results in generation of a hybridoma, which then produces chemically uniform immunoglobulins of the original specificity, and in whatever amounts are required. This method was developed by Koeler and Milstein in 1975, and is used to produce monoclonal antibodies (Fig. 2.10), which represent important tools for experimental immunology, diagnostics, and therapeutics. Many monoclonal antibodies are still produced in mouse and rat cells, making them xenogeneic for humans. Attempts to avoid the resulting rejection problems have involved the production of antibodies by human cells (which remains difficult), or the “humanization” of murine antibodies by recombinant insertion of the variable domains of a murine antibody adjacent to the constant domains of a human antibody. The generation of a transgenic mice, in which the Ig genes have been replaced by human genes, has made the production of hybridoma’s producing completely human antibodies possible

T-Independent B Cell Responses

B cells recognize antigens via the Ig receptor. However, if the antigen is in a monomeric, or oligomeric, soluble form the B cell can only mount a response if it undergoes the process of T-B collaboration. Many infectious pathogens carry surface antigens with polyclonal activation properties (e.g., lipopolysaccharide [LPS]) and/or crystal-like identical determinants, which are often repeated in a regular pattern (linear e.g., flagella, or two-dimensional e.g., viruses) with intervals of 5–10 nm. These paracrystalline-patterned antigens are capable of inducing B-cell responses without contact-dependent T cell help. This probably occurs by means of maximum Ig receptor cross-linking. Such B-cell responses are usually of the IgM type, since switching to different isotype classes is either impossible or very inefficient in the absence of T cell help. The IgM response is of a relatively brief duration (exhibiting a half-life of about 24 h), but can nonetheless be highly efficient. Examples of this efficiency include IgM responses induced by many viral envelope antigens which bear neutralizing (“protective”) determinants accessible to the corresponding antibodies, and responses to bacterial surface antigens (e.g., flagellae, lipopolysaccharides) or parasites

T Cells T-Cell Activation

There are two classes of T cells; T helper cells (CD4+) and cytotoxic T cells (CD8+). Table 2.5 summarizes the reliance of T-cell responses on the dose, localization, and duration of presence of antigen. T-cell stimulation via the TCR, accessory molecules and adhesion molecules results in the activation of various tyrosine kinases (Fig. 2.11) and mediates stringent and differential regulation of several signaling steps. T-cell induction and activation result from the activation of two signals. In addition to TCR activation (signal 1 = antigen), a costimulatory signal (signal 2) is usually required. Important costimulatory signals are delivered by the binding of B7 (B7.1 and B7.2) proteins (presentontheAPCorBcell)toligandsonthe Tcells (CD28protein,CTLA-4),or by CD40–CD40 ligand interactions. T-cell expansion is also enhanced by IL-2.

T-Cell Activation by Superantigens

In association with MHC class II molecules, a number of bacterial and possibly viral products can efficiently stimulate a large repertoire of CD4+ T cells at one time. This is often mediated by the binding of the bacterial or viral product to the constant segment of certain Vb chains (and possibly Va chains) with a low level of specificity (see Fig. 2.9a, p. 65). Superantigens are categorized as either exogenous or endogenous. Exogenous superantigens mainly include bacterial toxins (staphylococcus enterotoxin types A-E [SEA, SEB, etc.]), toxic shock syndrome toxin (TSST), toxins from Streptococcus pyogenes, and certain retroviruses. Endogenous superantigens are derived from components of certain retroviruses found in mice, and which display superantigen-like behavior (e.g., murine mammary tumor virus, MMTV). The function of superantigens during T cell activation can be compared to the effect of bacterial lipopolysaccharides on B cells, in that LPS-induced B cell activation is also polyclonal (although it functions by way of the LPS receptors instead of the Ig receptors (see below)).

Interactions between Cells of the Immune System T Helper Cells (CD4+ T Cells) and T-B Cell Collaboration

Mature T cells expressing CD4 are called T helper (Th) cells (see also p. 64f.), reflecting their role in co-operating with B cells. Foreign antigens, whose three-dimensional structures are recognized by B cells, also contain linear peptides. During the initial phase of the T helper cell response, these antigens are taken up by APCs, processed, and presented as peptides in association with MHC class II molecules—allowing their recognition by Th cells (see Fig. 2.8, p. 61 and Fig. 2.13, p. 76). Prior to our understanding of MHC restriction, B-cell epitopes were known as haptens, whilst those parts of the antigens which bore the T-cell epitope were known as carriers. 

Subpopulations of T Helper Cells 

Soluble signaling substances, cytokines (interleukins), released from T helper cells can also provide an inductive stimulus for B cells. Two subpopulations of T helper cells can be differentiated based on the patterns of cytokines produced (Fig. 2.14). Infections in general, but especially those by intracellular parasites, induce cytokine production by natural killer (NK) cells in addition to a strong T helper 1 (TH1) response. The response by these cells is characterized by early gamma interferon (IFNc) production, increased levels of phagocyte activity, elimination of the antigen by IFNc-activated macrophages, production of IgG2a and other complement-binding (opsonizing) antibodies (see the complement system, pp. 86ff.), and induction of cytotoxic T-cell responses. IL-12 functions as the most important promoter of TH1 cell function and additionally acts as an inhibitor of TH2 cells.

In contrast, worm infections or other parasitic diseases induce the early production of IL-4, and result in the development of a TH2 response. TH2 cells, in turn, recruit eosinophils and induce production of IgG1 and IgE antibodies. Persons suffering from allergies and atopic conditions show a pathologically excessive TH2 response potential. IL-4 not only promotes the TH2 response but also inhibits TH1 cells.

