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Evaluation of Immune Responses Robert Vogt and Paul A

Evaluation of Immune Responses Robert Vogt and Paul A

unit 3. assessing exposure to the environment

chapter 13. Evaluation of immune responses Robert Vogt and Paul A. Schulte Unit 3 Summary Introduction Chapter 13 Chapter This chapter will present some Over the past 15 years, familiarity in epidemiologic field studies and general background material on the with the has public health applications. At the cellular, biochemical, and genetic increased substantially among same time, the general principles mechanisms of the immune system, public health scientists, as well as advocated earlier remain fully then focus on specific examples that the public at large. Since the use relevant today, perhaps even illustrate the promise and pitfalls of immune biomarkers in molecular more so, given that the pace of of using immune biomarkers as epidemiology was first addressed technological development often tools for molecular epidemiologic (1), the essential role of the immune outstrips our ability to harness it in a research and public health practice. system in maintaining health has meaningful fashion. Some of the most exciting frontiers been brought to public attention This chapter will first update in medical science will be discussed: by the global HIV epidemic (2), the some of the general background early detection of cancer through composite burden of autoimmune material presented before (1) with ; malignancies that (3,4,5), the genetic respect to the cellular, biochemical, arise from the immune system itself; errors that lead to primary immune and genetic mechanisms of the newborn screening for lethal immune deficiencies (6), and the enigmatic immune system. Thereafter, the deficiencies and latent autoimmune relationship between and focus will be on specific examples disorders; and neurodevelopmental malignancy (7,8). During this period, that illustrate the promise and disabilities that could result from our understanding of the cellular and pitfalls of using immune biomarkers maternal immune responses, molecular processes that constitute as tools for translation research which protect the mother but harm the immune response has also and public health practice. Some the fetus. The chapter concludes increased in both scope and detail. of the most exciting frontiers in with some thoughts about current These advances open new avenues medical science will be discussed: challenges and future directions. for the use of immune biomarkers early detection of cancer through

Unit 3 • Chapter 13. Evaluation of immune responses 215 autoimmunity; environmental risk specific and those that are not Biological categories factors for malignancies that arise is often important, especially in of immune biomarkers from the immune system itself; exposure-related studies. The ability newborn screening for lethal immune of the immune system to recognize The distinction between - deficiencies and latent autoimmune foreign molecules is so discerning specific and non-specific disorders; and neurodevelopmental that it has even been likened to a biomarkers is fundamental and disabilities that could result from self-referential sensory organ (9). unique to the immune system. For maternal immune responses, convenience, this distinction can be which protect the mother but harm overlaid onto three major types of the fetus. The chapter concludes intrinsic biological markers: cellular, with some thoughts about current Whether innate or acquired, the biochemical and genetic (Table challenges and future directions. result of host defence activity is 13.1). These three categories are often inflammation. The cardinal arbitrary and somewhat artificial, Immune biomarkers signs of inflamed tissue were since biochemical and genetic as functional elements described by the ancient Greek markers originate in cells. In fact, and sentinel indicators physicians Celsus and Galen: calor many cells of the immune system are (heat), dolor (pain), rubor (redness), defined by the biochemical surface The benefits and limitations of using tumour (swelling), and receptors they express or the unique immune markers in epidemiologic (loss of function) (10). Our current rearrangements they contain. studies may be best appreciated by knowledge of the cellular and understanding their relationship to molecular basis of inflammation is Cellular biomarkers the basic of the host defence exhaustive, but the complexity of system: a complex network of cells the in situ inflammatory response Cellular immunology was for and mediators with recognition still lies beyond our complete many years a phenomenologic and response functions that occur understanding. Still, the cells and area of study, often confusing throughout most tissues of higher mediators of inflammation provide and contradictory. Two technical organisms (1). The primary functions essential biomarkers for medicine, breakthroughs combined to bring of the host defence system are biomedical research, and, more order to this area. Monoclonal repairing injured tissue, identifying recently, for epidemiologic studies. allowed the development and removing foreign substances, Inflammation is essential for of specific probes without a priori destroying or containing infectious host defence, as it brings cells and knowledge of the properties of agents, and, in some cases, mediators to the site of tissue injury the cellular target. Flow cytometry eradicating cancer cells. and , sequestering the allowed the cell-by-cell detection insult, destroying infectious agents of these targets using fluorescent- Innate (non-specific) and or the cells they have infected, labelled monoclonal antibodies and acquired (specific) immunity clearing the debris and promoting a dynamic streaming process that repair. However, it is a two-edged could analyse and sort thousands of Host defence functions are sword, and many of the symptoms cells per second (14,15). The sorted carried out through non-specific following injury or infection come cells could then be characterized mechanisms of innate immunity, not from the insult but from the host for their functional activities and and through specific mechanisms of response to it. Immunopathology is other properties, and linked to acquired (adaptive) immunity, which the study of how the immune system their respective target. The cellular develop as the organism encounters creates as well as prevents . targets, which are all proteins (or environmental agents (). From the classic animal models of glycoproteins) and are usually The term immune system is used in viral meningitis (11) and tuberculosis cell surface receptors, are most this chapter to refer to all components (12) to the recent revelation that often identified by their cluster of both non-specific innate immunity human and of differentiation (CD) number and antigen-specific acquired are largely inflammatory (16). International workshops are immunity, as their components and pathologies (13), biomarkers held periodically to update CD activities are invariably intertwined have shown that immunity and nomenclature (17); as of 2010, the (1). Nonetheless, the distinction inflammation are inexorably linked. list was up to CD363 (see http:// between markers that are antigen- www.hlda9.org/HLDA9Workshop/

216 Table 13.1. Examples of classifying immune biomarkers identified by the presence of certain markers in the absence of the B-cell Innate Acquired and T-cell markers.

Cellular Clonal expansion. Although (T-cells and B-cells) resting lymphocytes are quiescent Natural killer cells cells, when either B-cells or T-cells are stimulated in an appropriate Biochemical Complement Immunoglobulin fashion, they re-enter the mitotic cycle T-cell receptor and multiply into a family of related cells called a clone. All the cells of

Genetic MHC V-genes of Ig and Tcr a clone have the same antigenic specificity as the original . Clonal expansion accounts for tabid/60/Default.aspx). (Strictly they have become more commonly the two fundamental properties speaking, the CD number refers used (21), and our earlier cautions of acquired immunity recognized to the monoclonal antibodies that about methodological bias (1) may since ancient times: memory and recognize the cellular target and not now be somewhat mitigated. Still, specificity. Memory comes from the to the target itself, but this convention the less common tests, used mostly expanded population of lymphocytes is often ignored.) Many CD markers in research settings, may not have that persists after initial antigen identify stages in the maturation sufficient standardization to ensure exposure, which allows a more rapid from haematopoietic stem cells to comparability between methods and sizeable secondary response. the various mature forms. Targets and laboratories. In particular, Specificity comes from the fact that that identify all the cells in a lineage quantitative measurements of all the cells of a clone recognize and do not appear in any other types cell receptor expression are often the same antigen. However, clones of cells are called lineage specific subject to considerable bias do develop microheterogeneity as markers; they are relatively scarce between methods (22–24). mutations occur in progeny cells. This Unit 3 compared to targets that are found process is important for maturation

in various states of differentiation Lymphocytes of the specific immune response, 13 Chapter among more than one type of cell. and it is also relevant to autoimmune It is important to note that the Among cellular components, disease and lymphoproliferative functional state of a cell depends on lymphocytes are the antigen- malignancies. not simply the presence or absence specific cells of the acquired immune of receptors, but rather on the response. B-cells are lymphocytes Lymphocyte subsets. Both quantitative extent to which a given that differentiate into - T-cells and B-cells have subsets that receptor is expressed. Upregulation producing plasma cells. T-cells are may be identified by the presence of certain receptors, such as lymphocytes that perform regulatory of certain receptors or by functional CD69, is a typical consequence functions and differentiate into the assays. of lymphocyte activation, and this cytotoxic “killer cells”, which attack T-cells. Mature T-cells can be change in the degree of expression -infected and certain cancer identified by the lineage-specific can be a highly informative biomarker cells. A third type of lymphocyte, the CD3 receptor or by their antigen- (18,19). Neoplastic transformation is natural killer (NK) cell, does not show specific receptor (Tcr). The Tcr is also often accompanied by altered antigen specificity, but has a large coded by one of two gene families: receptor expression that may be role in innate immunity (recent studies α-β or gamma-delta. Alpha-β T-cells correlated with genetic and clinical have identified a hybrid form called are by far more common elements features (20). the NK T-cell that has limited antigen of the acquired immune response, Flow cytometry remains the specificity (25)). B-cells, T-cells while gamma-delta T-cells have a customary method for using CD and NK-cells are indistinguishable limited repertoire and are something markers to identify lineages, morphologically, but B-cells and of a bridge between innate and sublineages, and functional states of T-cells have surface receptors and acquired immunity (26). lymphocytes and other blood cells. genomic mutations that are lineage- T-cells are further divided into These methods have matured as specific, and NK-cells may be those bearing the CD4 receptor,

Unit 3 • Chapter 13. Evaluation of immune responses 217 those bearing the CD8 receptor, such as PCB and dioxins (38,39). molecules. All of the sIg molecules and a small fraction of those The TH1 response is associated on a particular B-cell have the same that bear both. Most of the CD4- with gamma and tumour antigen binding site, which gives bearing cells are helper T-cells necrosis factor (TNF-α), the B-cells their specificity. When B-cells

(Th) that upregulate the immune TH2 response with -4 are activated by antigen binding, response. The CD8-bearing T-cells and interleukin-13, and the TH17 they proliferate and redifferentiate were originally considered to be response with the IL-17 family of into antibody-producing cells. either cytotoxic (killer) cells (Tc) or six cytokines designated 17A-17F The endpoint in this secondary suppressor cells that downregulate (35). The relative elevation of these differentiation is the , the immune response. CD8 cytotoxic cytokines in tissue or serum is which in essence is a cellular factory T-cells are well characterized, but generally taken as evidence for the for making antibodies. Several other evidence for suppressor activity in respective type of in vivo response. receptors are expressed during this subset was never convincing. In However, the measurement of the various stages of progression 1995, the real suppressor population these factors (especially in serum) towards plasma cells or diversion was identified among CD4 T-cells as is not standardized, and their use in to memory cells (47); identification a small proportion that also bears epidemiologic field studies should be of these has long been a staple the CD25 receptor and contains approached cautiously. In particular, of diagnostic pathology for B-cell a high concentration of the Foxp3 artefacts of the immunoassays used malignancies (48). B-cells do not transcription factor (27). These to measure cytokines may produce have major functional subsets CD4-CD25 T-cells are now called spurious differences (40,41); analogous to CD4 and CD8 in regulatory T-cells (Treg); they are interestingly, such misleading T-cells. However, the presence or critical for preventing autoimmunity, artefacts may still have biologic and absence of CD5 (a receptor found on preserving secondary immunity immunologic validity (42). In any all T-cells) appears to define distinct (immune memory), and protecting case, the remarkable heterogeneity B-cell populations. CD5 B-cells are pregnancies (28). and plasticity of T helper cells (43) associated with chronic humoral Helper T-cells may also be can make interpretation of relevant responses, mucosal immunity, characterized in terms of their biomarkers enigmatic at best. autoimmunity and possibly with an regulatory roles. The original One other subclassification increased risk of transforming into a paradigm described a TH1 response, of T-cells deserves mention: the B-cell malignancy (49). which led to delayed hypersensitivity distinction between naive and mediated by cellular responses, and memory T-cells. Naive (virgin) Non-lymphoid cells a TH2 response, which led to humoral T-cells have not encountered immunity and mediated by antigen, while memory T-cells In addition to lymphocytes, several antibody production. The association arose by clonal expansion caused other types of cells are important of TH2 responses with both parasitic by antigen-driven activation. The participants in immune function; and has been surface receptor CD45 exists in two most of them spend at least part of well defined in laboratory and clinical isoforms: CD45RA is associated their life cycle in the bloodstream, studies (29–31). However, this with naive T-cells, while CD45RO where they (along with lymphocytes) simple picture has been replaced by is associated with memory T-cells. are collectively referred to as a more complex interaction involving While the two isoforms can be readily leukocytes or white blood cells the IL-17, which mediates a distinguished by flow cytometry, (WBC). The most numerous of third functional type called the TH17 the categorization is probably the bloodstream leukocytes are

T-cell (32). Research on the TH17 oversimplified, especially for CD8 granulocytes, end-stage cells with subset has progressed rapidly, and T-cells. However, the distinction short lifetimes whose granules it is now seen as having a central may provide some insight into the contain pre-formed mediators ready role in immune regulation (33), pathogenesis of immune-mediated for immediate release. Most of autoimmunity (34), inflammation disorders (44) and environmental them are , which migrate (35) and the link between innate and exposures (45,46). into inflamed tissue where they acquired immune activities (36,37). B-cells. Mature B-cells may be ingest (phagocytise) and destroy

The TH17 pathway may even explain identified by the lineage-specific . and the suspected immunotoxic effects CD19 receptor and by the presence are normally present in much of halogenated aryl hydrocarbons, of their surface immunoglobulin (sIg) smaller numbers; they are involved