Cytotoxic T Cells (CD8+ T Cells)

Mature CD8+ T cells perform the biologically important function of lysing target cells. Target cell recognition involves the association of MHC class I structures with peptides normally derived from endogenous sources, i.e., originating in the cells themselves or synthesized within them by intracellular parasites. Induction of cytotoxic CD8+ Tcell response often does not require helpe

Cytokines (Interleukins) and Adhesion

Cytokines are bioactive hormones, normally glycoproteins, which exercise a wide variety of biological effects on those cells which express the appropriate receptors (Table 2.6). Cytokines are designated by their cellular origin such that monokines include those interleukins produced by macrophages/ monocytes, whilst lymphokines include those interleukins produced by lymphocytes. The term interleukins is used for cytokines which mostly influence cellular interactions. All cytokines are cyto-regulatory proteins with molecular weights under 60 kDa (in most cases under 25 kDa). They are produced locally, have very short half-lives (a matter of seconds to minutes), and are effective at picomolar concentrations. The effects of cytokines may be paracrine (acting on cells near the production locus), or autocrine (the same cell both produces, and reacts to, the cytokine). By way of interaction with highly specific cell surface receptors, cytokines can induce cell-specific or more general effects (including mediator release, expression of differen tiation molecules and regulation of cell surface molecule expression). The functions of cytokines are usually pleiotropic, in that they display a number of effects of the same, or of a different, nature on one or more cell types. Below is a summary of cytokine functions:

 Promotion of inflammation: IL-1, IL-6, TNFa, chemokines (e.g., IL-8). 

& Inhibition of inflammation: IL-10, TGFb. 

& Promotion of hematopoiesis: GM-CSF, IL-3, G-CSF, M-CSF, IL-5, IL-7. 

& Activating B cells: CD40L, IL-6, IL-3, IL-4. & Activating T cells: IL-2, IL-4, IL-10, IL-13, IL-15. 

& Anti-infectious: IFNa, IFNb, IFNc, TNFa. 

& Anti-proliferative: IFNa, IFNb, TNFa, TGFb.

Cell adhesion molecules often play an essential role in cell-to-cell interactions. Two lympho-hematopoietic cells can only establish contact if one of them expresses surface molecules that interact with ligands expressed on the surface of the other cell. As for APC and T cell interactions, the result of such contact may be that a signal capable of inducing differentiation and functional changes will be induced. Adhesion proteins are usually comprised of several chains which can induce different effects when present in various combinations. Interaction of several cascades is often required for the final differentiation of a cell. Cell adhesion molecules normally form part of the Ig superfamily (e.g., ICAM, VCAM, CD2), integrin family (lymphocyte function antigen, LFA-1), selectin family, cadherin family, or various other families. Selectins and integrins also play an important role in interactions between leukocytes and the vascular wall, and thus mediate the migration of leukocytes from the bloodstream into inflamed tissues, or the entry of recirculating lymphocytes into the lymph node parenchyma through high endothelial venules (HEV).

Chemokines (chemoattractant cytokines) comprise a family of over 30 small (8–12 kDa) secreted proteins. These contribute to the recruitment of “inflammatory cells” (e.g., monocytes) into inflamed tissues, and influence the recirculation of all classes of leukocytes (Table 2.6). Some chemokines result in the activation of their target cell in addition to exerting chemotatic properties. Chemokines can be classified into three families based on their N terminus structure: CC chemokines feature two contiguous cysteine residues at the terminus; CXC chemokines have an amino acid between the two residues; and CX3C and C chemokines thus far comprise only one member each (fractalkine and lymphotactin, respectively). Although the N terminus carries bioactive determinants, using a chemokines amino acid sequence to predict its biological function is not reliable. The chemokine system forms a redundant network, or in other words, a single chemokine can often act upon a number of receptors, and the same receptor may recognize a number of different chemokines. Many of the chemokines also overlap in terms of biological function

Chemokines can be grouped in two functional classes: inflammatory chemokines which are secreted by inflamed or infected tissues as mediators of the nonspecific immune response; and constitutive chemokines which are produced in primary or secondary lymphoid organs. Together with endothelial adhesion molecules, inflammatory chemokines determine the cellular composition of the immigrating infiltrate. In contrast, the function of constitutive chemokines is to direct lymphocytes to precise locations within lymphoid compartments. Thus, chemokines play a major role in the establishment of inflammatory and lymphoid microenvironments. Chemokine receptors are G protein-coupled membrane receptors with seven transmembrane sequences. In keeping with the above nomenclature, they are designated as CCR, CXCR, or CX3CR plus consecutive numbering. Some viruses, for instance

Antibody-Dependent Cellular Immunity and Natural Killer Cells

Lymphocytes can nonspecifically bind IgG antibodies by means of Fc receptors, then specifically attack targets cells (e.g., infected or transformed cells) using the bound antibody. This phenomenon, known as antibody-dependent cellular cytotoxicity (ADCC), has been demonstrated in vitro—however its in-vivo function remains unclear. Natural killer (NK) cells also play a role in ADCC. The genesis of NK cells appears to be mainly thymus-independent. These cells can produce IFNc very early following activation and do not require a specific receptor. These cells are therefore early contributors to the IFNc-oriented TH1 immune response. NK cells can respond to cells that do not express MHC class I molecules, and are inactivated by contact with MHC molecules. This recognition process functions via special receptors that are not expressed in a clonal manner. NK cells probably play an important role in the early defensive stages of infectious diseases, although the exact nature of their role remains to be clarified (virus-induced IFNa and IFNb promote NK activation). NK cells also appear to contribute to rejection reactions, particularly the rejection of stem cells.

Humoral, Antibody-Dependent Effector Mechanisms

The objectives of the immune response include: the inactivation (neutralization) and removal of foreign substances, microorganisms, and viruses; the rejection of exogenous cells; and the prevention of proliferation of pathologically altered cells (tumors). The systems and mechanisms involved in these effector functions are largely non-specific. Specific immune recognition by B and T cells directs these effector mechanisms to specific targets. For instance, immunoglobulins opsonize microbes (e.g., pneumococci) which are equipped with polysaccharide capsules enabling them to resist phagocyte digestion. Opsonization involves the coating of such microbes with Fc-expressing antibodies which facilitates their phagocytosis by granulocytes. Many cells, particularly phagocytes (and interestingly enough also some bacteria like staphylococci), bear surface Fc receptors that interact with different Ig classes and subclasses. Mast cells and basophils bear IgE molecules, and undergo a process of degranulation following interaction with allergens against which the IgE molecules are directed. This induces the release of pharmacologically active biogenic amines (e.g., histamine). In turn, these amines represent the causative agent for physiological and clinical symptoms observed during allergic reactions 

The Complement System

The complement system (C system, Fig. 2.16) represents a non-specific defense system against pathogens, but can also be directed toward specific targets by antibodies. It is made up of a co-operative network of plasma proteins and cellular receptors, and is largely charged with the following tasks:

Opsonization of infectious pathogens and other foreign substances, with the aim of more efficient pathogen elimination. Bound complement factors can: enhance the binding of microbes to phagocytozing cells; result in the activation of inflammatory cells; mediate chemotaxis; induce release of inflammatory mediators; direct bactericidal effects; and induce cell lysis

Solubilization of otherwise insoluble antigen-antibody complexes.

Promotion of the transport of immune complexes, and their elimination and degradation.

Regulation of the immune response, achieved via their influence on antigen presentation and lymphocyte function.

Over 20 proteins of the complement system have been identified to date, and are classified as either activation or control proteins. These substances account for about 5% of the total plasma proteins (i.e., 3–4 g/l). C3 is not only present in the largest amount, but also represents a central structure for complement activation. A clear difference exists between “classic” antibody-induced complement activation and “alternative” activation via C3

During classic activation of complement, C1q must be bound by at least two antigen-antibody immune complexes, to which C4 and C2 then attach themselves. Together, these three components form a C3 convertase, which then splits C3. Pentameric IgM represents a particularly efficient C activator since at least two Ig Fc components in close proximity are required for C1q binding and activation.

During alternative activation of complement, the splitting of C3 occurs directly via the action of products derived from microorganisms, endotoxins, polysaccharides, or aggregated IgA. C3b, which is produced in both cases, is activated by the factors B and D, then itself acts as C3 convertase. Subsequent formation of the lytic complex, C5–C9 (C5–9), is identical for both classic and alternative activation, but is not necessarily essential since the released chemotaxins and opsonins are often alone enough to mediate the functions of microbe neutralization and elimination. Some viruses can activate the complement system without the intervention of antibodies by virtue of their ability to directly bind C1q. This appears to be largely restricted to retroviruses (including HIV). Importantly, without a stringent control mechanism complement would be activated in an uncontrolled manner, resulting in the lysis of the hosts own cells (for instance erythrocytes).

Those complement components with the most important biological effects include:

C3b, results in the opsonization of microorganisms and other antigens, either directly or in the form of immune complexes. “C-marked” microorganisms then bind to the appropriate receptors (R) (e.g., CRI on macrophages and erythrocytes, or CR2 on B cells)

C3a and C5a, contribute to the degranulation of basophils and mast cells and are therefore called anaphylatoxins. The secreted vasoactive amines (e.g., histamine) raise the level of vascular permeability, induce contraction of the smooth musculature, and stimulate arachidonic acid metabolism. C5a initiates the chemotactic recruitment of granulocytes and monocytes, promotes their aggregation, stimulates the oxidative processes, and promotes the release of the thrombocyte activating factor.

“Early” C factors, in particular C4, interact with immune complexes and inhibit their precipitation.

Terminal components (C5–9), together form the so-called membrane attack complex, MAC, which lyses microorganisms and other cells.

Immunological Cell Death

summarizes the mechanisms of cell death resulting from immunological cell interactions and differentiation processes, as they are understood to date

Immunological Tolerance

T-cell tolerance, as defined by a lack of immune reactivity can be due to a number of processes: Firstly, Negative selection in the thymus (referred to as deletion); secondly a simple lack of reactivity to antigen (self or nonself) as a result of the antigen having not been present in the secondary lymphoid organs in a sufficient quantity or for a sufficient amount of time; and thirdly an excessive stimulation of T-cells resulting from the ubiquitous presence of sufficient antigen resulting in T cell exhaustion.

T-Cell Tolerance

A distinction can be made between central tolerance, which develops in the thymus and is based on the negative selection (deletion) of T cells recognizing self antigens present in the thymus, and peripheral tolerance. Peripheral tolerance results in the same outcome as central tolerance, however, this formoftoleranceinvolvesantigenrecognitionbyantigen-reactiveperipheralT cells, followed by a process of clonal cell proliferation, end differentiation and death. The following mechanisms have been postulated, and in some cases confirmed, to account for a lack of peripheral T-cell responsiveness

T-cell indifference or ignorance. Both host and foreign antigens present only within peripheral epithelial, mesenchymal or neuroectodermal cells and tissues—and which do not migrate, or are not transported by APCs, in sufficient amounts to the organized lymphoid organs—are simply ignored by T and B cells. Most self-antigens, not present in the serum or in lymphohematopoietic cells, belong to this category and are ignored despite the fact that they are potentially immunogenic. Certain viruses, and their antigens, actually take advantage of this system of ignorance. For instance, the immune system ignores the rabies virus when it is restricted to axons, and papilloma viruses as long as the antigens are restricted to keratinocytes (warts). The main reason why many self antigens, and some foreign antigens, are ignored by T cells is that immune responses can only be induced within the spleen or in lymph nodes, and non-activated (or naive) T cells do not migrate into the periphery. It has also been postulated that those naive T and B cells which do encounter antigens in the periphery will become anergized, or inactivated, due to a lack of the so-called costimulatory or secondary signals at these sites. However, the evidence supporting this theory is still indirect. Experiments seeking to understand the “indifference” of T cells are summarized in the box on p. 92f. In all probability, a great many self-antigens (as well as peripheral tumors) are ignored by the immune system in this way. These self-antigens represent a potential target for autoimmunity. Both host and foreign antigens present only within peripheral epithelial, mesenchymal or neuroectodermal cells and tissues—and which do not migrate, or are not transported by APCs, in sufficient amounts to the organized lymphoid organs—are simply ignored by T and B cells. Most self-antigens, not present in the serum or in lymphohematopoietic cells, belong to this category and are ignored despite the fact that they are potentially immunogenic. Certain viruses, and their antigens, actually take advantage of this system of ignorance. For instance, the immune system ignores the rabies virus when it is restricted to axons, and papilloma viruses as long as the antigens are restricted to keratinocytes (warts). The main reason why many self antigens, and some foreign antigens, are ignored by T cells is that immune responses can only be induced within the spleen or in lymph nodes, and non-activated (or naive) T cells do not migrate into the periphery. It has also been postulated that those naive T and B cells which do encounter antigens in the periphery will become anergized, or inactivated, due to a lack of the so-called costimulatory or secondary signals at these sites. However, the evidence supporting this theory is still indirect. Experiments seeking to understand the “indifference” of T cells are summarized in the box on p. 92f. In all probability, a great many self-antigens (as well as peripheral tumors) are ignored by the immune system in this way. These self-antigens represent a potential target for autoimmunity