218 in allergy and the host response these markers, particularly the achieved (63). RNA microarrays to parasitic infections. Although macromolecules. Proteins are often have shown considerable promise granulocytes are endstage, they do detected and quantified by antibody- in some clinical applications (64), have some limited ability to modify binding methods, which may be but again, a careful approach to their functional status. For instance, subject to cross-reactivities or other method evaluation and study design activated neutrophils upregulate the interferences that cause spurious is required for meaningful results. expression of the CD64 receptor (50), results (57). The use of mass a response that is now used clinically spectroscopy for protein analysis Antigen-specific biochemical as a sign of occult infection, and has increased, particularly as a markers activated eosinophils upregulate co- biomarker discovery tool (58–60). stimulatory and adhesion molecules Some of the initial, promising results Among the wide range of in response to parasitic infection (51). obtained this way have turned out biochemicals involved with immunity, Resident cells in the connective to be disappointing (61); a careful only antibodies (also called tissue underlying the , mucosa approach to method evaluation immunoglobulins (Ig)) and T-cell and internal are also critical and study design is required for receptors (Tcr) are antigen-specific. to immune function. Macrophages meaningful results (62). RNA is Immunoglobulins. Ig molecules ingest, process and package generally detected and quantified are rather large heterodimeric antigens for presentation to T-cells, by hybridization reactions, often proteins that have a basic Y-shaped a process mediated by a transient in an expression microarray with structure with two symmetrical intercellular macromolecular thousands of targets. These methods antigen binding sites connected to complex recently termed the immune are also subject to technical vagaries, a common stem through a “hinge” synapse (52,53). Other accessory but some standardization has been region (Figure 13.1). The basic Ig cells, such as dendritic cells (54), are also involved in antigen Figure 13.1. This figure shows a space-filling molecular model of the human IgG1 presentation. Mast cells contain antibody molecule. The constant region of the two heavy chains are shown in red and their variable region in yellow. The constant region of the two light chains is and other mediators of shown in blue and their variable region in grey. The two antigen binding sites are at allergy in pre-formed granules ready the top of model, where the yellow and grey regions come together. The lower part of Unit 3 for immediate release. They have the model shows the region where various antibody functions, such as complement fixation, reside. Carbohydrate molecules that bind to this region are shown in violet, surface receptors that bind very 13 Chapter and areas specialized to the IgG1 subclass are shown in white. Copyright Mike Clark, strongly to IgE antibodies, sensitizing adapted with permission from http://www.path.cam.ac.uk/~mrc7/mikeimages.html them (and the individual they inhabit) to recognized by the IgE. When allergens interact with their surface-bound IgE, activation and degranulation lead to immediate hypersensitivity (55). Mast cells also mediate signalling between the peripheral nerves and local immune activity, one reason that immediate hypersensitivity responses can be induced rather easily by Pavlovian conditioning (56).

Biochemical biomarkers

Biochemical biomarkers (excluding genomic DNA) include protein and RNA macromolecules as well as smaller molecules, such as steroid hormones and . Technical issues attend all the methods used to measure

Unit 3 • Chapter 13. Evaluation of immune responses 219 unit is composed of two identical cause of common allergy and a molecules to effect and regulate copies of each of two peptide major factor in the pathogenesis of host defence. chains, the light chain and the heavy asthma (68). , cytokines and chain. Light chains come in two In terms of measurement, earlier . Lymphokines, which varieties, kappa and lambda, coded difficulties in the standardization are produced by lymphocytes, are by different genes from different of assays to measure the major Ig a subset of the localized cellular loci. Heavy chains are coded by isotypes in serum (1) have been peptide mediators called cytokines only one genetic locus, but somatic largely resolved (69). However, elaborated by a variety of cells. recombination within the locus can measurement of isotype subclasses The interleukins are cytokines produce different variants called is not as well standardized. IgE particularly involved with signalling isotypes through a process called concentrations in serum are much among leukocytes. The first class-switching. lower than the other secreted isotypes interleukins (IL-1 and IL-2) were Immunoglobulins have two and must be measured by more labouriously identified as functional functions in the immune response: sensitive methods. Measurement of activation and growth factors from membrane-bound Ig molecules are antigen-specific IgE is an important cell culture supernatants (74). One the antigen-specific receptors on marker for allergy in diagnostic, of the most recent (IL-34) was B-cells, and secreted Ig molecules occupational, and research settings. uncovered by a systematic search have a variety of effector functions Most of these assays are well of the extracellular proteome critical to host defence including standardized (70), but customized expressed by a set of recombinant complement fixation, opsonization tests for IgE to novel allergens must secreted proteins, using a suite of (which promotes phagocytosis) and be carefully characterized to assure assays that measured metabolic, viral inactivation. When antigen sensitivity and specificity. growth or transcriptional responses binds to Ig receptors on B-cells, they T-cell receptors (Tcr). Tcr in diverse cell types (75). proliferate and secrete antibody of proteins have a molecular structure All cytokines are localized tissue the same specificity as the original and antigen specificity analogous mediators, and their concentration Ig receptor. Although the secreted to, but somewhat different than, that in peripheral blood is normally antibody has the same antigen of antibodies. Tcr molecules are extremely low or undetectable. binding site as the B-cell receptor, not secreted and have no effector Increased concentrations due it may differ in other portions of function. There are two major types to spillover from tissue sites of the molecule, which accounts for of Tcr: α-β and gamma-delta. These inflammation may sometimes be the different isotypes: IgM, IgG, types may in turn be grouped into detected in serum, but the assays IgA, IgD, and IgE. As an effector families that can be differentiated used to measure them are not molecule, IgM is the first isotype to by monoclonal antibodies (71) standardized and are subject to be produced in a primary immune or genetic analysis (see below). interferences, matrix effects and response; it is especially good at Like surface receptor antibodies considerable bias between methods. binding antigen into complexes on B-cells, the Tcr receptors on Genetic polymorphisms in and activating complement. IgD T-cells allow them to respond to cytokines and cytokine receptors functions only as a B-cell receptor antigen binding by proliferating and have been shown to be useful and is not normally secreted. IgG is secreting effector molecules, in biomarkers of susceptibility for the most common isotype in serum, this case peptide regulators called lymphoid malignancies (76), other normally accounting for up to one- lymphokines (13,72). Cytotoxic cancers (77–80), oral diseases third of total serum protein. It exists (killer) T-cells use their Tcr receptors (81), allergies (82) and autoimmune in four subclasses, which differ to identify their cellular targets: viral- disorders (83,84). Soluble in functions such as complement infected cells or, in some cases, cytokine receptors, which are fixation and placental transfer. IgA cells that have undergone malignant deliberately released from cells by is responsible for mucosal immunity transformation (73). a variety of specific mechanisms (65,66) and is secreted from (85), are important mediators of epithelial surfaces in the airways Non-specific biochemical inflammation (86). While serum and the gut; a variant form is present markers concentrations of soluble receptors in serum (67). IgE is (in humans) and receptor-cytokine complexes uniquely involved in immediate The immune system uses a variety are good candidate biomarkers hypersensitivity, best known as the of proteins, peptides, and smaller for inflammation-related disorders,