Complete, exhaustive T-cell induction. When an antigen, self or non-self, enters a lymphoid organ it encounters many APCs and T cells, resulting in the extremely efficient activation those T cells carrying the appropriate TCR. During such a scenario the responding T cells differentiate into shortlived effector cells which only survive for two to four days. This induction phase may actually correspond to the postulated phenomenon of anergy (see Table 2.5, p. 71). Should this be the case, anergy—defined as the inability of T cells to react to antigen stimulation in vitro—may in fact be explained by the responding cells having already entered a pathway of cell death (apoptosis) (see Fig. 2.17, p. 88). Once all the terminally differentiated effector T cells have died, immune reactivity against the stimulating antigen ends. Tolerance is hereafter maintained, as should the responsible antigen have entered into the thymus those newly maturing thymocytes will be subjected to the process of negative selection (e.g., as seen in chronic systemic (viremic) infections with noncytopathic viruses). Moreover, those newly matured T cells which may have escaped negative selection and emigrated into the periphery will continuously be induced to undergo activation and exhaustion within the secondary lymphoid organs.

B-Cell Tolerance

In contrast to classic central T-cell tolerance, B cells capable of recognizing self-antigens appear unlikely to be subjected to negative selection (Table 2.7). B-cell regeneration in the bone marrow is a very intensive process, during which antigen selection probably does not play an important role. Although negative selection of bone marrow B cells can be demonstrated experimentally for highly-expressed membrane-bound MHC molecules (in antibody-transgenic mice)—this apparently does not occur for more rare membrane-bound antigens, or for most soluble self-antigens. As a general rule, these potentially self-reactive B cells are not stimulated to produce an immune response because the necessary T helper cells are not present as a result of having being subjected to negative selection in the thymus. B cell and antibody tolerance is therefore largely a result of T cell tolerance which results in the absence of T help.

Immunological Memory

Immunological memory is usually defined by an earlier and better immune response, mediated by increased frequencies of specific B or T cells as determined by in vitro or adoptive transfer experiments. B-cell immunological memory is more completely described as the ability to mediate protective immunity by means of increased antibody concentrations. Higher frequencies of specific B and T lymphocytes alone, appears to only or no protection. Instead, immunological protection requires antigen-dependent activation of B and Tcells, which then produce antibodies continuously or can rapidly mediate effector T functions and can rapidly migrate into peripheral tissues to control virus infections.

Usually the second time a host encounters the same antigen its immune response is both accelerated and augmented. This secondary immune response is certainly different from the primary response, however, it is still a matter of debate as to whether these parameters alone correlate with immune protection. It is not yet clear whether the difference between a primary and secondary immune response results solely from the increased numbers of antigen-specific B and T cells and their acquisition of “memory qualities”, or whether immune protection is simply due to continuous antigen-induced activation

B-Cell Memory

It is important to differentiate between the characteristics of memory T and B cells as detected in vitro, and the salient in-vivo attributes of improved immune defenses. Following a primary immune response, increased numbers of memory B cells can of course be detected using in vitro assays or by murine experiments involving the transfer of cells into naive recipients. However, these increased B cell frequencies do not necessarily ensure immune protection against, for instance, viral re-infection. Such protection requires the existence of an increased titer of protective antibodies within the host.

T-Cell Memory

As with B cells and antibodies, enhanced defenses againstintracellular pathogens (especially viruses and intracellular bacteria) does not solely depend on increased numbers of specific T cells, but rather is determined by the activation status of T cells. Here again it must be emphasized that protective immunological memory against most bacteria, bacterial toxins, and viruses, is mediated by antibodies! Memory T cells are nonetheless important in the control of intracellular bacterial infections (e.g., tuberculosis [TB], leprosy), as well as persistent noncytopathic viruses such as hepatitis B and HIV (see also p.106). It has been demonstrated, at least in mouse models, that a higher number of T cells alone is often insufficient for the protection of the host against the immunopathological consequences of a defensive CD8+ T-cell response. Yet such T cell responses must be activated in order to provide immunity. In the case of tuberculosis, sustained activation of a controlled T-cell response by minimal infection foci was postulated, and confirmed, in the 1960s as constituting infection immunity—i.e.

Immune Defenses against Infection and Tumor Immunity

Protection against infections can be mediated by either; non-specific defense mechanisms (interferons, NK cells), or specific immunity in the form of antibodies and T cells which release cytokines and mediate contactand perforin-dependent cell lysis. Control of cytopathic viruses requires soluble factors (antibodies, cytokines), whilst control of noncytopathic viruses requires and tumors is more likely to be mediated via perforins and cytolysis. However, cytotoxic immune responses can also cause disease, especially during noncytopathic infections. Development of an evolutionary balance between infectious agents and immune responses is an ongoing process, as reflected by the numerous mechanisms employed by pathogens and tumors to evade immune-mediated defenses.

General Rules Applying to Infection Defenses

Non-specific defenses are very important (e.g., Toll-like receptors, IFNa/b), and ’natural immunity’ (meaning not intentionally or specifically induced) represented by natural antibodies, direct complement activation, NK cell and phagocytes, plays a significant role in all infections. However, much remains to be learned about their roles

& Antibodies represent potent effector molecules against acute bacterial infections, bacterial toxins, viral re-infections, and in many cases against acute cytopathic primary viral infections (e.g., rabies and influenza). Antibodies are also likely to make a major contribution to the host-parasite balance occurring during chronic parasitic infections. IgA is the most important defense mechanism at mucosal surfaces

Perforin-dependent cytotoxicity in CD8+ T cells is important for defense against noncytopathic viruses, for the release of chronic intracellular bacteria, and for protection against intracellular stages of certain parasites.

Nonlytic T-cell responsesprovideprotectioninthe formof cytokines (very important cytokines include IFNc and TNFa), which promote the enhanced digestion and destruction of intracellular bacteria and parasites (e.g., listeria, leishmania, etc.), and in some situations enhance immunity against complex viruses (e.g., the smallpox virus) (Fig. 2.15, p. 79). Infectious agents apparently induce cytokines within a matter of hours (for instance IFNc, IL-12 , and IL-4), and this early cytokine production in turn functions to define the ensuing T cell response as type 1 or type 2

IgE-mediated defense is important, along with IgA, in enhancing the elimination of gastrointestinal, pulmonary, and dermal parasites. Although details of the process are still sketchy, IgE-dependent basophil and eosinophil defense mechanisms have been described for model schistosomal infections.

Avoidance strategies. Infectious agents have developed a variety of strategies by which they can sometimes succeed in circumventing or escaping immune responses, often by inhibiting cytokine action.

Immune Protection and Immunopathology

Whether the consequences of an immune response are protective or harmful depends on the balance between infectious spread and the strength of the ensuing immune response. As for most biological systems, the immune defense system is optimized to succeed in 50–90% of cases, not for 100% of cases. For example, immune destruction of virus-infested host cells during the eclipse phase of a virus infection represents a potent means of preventing virus replication (Fig. 2.15, p. 79). From this point of view, lytic CD8+ T-cell responses make good sense as the host will die if proliferation of a cytopathic virus is not halted early on. If a noncytopathic virus is not brought under immediate control, the primary illness is not severe—however, the delayed cytotoxic response may then lead to the destruction of very large numbers of infected host cells and thus exacerbate disease .\

Influence of Prophylactic Immunization on the Immune Defenses

Vaccines provide protection from diseases, but in most cases cannot entirely prevent re-infection. Vaccination normally results in a limited infection by an attenuated pathogen, or induces immunity through the use of killed pathogens or toxoids. The former type of vaccine produces a very mild infection or illness capable of inducing an immune response and which subsequently protects the host against re-infection. The successful eradication of smallpox in the seventies so far represents the greatest success story in the history of vaccination. The fact is that vaccinations never offer absolute security, but instead improve the chances of survival by a factor of 100 to 10 000. A special situation applies to infections with noncytopathic agents in which disease results from the immune response itself (see above). Under certain circumstances, and in a small number of vaccinated persons, the vaccination procedure may therefore shift the balance between immune defense and infection towards an unfavorable outcome, such that the vaccination will actually strengthen the disease. Rare examples of this phenomenon may include the use of inactivated vaccines against the respiratory syncytial virus (RSV) in the sixties, and experience with certain so-called subunit vaccines and recombinant vaccines against noncytopathic viral infections in rare model situations. Generally, it should be kept in mind that most of the successful immunization programs developed to date have mediated protection via antibodies. This particularly applies to the classic protective vaccines listed in Table 1.13 (p. 33) for children, and explains why antibodies not only are responsible for the protection of neonates during the immuno-incompetent early postnatal period where immunological experience is passed onfromthe mother via antibodies, but also attenuate early childhood infections to become vaccine-like. This explains why successful vaccines all protect via neutralizing antibodies, because this pathway has been selected by co-evolution. As mentioned earlier, with regard to immunological memory, memory T cells appear to be essential to host immune protection, particularly in those situations when antigen persistence is controlled efficiently by means of infection-immunity (e.g., tuberculosis, HIV)

Tumor Immunity

Our knowledge concerning the immune control of tumors is still modest. Some tumor types bear defined tumor-associated, or tumor-specific, antigens. However this is apparently not sufficient for induction of an efficient immune defense. There is also the problem of tumor diagnosis; the presence of tumors is sometimes confirmed using a functional or immunological basis, yet the tumor cannot be located because conventional examinations are often unable to discover them until they reach a size of about 109 cells (i.e., about 1 ml) of tumor tissue.

Factors important in immune defense reactions include the location and rate of proliferation, vascularization or the lack thereof, and necrosis with phagocytosis of disintegrating tumor tissue. We never actually get to see those rare tumors against which immune control might have been successfully elicited, instead we only see those clinically relevant tumors that have unfortunately become successful tumors which have escaped immune control.

Evidence of the immune system’s role in tumor control includes:

Greater than 85% of all tumors are carcinomas and sarcomas, that is nonlymphohematopoietic tumors which arise in the periphery, outside of organized lymphoid tissues. The immune system, in a manner similar to that seen for many strictly extra-lymphatic self antigens, ignores such tumors at first.

Lymphohematopoietic tumors often present immunological oddities such as unusually low, or entirely absent, MHC and/or low tumor antigen concentrations, plus they frequently lack accessory molecules and signals

Congenital or acquired immunodeficiency—whether caused by anti-lymphocytic sera, cytostatic drugs, gamma irradiation, UV irradiation, or infection—usually encourages tumor growth, especially for lymphohematopoietic tumors. Carcinomas and sarcomas show little or no increased susceptibility. Interestingly, experimental carcinogens are frequently also immunosuppressive.

Surgical removal of a large primary tumor may result in the disappearance (or rarely in rapid growth) of metastases within the lymph nodes.

Tumor cells often display modulated MHC expression—some tumors lack MHC class I molecules entirely—or in some cases tumors selectively downmodulate the only MHC allele capable of presenting a specific tumor-associated peptide (e.g., the colon adenocarcinomas). Other tumors side-step immune defenses by down-regulating tumor-specific antigens.

The immune response may fail if tumor differentiation antigens are expressed, against which the host exhibits an immunological tolerance (e.g., carcinoembryonic antigen [CEA], T-cell leukemia antigen).

Blockade of the reticuloendothelial system may encourage the development of lymphohematopoietic tumors. For instance, chronic parasitic infections or infection by malaria can result in the development of Burkitt lymphoma, a B-cell malignancy

The Pathological Immune Response

An immune response can also cause disease. Such responses can be classified into the following types: Type I: allergic IgE-dependent diseases; Type II: antibody-dependent responses to cell membranes, blood group antigens or other auto-antigens; Type III: immune complex-initiated diseases whereby surplus antigen-antibody complexes are deposited on basement membranes, resulting in development of chronic disease via complement activation and inflammatory reactions; Type IV: cellular immunopathology resulting from excessive T-cell responses against infections that otherwise exhibit low cytopathogenicity, or against allogenic organ transplants.

Type I: IgE-Triggered Anaphylaxis

This type of immediate hypersensitivity reaction occurs within minutes in allergically sensitized individuals. Although serum IgE has a short half-life (one to two days), IgE antibodies bound to the Fce receptor on basophils and mast cells have a half-life of several months and when bound by the specific allergen mediate cellular degranulation and the release of biogenic amines (e.g., histamine, serotonin). These mediators can influence the smooth musculature, and mainly result in the constriction of the pulmonaryand broncho-postcapillary venules, together with arteriole dilation. The local manifestations of IgE-triggered anaphylaxis include whealing of the skin (urticaria), diarrhea for food allergies, rhinitis or asthma for pollen allergies, or a generalized anaphylactic shock. IgE reactions are usually measured in vitro using RIA (radioimmunoassay), RIST (radioimmunosorbent test) or RAST (radioallergosorbent test) (see Fig. 2.28 and Fig. 2.29, p.131f.) Frequent causal agents of IgE allergies in humans include pollen, animal hair, house dust (mites), insect bites and stings, penicillin, and foods. Examples of allergic diseases include local allergic rhinitis and conjunctivitis, allergic bronchial asthma, systemic anaphylactic shock, insect toxin allergies, house dust (mite) and food allergies, urticaria, and angioedemas.