220 the assays to measure them are to T-cells. Class I proteins present Toll-like receptors (TLRs). This not standardized, and results from antigens to CD8 cytotoxic T-cells, class of receptor, named for its different methods may not give and class II proteins present similarity to the Toll receptor of concordant results. antigens to CD4 helper T-cells. All of Drosophila, is largely responsible for Other non-antigenic-specific mediators. the HLA loci are highly polymorphic initiating the inflammatory response In addition to lymphokines, many within a species; the four major to microbes and for the host other non-specific biochemical loci in humans include over 2500 perception of microbes in general mediators are used in host different alleles, leading to the (95). TLRs are evolutionarily ancient defence activities. They often difficulty in finding matches between proteins, and most mammalian involve inflammation and include organ donors and recipients. species have about a dozen acute-phase reactant proteins, Different allotypes may confer different types; some shared across such as complement (a family of relative susceptibility or resistance species and others unique. TLRs proteins that react in a cascading to autoimmune diseases and certain account for much of the protective fashion) and C-reactive protein, infectious agents (89). HLA protein effects of the host response to as well as small molecules such allotypes can be identified by infection, such as the induction of as histamine, prostaglandins and reactions with allo-specific antibody lasting specific immunity, as well endocrine hormones. Although reagents, and alloantibodies in as its pathological effects (e.g. these substances may serve as previously sensitized individuals can systemic inflammation and shock) biomarkers of immune function, be detected by binding assays (90). (95). Their discovery illustrates the their non-specific nature and highly Alloreactivity between tissues from use of forward genetic methods in interactive functional pathways two individuals may be detected by identifying genes for biomarkers often make it difficult to distinguish mixing lymphocytes from the two that are constitutively expressed cause from effect. The importance sources and measuring functional but conditionally functional (96). of neuropeptides and nerve growth responses (e.g. proliferation, Originally considered as effectors of factors in the immune response secretion or mRNA innate immunity, TLRs are now seen and inflammation has become production) (91). These types of to have important roles in acquired increasingly apparent (87,88). assays are used clinically, but immunity as well. They are involved Unit 3 Non-antigen-specific are highly specialized and should in the class-switching maturation

cell receptors. Besides Ig and be performed by experienced of B-cells, as the immune system 13 Chapter Tcr, several other cell surface histocompatibility laboratories. T-cell transitions from the primary receptor protein families are critical antigen recognition normally involves response dominated by IgM to the components of immune function presentation by viral-infected secondary responses dominated by even though they are not antigen- epithelial cells or by accessory IgG and IgA (97). They also appear specific. Two of the most important cells, such as macrophages that to interact with the superantigen- are the receptors coded by the have packaged the antigen with mediated polyclonal activation of genes of the major histocompatibility HLA proteins. The detection of T-cells responsible for toxic shock complex (MHC), and the Toll-Like antigen-specific T-cells using in syndrome (98), an often fatal Receptors (TLR). vitro stimulation assays is greatly condition associated with the use Major histocompatibility complex enhanced by pre-packaging the of tampons that reached epidemic (MHC) (transplantation) antigens. The antigen with a suitable HLA protein proportions in the United States MHC receptors come in three primary into a complex called a tetramer (92). around 1980 (99). classes; classes I and II comprise These assays are highly specialized TLRs belong to a larger group the human leukocyte antigens (HLA). and used primarily in research of molecules called the pattern (The class III MHC region contains a settings, although some degree of recognition receptors (PRRs) that diverse set of genes, some of which standardization has been achieved recognize conserved molecular code for certain immune-related (93). The use of tetramers to identify motifs from pathogenic microbes: proteins like cytokines and complement CD8 cytotoxic T-cells specific for -associated molecular components.) Although they were viral or tumour antigens has been patterns (PAMP). A PAMP database discovered because they caused quite successful, but identification of has been established that contains rejection of organ transplants, their antigen-specific CD4 helper T-cells about 500 patterns, including 177 normal biological function involves remains problematic (94). recognized by TLRs (http://www. packaging antigens for presentation imtech.res.in/raghava/prrdb/) (100).

Unit 3 • Chapter 13. Evaluation of immune responses 221 The biology of TLRs has just started involved in antigen recognition. In Major histocompatibility complex to impact clinical medicine and a differentiation process unique to (MHC) genes public health (101–106), and aberrant lymphocytes, somatic recombination variations in TLRs expression removes the intervening sequence The other major gene family involved caused by genetic polymorphisms, between the V-genes and C-genes in the immune response is the major mutations or dysregulation will to form a new gene that codes for histocompatibility complex (MHC), become increasingly important the intact Ig or Tcr protein. This which comprises 26 different genes biomarkers. recombination persists as a genetic including those that code for the fingerprint in all the clonal progeny human HLA proteins (see http:// Genetic biomarkers that subsequently arise from the www.ebi.ac.uk/imgt/hla/). These lymphocyte undergoing the original genes do not influence antigen Immunoglobulin recombination. The details differ specificity directly, but they do have and T-cell receptor genes for each locus, and Ig heavy chain notable effects on antigen-specific genes have a special feature of immune responses and are often Immunoglobulin and T-cell receptor multiple C-genes that sequentially used as biomarkers of susceptibility. genes code for the only antigen- recombine with the chosen V-gene In particular, the association of MHC specific proteins in the immune to form different antibody isotypes polymorphisms has been a sine qua system. The three genes that with the same antigen specificity. non for since produce Ig molecules are located on The V-genes that code for it was first uncovered in a mouse different : the kappa antigen binding sites might at first model of autoimmune thyroiditis light chain gene ( be considered precise markers for (108,109). 2), the lambda light chain gene antigen specificity; however, immune The high degree of genetic (chromosome 22), and the heavy specificity is a selective process. polymorphism in the MHC chain gene (chromosome 14). The antigen binding site in Ig or presents difficulties for genetic During differentiation, B-cells Tcr molecules is not designed to fit analysis, which can be done at determine whether they will use the a particular antigen; rather, binding low resolution for modest costs or kappa or lambda gene, and that sites are created stochastically, higher resolution for higher costs. choice is maintained by all progeny and those that happen to react with The technical issues revolve first of the clone. The two light chains antigens are selected for clonal around the regions and primers are never expressed together in expansion. In fact, each antigen selected for PCR amplification. the same cell. The T-cell receptor, binding site is polyspecific (107), and Thereafter, the amplified product which has an analogous genetic immune specificity to most antigens can be tested by probes, but they basis, is coded for by either alpha depends on the wide repertoire of too may cross-react with different and beta genes or by gamma and specificities that reinforces common alleles. Selected regions associated delta genes; like Ig light chains, the reactivities and dilutes out the with the allotypes (which may lie two pairs are never expressed in others. The relationship between in intron-exon boundaries) can be the same cell. The beta and gamma V-genes and antigen specificity is sequenced, but even sequence- genes are on chromosome 7, while therefore indirect and ultimately based typing cannot rule out a the alpha and delta genes are on must be determined by antigen polymorphism that lies outside the chromosome 17. binding, not gene sequences. sequenced region. As with HLA All seven of the Ig and Tcr Because of the unique protein analysis, these analyses genetic loci contain two distinct recombination of V-genes and should be done by experienced regions separated by an intervening the selective clonal expansion laboratories, and epidemiologic sequence of nucleotides. One or diminution of the lymphocytes investigators should understand the region contains multiple sequences, containing them, the repertoire of limitations of methods used. referred to as V-genes, that code for V-genes differs between individuals the N-terminal portion of the peptide and within individuals over time. They Other genes which will form the antigen binding can therefore serve as biomarkers site. The other region, referred to of exposure and effect, as well as Many other genes may be as the C-gene, contains sequences biomarkers of susceptibility. biomarkers of susceptibility or that code for the remainder of effect modifiers for immune and the peptide chain, which is not inflammatory pathologies. Some