Type II: Cytotoxic Humoral Immune Responses

These are pathological immune responses induced by the binding of IgM or IgG antibodies to antigens present on a cell surface (including viral products or haptens), or within tissue components. The mediators responsible for such tissue damage are usually components of the complement system,     

Autoantibody Responses

Some clinically important autoantibodies are directed against hormone receptors, for example thyrotoxicosis in Basedow’s disease is caused by autoantibodies that stimulate the TSH receptor, and myasthenia gravis is caused by blockage of the acetylcholine receptor by specific autoantibodies. Other antibody-induced diseases mediated by antibodies, directed against hormones and other cellular self antigens, include Hashimoto thyroiditis (induced by anti-thyroglobulin and anti-mitochondrial autoantibodies), pernicious anemia (anti-intrinsic factor), pemphigus vulgaris (anti-desmosome) Guillain-Barre´ syndrome (ascending paralysis caused by specific myelin autoantibodies), and scleroderma (involving anti-collagen antibodies). Other immunopathologies involving autoantibodies include transplant rejection as a result of endothelial damage (especially in xenogeneic transplants), and tumor rejection caused by antibodies against tumor-associated antigens present on neoplastic cells (especially relevant for lymphohematopoietic tumors). However, in general the detection of autoantibodies does not necessarily correlate with evidence of pathological changes or processes. In fact, our detection methods often measure low-avidity autoantibodies that may have no direct disease-causing effects.

Anti-blood Group Antibody Reactions

ABO system. These B-cell epitopes consist of sugar groups present in the membranes of red blood cells. The four classic blood groups are determined by one gene with three alleles. This gene controls glycosylation. The O allele codes only for a basic cell surface structure (H substance) with the terminal sugars galactose and fucose. The A allele adds N-acetylgalactosamine to this basic structure, the B allele adds galactose. This results in epitopes, which are also seen frequently in nature largely as components of intestinal bacteria. Individuals who carry the A allele are tolerant to the A-coded epitope, whilst individuals with the B allele are tolerant to the B epitope. Individuals who carry both of these alleles (genotype AB) are tolerant to both epitopes, whereas persons who are homozygotes for the O allele are not tolerant to either A or B. Following birth, the intestinal tract is colonized by bacteria containing large numbers of epitopes similar to the A and B epitopes. During the first months of life, people with blood group O (homozygous for the O allele) produce both anti-A and anti-B antibodies, people with blood group A (genotype AO or AA) produce only anti-B antibodies, people with blood group B (genotype BO or BB) produce only anti-A antibodies, and people with blood group AB produce neither anti-A or anti-B antibodies.

These so-called “natural” antibodies (meaning these antibodies are produced without a recognizable immunization process) are of the IgM class; there is usually no switch to IgG, probably resulting from a lack of necessary helper T-cell epitopes. The presence of the blood group antibodies makes blood transfusions between non-matched individuals extremely risky, necessitating that the blood group of both the donor and recipient is determined before the blood transfusion takes place. Nevertheless, the antibodies in the donor blood are not so important because they are diluted. The O genotype is therefore a universal donor. Note that IgM antibodies to blood groups present no danger to the fetus since they cannot pass through the placental barrier

Other blood group systems. There are other additional blood group systems against which antibodies may be produced, and which can present a risk during transfusions. Thus, the crossmatch test represents an important measure in the avoidance of transfusion problems. Immediately prior to a planned transfusion, serum from the prospective recipient is mixed with erythrocytes from the prospective donor, and serum from the prospective donor is mixed with erythrocytes from the prospective recipient. To ensure no reaction following transfusion, there should be no agglutination present in either mixture. Some potentially dangerous serum antibodies may bind to the erythrocytes causing opsonization, but not necessarily inducing agglutination. To check for the presence of such antibodies, anti-human immunoglobulin serum is added and should it crosslink such antibodies agglutination will result.

Type III: Diseases Caused by Immune Complexes

Pathologies initiated by immune complexes result from the deposition of small, soluble, antigen-antibody complexes within tissues. The main hallmark of such reactions is inflammation with the involvement of complement. Normally, large antigen-antibody complexes (that is, those produced in equivalence) are readily removed by the phagocytes of the reticuloendothelial system. Occasionally, however—especially in the presence of persistent bacterial, viral, or environmental, antigens (e.g., fungal spores, vegetable or animal materials), or during autoimmune diseases directed against autoantigens (e.g., DNA, hormones, collagen, IgG) where autoantibodies to the body’s own antigens are produced continuously—deposition of antigen-antibody complexes may become widespread often being present on active secretory membranes and within smaller vessels. Such processes are mainly observed within infected organs, but can also occur within kidneys, joints, arteries, skin and lung, or within the brain’s plexus choroideus. The resulting inflammation causes local tissue damage. Most importantly, activation of complement by such complexes results in production of inflammatory C components (C3a and C5a). Some of these anaphylatoxins cause the release of vasoactive amines which increase vascular permeability (see also p. 103f.). Additional chemotactic activities attracts granulocytes which attempt to phagocytize the complexes. When these phagocytes die, their lysosomal hydrolytic enzymes are released and cause further tissue damage. This process can result in long-term chronic inflammatory reactions.

There are two basic patterns of immune complex pathogenesis:

Immune complexes in the presence of antigen excess. The acute form of this disease results in serum sickness, the chronic form leads to the development of arthritis or glomerulonephritis. Serum sickness often resulted from serum therapy used during the pre-antibiotic era, but now only occurs rarely. Inoculation with equine antibodies directed against human pathogens, or bacterial toxins, often induced the production of host (human) antibodies against the equine serum. Because relatively large amounts of equine serum were administered for such therapeutic purposes, such therapy would result in the induction of antigen-antibody complexes—some of which were formed in the presence of antigen excess—and occasionally induced a state of shock.

Immune complexes in the presence of antibody excess. The so-called Arthus reaction is observed when an individual is exposed to repeated small doses of an antigen over a long period of time, resulting in the induction of complexes and an antibody excess. Further exposure to the antigen, particularly dermal exposure, induces a typical reaction of edema and erythema which peaks after three to eight hours and disappears within 48 hours, but which sometimes leads to necrosis. Arthus-type reactions often represent occupational diseases in people exposed to repeated doses of environmental antigens: farmer’s lung (thermophilic Actinomyces in moldy hay), pigeon breeder’s lung (protein in the dust of dried feces of birds), cheese worker’s lung (spores of Penicillium casei), furrier’s lung (proteins from pelt hairs), malt-worker’s lung (spores of Aspergillus clavatus and A. fumigatus).

Type IV: Hypersensitivity or Delayed Type, Cell-Mediated Hypersensitivity

Intracutaneous injection of a soluble antigen derived from an infectious pathogen induces a delayed dermal thickening reaction in those people who have suffered a previous infection. This delayed skin reaction can serve as a test to confirm immunity against intracellular bacteria or parasites.

For most cases, the time between administration of the antigen and the swelling reaction is 48–72 hours—as described above for cellular delayed type hypersensitivity (DTH) reactions in the skin (p. 99). As observed for antibodydependent hyper-reactions of types I-III, the type IV response is pathogenic and differs from protective immune responses only in terms of the extent and consequences of the tissue damage, but not in terms of the mechanism of action. The balance between autoimmune disease and type IV immunopathology in such cases is readily illustrated by type IV reactions (e.g., aggressive hepatitis in humans or lymphocytic choriomeningitis in mice). Should the causal infectious pathogen be known, the response is termed a type IV reaction, if the causal agent is unknown (or not yet determined) the same condition may be termed “autoimmune disease.”

Autoimmune T cells are usually directed against autoantigens that would otherwise be ignored (since they are only expressed in the extralymphatic periphery). Autoaggressive CD4+ T cells apparently respond against myelin basic protein in multiple sclerosis, against collagen determinants in polyarthritis, and against islet cell components in diabetes.

Transplantation Immunity

The strong transplantation antigens are encoded within the MHC complex (see p. 58ff.), whilst the weak antigens constitute the MHC-presented allelic differences of non MHC-encoded host proteins or peptides. It is possible to differentiate between the host-versus-graft (HVG) reaction of the recipient against a genetically foreign tissue or organ, and the graft-versus-host (GVH) reaction.

The GVH reaction. This type of reaction results when immunologically responsive donor T cells are transferred to an allogeneic recipient who is unable to reject them (e.g., following a bone marrow transplant into an immuno-incompetent or immuno-suppressed recipient). The targets against which the transplanted T cells generate an immune response include the MHC class I and II molecules of the recipient. The recipient’s transplantation antigens also present allelic variants of recipient self-peptides, which can be recognized by donor T cells as weak transplantation antigens when presented by common MHC alleles (it is conceivable that strong recipient transplantation antigens could be accepted and processed by donor APCs, however even if this did occur it would be of limited functional consequence as they would not be presented by the recipient APC in the correct antigen configuration). Weak histocompatibility antigens—for instance those peptide variants recognized as nonself when presented in combination with essentially histocompatible MHC molecules—play a more significant role in bone marrow transplants. The existence, and pathological role, of weak transplantation antigens has only been demonstrated in completely histocompatible siblings or within inbred animal strains with identical MHC.

HVG reactions, that is immune responses of the recipient against transplanted cells or organs, are not generated in autotransplants (for instance transplantation of skin from one part of the body to another on the same individual). This also applies to transplants between monozygotic twins or genetically identical animals (syngeneic transplants). However, transplants between non-related or non-inbred animals of the same species (allogeneic transplants), and transplants between individuals of different species (xenogeneic transplants) are immunologically rejected. Because T cells recognition is subject to MHC restriction, cellular rejection within a species is even more pronounced than between different species, although the latter procedure involves other transplantation complications. These include the occurrence of natural cross-reactive antibodies, and a lack of complement inactivation by anti-complement factors (which are often species-incompatible and therefore absent in xenogeneic transplants), which together often results in hyperacute rejection within minutes, hours, or a few days—that is before any specific immune responses can even be induced.

Hyperacute rejection of vascularized transplants, occurring within minutes to hours and resulting from preformed recipient antibodies reacting against antigens present on the donor endothelium, resulting in coagulation, thromboses, and infarctions with extensive necrosis.

Acute rejection, occurring within days or weeks. This is accompanied by a perivascular and prominent occurrence of T lymphocyte infiltrates. Acute rejection can be prevented by immunosuppress

Acute rejection, occurring within days or weeks. This is accompanied by a perivascular and prominent occurrence of T lymphocyte infiltrates. Acute rejection can be prevented by immunosuppress

Methods of measurement. The main methods used for follow-up analysis of HVG and GVH reactions are biopsies and histological evaluation, evaluation of blood cells and in-vitro mixed lymphocyte reactions

Immune Defects and Immune Response Modulation

Immune defects are frequently acquired by therapy or viral infections, or as a consequence of advanced age. In rare cases immune defects can also result from congenital defects, these include severe combined immunodeficiency’s (SCID) or transient partial immune defects (mainly involving IgA responses). Immunomodulation can be attempted using interleukins or monoclonal antibodies directed against lymphocyte surface molecules or antigenic peptides. Immunostimulation is achieved using adjuvants or the genetically engineered insertion of costimulatory molecules into tumor cells. Immunosuppression can be induced globally using drugs, or specifically using antibodies, interleukins or soluble interleukin receptors; this can also be achieved by means of tolerance induction with proteins, peptides, or cell chimerism.

Immune Defects

The most important and frequent immune defects are acquired, e.g., iatrogenic (cytostatics, cortisone, irradiation, etc.), age-induced, or the result of viral infections (above all HIV). Congenital defects are rare; examples include Bruton’s X-chromosome-linked B-cell defect, thymic hypoplasia (DiGeorge), and combined T- and B-cell deficiency resulting from MHC defects (bare lymphocyte syndrome) or from enzyme defects (adenosine deaminase [ADA] deficiency or purine nucleoside phosphorylase [PNP] deficiency). These defects can also be repaired by reconstitution (thymic transplants), or in some cases through the use of stem cells (gene therapy; one of the very first successful gene therapies was the treatment of ADA deficiency). More frequent congenital defects involve selective deficiencies, for example a relative-to-absolute IgA deficiency, normally being more prominent in infants than later in life. Children with such deficiencies are more susceptible to infection with Haemophilus influenzae, pneumococci, and meningococci. General consequences of immune defects include recurring and unusual infections, eczemas, and diarrhea.