222 of these genes code for other non- regulator that controls the expression such as increases in the incidence specific immune mediators, while of tissue-specific antigens, may of mild infections, to life-threatening, others have no direct relationship to have a profound impact on immune such as overwhelming sepsis. the immune system. Polymorphisms tolerance and autoimmunity Immune deficiencies may be in cytokine genes, such as TNF-α (124). Genes concerned with the indicated by low or absent levels and IL-8, have been associated with regulation of cell growth, such as of serum immunoglobulins, low or several clinical endpoints including BCL2, are especially important in absent numbers of immune cells, or severity of (110), lymphocytes, given their propensity decreased functional responses. incident cardiovascular disease for clonal expansion, and the non- (111), inflammatory bowel disease coding regulatory microRNAs Immune reactive disorders and cancer (112), type 2 diabetes that are involved with cell growth (113) and thrombotic disease in pathways may be more important Immune reactive disorders are due children (114). They have also been markers than many coding genes to inappropriate or poorly regulated associated with effect modification (125). responses in which the ensuing in chemical exposures (115) inflammation damages host tissues. and nutritional biomarkers (116). Special considerations Autoimmune and allergic diseases However, such associations may not for using immune biomarkers are the major types of reactive be apparent when tested in large- in epidemiologic studies disorders. Depending on their cause scale, longitudinal studies (117). and nature, they can range from Genes that have no direct Immune biomarkers may be mild to severe. relationship to the immune system, used to evaluate populations for Common allergies are caused such as those involved with disorders of the immune system by inappropriate responses to metabolism, can also influence itself, for immunogenic exposures, environmental antigens (usually immunity and immunopathology. or for pathological conditions in referred to as allergens) that An association between oxidative other organ systems that provoke release histamine and lipid-derived metabolites of therapeutic drugs and changes in immune status. mediators. These allergic reactions the autoimmune disease systemic are often directed against airborne Unit 3 lupus erythematosis (118) was Disorders of the immune antigens and often contribute to long attributed to a slow-acetylator system the pathogenesis of asthma. Their 13 Chapter polymorphism of the arylamine-N- severity ranges from mild localized acetyltransferase-2 gene. While Three general types of disorders symptoms, such as rhinitis, to life- subsequent epidemiological studies of the immune system may have threatening systemic anaphylaxis. have cast doubt on the relationship adverse health consequences: Depending on the causative antigen, with clinical disease (119,120), a immune deficiencies, inappropriate in vitro tests for -specific relationship with autoimmunity may immune reactivities, and unregulated (IgE) serum still exist. Observations in a mouse proliferation leading to lymphoid antibodies are often good markers model of an association between malignancies (1). for exposure to the antigens that expression of the aryl hydrocarbon evoke allergies (70). receptor (AHR) and the TH17 T-cell Immune deficiency disorders Autoimmune disorders are often subset (38) suggest that exposure debilitating diseases in which the to aryl hydrocarbons, modified Immune deficiency disorders are immune system reacts against its by AHR polymorphisms, may be those in which the immune system own host tissues. Autoimmune associated with autoimmunity and fails to mount adequate protective reactions can damage the skin, liver, perhaps with B-cell malignancies responses against infection or kidneys, various glands, joints and (121). Epigenetic changes could certain forms of cancer. Deficiencies other tissues, leading to diseases also account for differences may be primary (caused by inherited such as rheumatoid arthritis, between individuals in the way their genetic traits or spontaneous ankylosing spondylitis, systemic gene–environment interactions mutations) or secondary (caused lupus erythematosis, thyroiditis, lead to acquired susceptibility or by exposures or infections, such as multiple sclerosis, myasthenia gravis resistance for autoimmunity (122) HIV). Depending on the nature of the and type 1 diabetes. Autoimmune or lymphocyte malignancies (123). deficiency, the health consequences diseases are almost always Genes, such as the autoimmune can range from almost unnoticeable, associated with antibodies that

Unit 3 • Chapter 13. Evaluation of immune responses 223 react to self-proteins in particular Disorders of other organ immune biomarkers in public health tissues or cell components, and systems investigations compared to most these autoantibodies can serve as basic research investigations (1). predictive markers (108). Infections Animal models in general, and mice in particular, have been the mainstay Immune proliferative disorders Infectious diseases that involve any of basic immunology research. tissues are likely to cause changes Central lymphoid tissues, such as Immune proliferative disorders in the host defence system; in fact, spleen, are readily harvested from include , multiple many of the symptoms associated mice; however, useful quantities myeloma, and chronic lymphocytic with infections are caused not by the of peripheral blood are difficult to leukaemia. Like other forms of infectious agents themselves, but by obtain. Conversely, human studies cancer, they involve the uncontrolled cellular and molecular activities of are often limited to sampling expansion of one family (clone) of the host response. Antibodies and peripheral blood. Although it does cells. Immune proliferative disorders antigen-specific T-cells can provide provide a convenient source of both have unique clonal characteristics in markers for specific infectious cells and mediators, peripheral both their receptor phenotypes and agents, while elevations in acute blood is by no means representative molecular genotypes, which can phase serum proteins, some of the immune system as a whole. often serve as excellent biomarkers. cytokines, and certain cell surface Host defence activities take place receptors (50) are non-specific in the central lymphoid tissues Immunogenic exposures markers that suggest infection. (spleen, lymph nodes, epithelial- associated lymphoid tissues) and An acquired immune response Malignancies in interstitial tissue at local sites of can provide biomarkers of specific injury and infection. Cell traffic and exposure to infectious agents or Some solid tumours that release recirculation through the blood is sensitizing chemicals. Antibodies tumour-specific antigens may elicit carefully regulated: activated cells are commonly employed in immunogenic responses that serve and molecules are quickly removed, seroprevalence studies for as markers of the malignancy. while some cells and mediators (126), bacteria (127) and parasites These markers may manifest as persist outside the bloodstream for (128). IgE antibodies can reveal tumour-associated autoantibodies days and even years. With these exposure to allergy-inducing or T-cell responses. points in mind, epidemiologists antigens and their association with should appreciate the challenge asthma. Antibodies to sensitizing Other conditions of adapting findings from animal chemicals, such as toluene models of immune function to the diisocyanate, can serve as markers Malnutrition, chronic disease, stress, design of epidemiologic studies. of exposure and susceptibility pregnancy and a variety of other in occupational settings (129). factors can all influence and be Summary of basic concepts Functional assays, such as T-cell influenced by the immune system. proliferation, are more difficult Immune markers could be used Almost all markers used as tests to perform, but can be useful as indicators of these conditions; of immune status are active with particular exposures; for conversely, these effects can be participants in protective, regulatory example, the lymphoproliferative confounding variables when immune or pathogenic processes of the test for beryllium has been markers are used in attempts to immune system. This direct carefully evaluated as a marker characterize the host defence biological relevance provides for sensitization (130). It should be system itself (1). special opportunities to learn about emphasized that any biomarker the mechanisms of host injury and of acquired immunity requires Immune biomarkers in animal response through tests for immune sensitization and therefore cannot models and epidemiologic components. However, it can also rule out a non-sensitizing exposure. studies make interpretation more difficult, since physiologic interactions The type of samples used to test for among markers can mask changes immune components illustrates a or create internal confounders. major difference between the use of Moreover, the continual changes