Immunoregulation

This area of immunology is difficult to define and remains elusive. Antigens represent the most important positive regulator of immunity; since there is simply no immune stimulation when antigens have been eliminated or are absent. Other important regulators include interferon gamma (IFNc) for TH1 responses, and IL-4 for TH2 responses. Further IL-dependent regulatory functions are in the process of being defined. The existence of specific CD8+ T suppressor cells, capable of downregulating immune responses, has been postulated and their role was assumed to be that of counteracting the inflammatory CD4+ T cell response. However, to date there has been no convincing proof of their existence. The term CD8+ T suppressor cells, which is used frequently, is therefore misleading and inaccurate. In relatively rare cases, cyto toxic CD8+ T cells do exercise a regulatory effect by lysing infected APCs or B cells (see also p. 106). It is unclear whether CD4+ T cells could have similar effects. Regulation via idiotypic/anti-idiotypic antibody networks (i.e., antibodies directed against the ABS of other antibodies), or anti-TCR networks, have also been postulated—but remain hypothetical. Although attractive hypothesis, for most cases such regulatory pathways have only proved disappointing theoretical concepts, and as such should no longer be employed in the explanation of immunoregulation. In isolated cases, anti-idiotypic, or anti-TCR peptide-specific feedback, mechanisms can be modeled under forced experimental conditions. However such conditions probably fail to model normal situations, therefore they cannot accurately indicate whether these feedback mechanisms have a role in regulating the immune system as a whole.

Immunostimulant

The aim of immunological treatment of infections and tumors is to enhance immune responsiveness via the use of thymic hormones (thymopoietin, pentapeptides), leukocyte extracts, or interferons. Derivatives or synthetic analogs of microorganisms such as BCG, components of Corynebacterium parvum and peptidoglycans (e.g., muramyl peptide), or oligonucleic acids (CpG), are used as adjuvants. Components of streptococci and Streptomyces, eluates and fractions of bacterial mixtures, and the related synthetic substance levamisole are also used. The role of Toll-like receptors in these adjuvant effects is becoming increasingly understood, with a major role of these molecules being to link non-specific innate resistance to specific immunity

Immunosuppression

Generalized immunosuppression; glucocorticoids (inhibition of inflammatory cells), cytostatic drugs (endoxan, DNA alkylating agents, methotrexate, antimetabolites), and more specific immunosuppressants, e.g., cyclosporine A, FK506, rapamycin (inhibition of signal transduction in T cells, se

Immunosuppression by antibodies, soluble cytokine receptors, deletion of T cells or T-cell sub-populations (anti-CD4, anti-CD8, anti-CD3, anti-Thy1, etc.). Administration of monoclonal antibodies directed against adhesion molecules and accessory molecules or cytokines and cytokine receptors. Administration of soluble cytokine receptors, or soluble CTLA4, in order to block B7- 1 and B7-2 (important costimulators,

Specific tolerance induction or “negative immunization.” Massive and depletive T-cell activation brought about by systemic administration of large amounts of peptides, proteins (risk of immunopathology), or cells (chimerism).

Complete neutralization and elimination of the antigen with the purpose of preventing induction of an antibody response. Example; rhesus prophylaxis with hyperimmune serum.

Adaptive Immunotherapy

This involves in-vitro antigen stimulation, and consequent proliferation, of patient T-cell effector clones or populations (CD8+ T cells or less specific lymphokine-activated killer cells, LAK cells), followed by transfusion of these cells back into the patient. This method is sometimes used as a means of limiting cytomegaly or Epstein-Barr virus infection of bone marrow recipients. The LAK cells also include less specific NK-like cells, which can be expanded with IL-2 in the absence of antigen stimulation.

Toxic antibodies are monoclonal antibodies to which toxins have been coupled. These are used as specific toxin transporters, administered directly, or with liposomes bearing anchored antibodies and containing a toxin or cytostatic drug.

Lymphocyte Function Tests

Certain functions of isolated lymphocyte populations can be determined by a number of methods:

Determination of the number of cells producing antibodies, e.g., the hemolytic plaque assay in which antibody production is tested by adding antigencoupled erythrocytes. In the vicinity of antibody-secreting cells, the erythrocytes are covered with antibodies and can be lysed by addition of complement. Today, ELISA methods are more often used than erythrocytes (ELISPOT).

ELISPOT ASSAY: used to measure antibody-producing, or IL-releasing, lymphocytes. The antigen or anti-IL antibody is fixed on a plastic surface. Lymphocytes are then placed over this, within a thin layer of agar medium. When the cells are incubated at 37 8C, they may secrete the antibodies or IL recognized by the corresponding test substances. After a certain period of time, the cell layer is shaken off and the preparation is thoroughly washed. The bound material can then be developed using an overlaid semisolid agar, as for the ELISA method. The enzyme reaction generates spots of color, each of which corresponds to a cell, and which can be counted

Measurement of the release capacity of cytokines, or detection of mRNA, is also possible with the ELISPOT assay.

Lymphocyte stimulation assay: isolated lymphocytes are incubated with antigen in culture medium. Measuring the 3H-thymidine incorporation,

interleukin release, or a pH transition, can determine whether antigen-specific lymphocytes are present or whether polyclonal T-cell responses (concanavalin [ConA], phytohemagglutinin [PHA]) or B-cell responses (lipopolysaccharide [LPS], pokeweed mitogen [PMA]) were induced.

Chromium release assay measures cytolytic activity, mainly by CD8+ Tcells, directed against allogeneic, virus-infected, or peptide-loaded target cells. The target cells are incubated with 51Cr which the cells incorporate. They are then cultivated with effector cells for 4–6 hours. When the target cells are lysed chromium is released into the culture medium, following which it can be quantitatively measured.

Assay of intracellular cytokines. Following a brief stimulatory culture (six hours), the cells are rendered permeable using a mild detergent so that specifically labeled antibodies can diffuse into the cells. Labeled cells can then be analyzed by FACS equipment (or by a microscope).