224 that occur as the immune Examples of using immune lived up to its presumed promise, but system senses and responds to biomarkers in epidemiology newly-uncovered biomarkers have environmental influences makes and public health revitalized efforts aimed at both the normal ranges of variability for therapy (131) and early detection. immune constituents very large Autoantibodies as pre-clinical In part, the resurgence involved a between individuals, and even within markers of cancer more realistic perspective on the use individuals over time. Finally, the of biomarkers. As studies showed immune system of each individual Cancer cells may express proteins that many so-called TSAs could be continues to evolve throughout that are not expressed in normal detected in persons without cancer, life, its course determined by a tissue or are expressed only at very the term has largely been replaced combination of inherited traits and low levels, and are therefore seen by the more appropriate tumour- acquired exposures. Since human as foreign antigens by the immune associated antigen (TAA). beings are generally outbred and system (Figure 13.2) (131). Such The first human TAA identified often exposed to a great variety of tumour-specific antigens (TSAs) was carcinoembryonic antigen environmental stimuli, the diversity were first uncovered in rodent (CEA) produced by colon cancer among individual immune systems tumours induced by coal tar dyes (133), and it remains useful as a is far greater than that among (132) and were later found in certain marker for tumour recurrence and any organ system other than the naturally-occurring human cancers progression (134). While serum neurobehavioural. The numerous (133). These discoveries augured CEA levels did provide statistically confounding factors that can two attractive concepts: first, that significant predictive value when influence the immune response tumour defence was a primary used in prospective population must also be taken into account (1). function of the immune system that studies (135), neither the sensitivity might be harnessed therapeutically; nor specificity of the test justified second, that testing for TSAs would its use in general screening (136). allow early detection of cancer. For Prostate-specific antigen (PSA) has several decades, neither concept proven somewhat more serviceable, Unit 3 Figure 13.2. Three ways for self antigens to become tumour antigens. Peptides from three normal self proteins (yellow, blue, and green) are presented on the cell surface as normal self peptides (yellow, blue, and green) in major histocompatibility complex (MHC) molecules. In cases of mutation (A), failure of the tumour cell to repair DNA damage can result in a mutation (red) 13 Chapter in a normal protein and, consequently, presentation of mutated peptides (red) on the surface of tumour cells. Because of a mutation, or factors that regulate its expression, a normal protein (green) can be overexpressed in a tumour cell and its peptides presented on the cell surface at highly abnormal levels (B). In cases of post-translational modification (C), a normal protein can be abnormally processed (spliced, glycosylated, phosphorylated, or lipidated) post-translationally (green stripes), resulting in an abnormal repertoire of peptides on the surface of the tumour cell. Used directly from (131) by permission from the Massachusetts Medical Society.

Unit 3 • Chapter 13. Evaluation of immune responses 225 though still problematic, as a screen In the Western hemisphere, T-cell lymphocytic leukaemia, and multiple for prostate cancer (137), but in malignancies are rare, but B-cell myeloma/plasmocytoma. B-cell general blood screening for TAA has malignancies represent a major acute lymphoblastic leukaemia, the not improved the early detection. proportion of cancers not related to most common form of childhood The paradigm shift that has the obvious risk factors of smoking leukaemia, originates so early in the occurred over the last several and diet. B-cell differentiation pathway that years focused attention not on B-cells arise from haematopoietic it is usually considered a stem cell TAAs themselves, but rather on stem cells and undergo multiple malignancy. The remaining B-cell the immune responses to them. stages of differentiation, terminating malignancies all arise from B-cells In essence, TAAs can act as as antibody-secreting plasma in later stages of differentiation autoantigens, producing a weak cells. Four major classes of cancer (Figure 13.3) (147). The genetic but detectable antibody or T-cell arise from these various stages: and environmental risk factors for response. Although this is not a new acute lymphoblastic leukaemia, these B-cell malignancies remain idea (138,139), modern methods of non-, chronic surprisingly elusive, but information molecular engineering allow TAA genes to be cloned and transfected, Figure 13.3. A Venn Diagram illustrating the hypothetical relationship among the MBL, MGUS, and the malignant B-cell diseases to which they may progress. The providing a ready supply of diagram is based on the spectrum of progression endpoints for MBL and MGUS antigen to use in high-throughput and the interrelationships of their respective biomarkers. The overlapping areas multiplexed assays with attomolar indicate some extent of shared biomarkers or clinical endpoints. Since the definition sensitivity (140,141). Promising of MBL excludes any haematolymphoid disorder, areas of overlap between MBL and a malignancy is meant to convey shared biomarkers or progression from MBL. MBL results have been obtained for the and MGUS usually appear independently, but may appear together. Both conditions detection of autoantibodies in lung can remain quiescent over the lifespan of the individuals in whom they are found, or cancer (142), liver cancer (143), they can progress to clinical disease. CLL is shown as a complete subset of MBL, prostate cancer (141) and ovarian and MM/WM/SMM as nearly complete subsets of MGUS, under the presumption that all CLL is preceded by MBL and nearly all MM and WM is preceded by MGUS cancer (144). Assays for detecting (although the precedent conditions may not be detected before the clinical disease TAA-specific T-cells have been endpoints are diagnosed). SMM frequently, but not always, progresses to MM. MGUS applied mostly to studies of tumour cases may infrequently develop CLL or NHL. At least one case with a combination or (145), of MBL and MGUS that developed into WM has been reported. MBL is detectable in a subset of already-diagnosed NHL cases (but to date there have been no reports of but exploratory studies show that MBL developing into NHL). At least one type of NHL (small lymphocytic lymphoma) is such T-cells can be detected in considered to be a variant of CLL. AL may be associated with MGUS, SMM, MM, or breast cancer patients naive to WM and rarely with PC, CLL, or NHL. MBL progression directly to AL has not been immunotherapy (146). Immune reported to date. Used directly from (147) with permission biomarkers may yet prove to be useful tools in the early detection of cancer.

Genetic and environmental risk factors for B-cell malignancies

While the role of the mammalian immune system in protection against cancer remains enigmatic, it is clear that lymphocytes themselves can lose control of their proliferative potential and expand uncontrollably into lymphoid malignancies. In the Eastern hemisphere, most lymphoid malignancies arise from T-cells, a phenomenon directly related to the endemic presence of human T-cell AL, immunoglobulin light chain amyloidosis; CLL, chronic lymphocytic leukaemia; MBL, monoclonal B-cell lymphocytotrophic viruses (HTLV). ; MGUS, of undetermined significance; MM, ; NHL, non- Hodgkin lymphoma; PC, plasmacytoma; SMM, smouldering multiple myeloma; WM, Waldenstrom .

226 revealed by population studies and normal haematopoietic cells in the isolate B-cell subsets with distinct immune biomarkers has allowed a . While most cases of phenotypic characteristics. Once much better understanding of their CLL are sporadic, a familial variant a distinct population has been natural history. has been recognized for many years identified, MBL can be identified by The clonal expansion of (152). light chain restriction. Antibodies lymphocytes must be carefully The transition from a pre- may have either kappa or lambda regulated to avoid overwhelming clinical B-cell proliferative disorder light chains, but all the cells of a the host. Protective immunization to CLL has been documented in a particular clone must make the generally involves the controlled succession of studies made possible antibodies with the same light chain. proliferation of many different by the advent of flow cytometry A phenotypic cluster that shows only lymphocyte families, leading to (15). The term monoclonal B-cell one type of light chain may therefore a polyclonal response. When lymphocytosis (MBL) is now used be considered monoclonal. The proliferation is dominated by a single to describe the pre-clinical state complexity of cell preparation, flow clone, the result is a monoclonal (153). One of the first systematic cytometry and data analysis make response. Monoclonal proliferation studies of MBL originated from standardization of methods to detect is the first step in a progression that environmental public health studies clonality critical for epidemiological may lead to a lymphoid malignancy in which 13 individuals with MBL assessment. Fortunately, the raw (148,149). were detected (prevalence of data from flow cytometric analysis Chronic lymphocytic leukaemia 0.9% among participants age 40 can be captured and re-analysed (CLL) is a classic example of a or above) (154). These individuals using different gating strategies, B-cell malignancy arising from were followed for up to 12 years, allowing retrospective analysis of monoclonal expansion (150). along with other study participants existing data (158). Cellular, biochemical and genetic who had high B-cell counts without Long-term studies of MBL and biomarkers have all contributed MBL. The majority of MBL cases CLL using established and newly- to our increased understanding remained stable or died of unrelated uncovered biomarkers, such as of the natural history of CLL. The causes, but progression to a B-cell microRNAs, will be required to disease process probably begins malignancy was observed in two of sort out environmental risk factors, Unit 3 with chronic immune stimulation the 13: one case of CLL and one case innate susceptibility and biomarkers

by infectious agents, other external of Waldenstrom macroglobulinemia, of progression. 13 Chapter antigens, or autoantigens. As normal a related disease. Interestingly, the B-cell clones expand in response high B-cell counts in individuals who Newborn screening for to antigen stimulation, the chance did not develop MBL regressed to immune disorders of individual cells acquiring genetic normal over the follow-up period. defects increases. Some of these The other seminal MBL study Newborn infants that appear defects cause the cell to escape involved familial CLL, where 18% of healthy may actually have serious regulatory control of proliferation: first-degree family members without latent disorders that will cause the best example to date is the loss CLL were found to have MBL future disease, disabilities or of the microRNAs miR-15a and (155). This striking increase over even premature death. Newborn miR-16–1, critical regulatory factors the general population prevalence bloodspot screening (NBS) is in the bcl-2 pathway for apoptosis suggests the familial risk for CLL is designed to identify such infants (151). Continued expansion of reflected in the risk for MBL. Since quickly so that medical intervention the deregulated clone promotes these studies, other population can begin before they fall victim to opportunity for other genetic lesions surveys have shown that the such disorders. A small amount of to accumulate, including epigenetic prevalence of MBL increases with blood from a heel stick is collected changes (123). At some point, the age, approaching 3–5% in older, on filter paper to form a dried blood damaged clone exhibits phenotypic otherwise healthy, adults (156,157). spot (DBS). DBS samples are sent changes, typically an increased Even with the power of to central laboratories where they expression of CD5 and decreased multiparameter flow cytometry, are analysed by various methods to expression of CD20. Eventually the detection of MBL is not trivial, detect biomarkers of latent disorders the clonal proliferation causes since it depends on subjective (159). The first conditions detected clinical disease by accumulating assessment and sequential by NBS were metabolic or endocrine in lymphoid tissues and displacing selection (“gating”) strategies that disorders, and they remain the

Unit 3 • Chapter 13. Evaluation of immune responses 227 dominant type screened for in that, although SCID is expressed as scattered throughout the exons, so current NBS programs. However, a combined deficiency involving both screening by conventional genetic interest in screening for other types humoral (B-cell) and cell-mediated tests is not feasible. of disorders is growing rapidly (T-cell) immunity, it is actually a The third consideration came (160), and the idea of screening for defect in T-cell development. B-cell from the knowledge of T-cell risk factors of future disorders, in counts in SCID babies are normal development (Figure 13.4) (164), addition to screening for established or even elevated, and they are which led to a unique marker of (though occult) conditions, has been fully functional. However, without T-cells that could be measured gaining attention. Two immune functional T-cells to provide help, in DBS (165). When lymphocytes disorders typify these two trends: even humoral responses are rearrange the genes that form severe combined immune deficiency deficient, giving the phenotype of their antigen receptors, a small (SCID), and type 1 (juvenile) a combined immune deficiency. segment of DNA is removed to diabetes. The second realization was that juxtapose two previously separated mutations at any one of several segments. In T-cells, the excised Severe combined immune unrelated genetic loci could result in segment is removed as a circular deficiency failure of T-cell development: SCID fragment called a T-cell receptor was a single gene defect in each excision circle (TREC). TRECs Severe combined immune deficiency individual case, but with multiple are produced only in the original (SCID) is a lethal congenital failure genetic causes overall (Table 13.2). recombination event and are not of immune development (161). It is Other loci in which mutations could duplicated in subsequent cell often called “bubble boy disease” cause SCID may yet be uncovered division, so fewer T-cells contain because of early attempts to prevent (163). Moreover, the mutations TRECs as the immune system infection through sequestering the at these various loci are widely develops. The newborn infant, child from the natural environment. Because of the persistence of Figure 13.4. Defects in human T-cell development resulting in SCID phenotype. placentally-transferred maternal A simplified depiction of lymphocyte differentiation is shown. B-cells and NK cells mature in the bone marrow, whereas T-cells mature in the thymus. Normally, only the antibodies, SCID remains concealed mature forms of these cells are released into the peripheral blood. Various stages for several weeks after birth, but in NK and T-cell development that are blocked by mutations in the genes known to without a functional immune system, cause SCID (IL2RG, JAK3, ADA, IL7R, RAG1, RAG1, ARTEMIS, and CD45) are babies soon become infected and indicated by X and dashed lines. Presence or absence of T-cell-specific antigenic markers (CD4,CD8TCR-α/β, and TCR-gamma/delta) is also indicated. Effects of typically die in infancy. A series of these mutations on B-cell development is not shown. Used directly from (164) with landmark studies have shown that permission newborns with SCID can be rescued before they become symptomatic by transplanting bone marrow progenitor cells (162). However, such rescue is difficult or impossible after SCID babies become infected. SCID thus meets the ideal criteria for NBS: a lethal disorder with a latent onset that can be prevented by medical intervention. The birth prevalence of SCID is not certain, but is estimated to be in the range of 1 to 4 per 100 000. The process of finding an immune biomarker for SCID that can be measured on a newborn DBS illustrates how far-reaching our knowledge of the immune system and our abilities to probe it have come. First came the understanding

228 Table 13.2. Genetic loci associated with severe combined immune deficiency (SCID)

Characteristics of SCID

Presence of

No. unique Mode of Gene Locus Gene product/function T-cell B-cell NK-cell mutations OMIM No. inheritance* identified IL7R 5p13 IL7 receptor. Needed for T-cell - + + AR 5 146661 development. Activates JAK3 kinase

CD45 1q31-q32 Protein tyrosine phosphatase. - + + AR 3 151460 Regulates Src kinases required for T-cell and B-cell antigen receptor signal transduction

IL2RG Xq13.1 Gamma-c chain of IL2, 4, 7, 9, - + - XLR 169 308380 15 cytokine receptors. Needed to activate JAK3 for intracellular signalling

JAK3 19p13.1 Tyrosine kinase. Needed for - + - AR 27 600173 differentiation of haematopoietic cells

RAG1 11p13 DNA recombinase. RAG1/RAG2 - - + AR 44 179615 mediate DNA recombination during B-cell and T-cell development

RAG2 11p13 DNA recombinase. RAG1/RAG2 - - + AR 18 179616 mediate DNA recombination during B-cell and T-cell development

ARTEMIS 10p Involved in DNA repair during V(D)J - - + AR 9 605988 recombination Unit 3 ADA 20q13.11 Part of the purine salvage and --- AR 54 102700 methylation pathways. Needed for

removal of toxic metabolites (e.g. 13 Chapter ATP, S-adenosyl homocysteine) that inhibit lymphoid cells

*Mode of inheritance: AR, autosomal recessive; XLR, X-linked recessive. Adapted from (164). however, is still producing new The Q-PCR assay for TRECs Syndrome (167), or acquired T-cells at a rapid pace, so about has been tested on several thousand disorders, such as congenital HIV 10% of the peripheral blood T-cells anonymized newborn DBS and infection (168,169). Given the rarity contain TRECs. These cells may some 18 DBS from newborns with of all of these disorders, large-scale be measured by quantitative real- SCID. About 99% of the anonymized population studies where NBS time polymerase chain reaction DBS samples from newborns fall for SCID is performed under the (Q-PCR), a technique in which within the expected range of PCR aegis of translation research will be each cycle of DNA amplification is amplification. In contrast, all of the necessary to evaluate and refine monitored for the appearance of DBS from newborns with SCID testing protocols. fluorescence from a probe released failed to show any amplification during the polymerase-mediated (165). The assay is therefore highly Type 1 (-dependent) extension process (166). The sensitive and specific, but given the diabetes greater the amount of DNA in the rarity of SCID, its positive predictive original sample, the more quickly the value is still low (0.1–1%). Besides Type 1 diabetes (T1D), formerly fluorescence signal increases. This SCID, the newborn DBS that fail known as juvenile or type 1 diabetes, allows construction of a calibration to amplify may be due to technical is the major cause of diabetes in curve relating fluorescence to the problems with the assay or to children. T1D is generally caused number of TRECs. other T-cell deficiencies caused by by the autoimmune destruction genetic disorders, such as DiGeorge of insulin-producing β cells of the

Unit 3 • Chapter 13. Evaluation of immune responses 229 pancreas (170). The autoimmune paradigm (Figure 13.5) for the long-term study cohort (175,176). To pathogenesis of T1D was revealed progression from innate risk to islet maximize the proportion of recruited by two biomarkers, one genetic cell autoimmunity and ultimately children who will develop T1D, the and one acquired. The genetic to T1D. The major remaining highest genetic risk, defined as one biomarker is linkage with certain puzzle is the role of environmental of four MHC class II haplotypes alleles of the MHC genes that code exposures in triggering or (Table 13.3), is required for eligibility for the human leukocyte antigens advancing the autoimmune in the general population. However, (HLA), a risk locus shared by all process (174). The candidates for since familial risk contributes autoimmune disorders. About half such exposures include bacterial independently, six additional MHC of the attributable risk for T1D is and viral infections (particularly haplotypes are eligible in families genetic, and about half of that risk enteroviruses and rhinoviruses), where a first-degree relative of the is contained in the HLA genes. The food antigens, xenobiotic chemicals, prospective recruit already has T1D genetic risk for T1D is associated allergens, ultraviolet light, and (Table 13.3). particularly with the class II MHC the immunomodulatory effects of Because risk from environmental genes that code for the HLA-D stress. Clearly, the identification of exposures may begin very early, antigens (171). Some alleles confer environmental risk factors would perhaps even in utero (177), TEDDY susceptibility, while others confer open new possibilities for prevention collects the first samples to look resistance. Interestingly, resistance and intervention. for environmental factors at three is dominant, which allows more cost- With this goal in mind, a months of age. With such a short effective screening approaches prospective multisite natural window to identify and recruit that identify protective alleles and history study has been initiated to participants, TEDDY investigators eliminate them from further testing. address comprehensively the role seek informed consent for the initial The acquired biomarker for of environmental exposures in T1D. genetic screen from the parents of T1D is a group of autoantibodies Called TEDDY (The Environmental newborns, making it a research that react with pancreatic islet cell Determinants of Diabetes in the application of newborn screening. antigens (172). Autoantibodies Young), this study is recruiting infants By the close of the screening phase, are the other essential biomarker at higher genetic risk for T1D (as well some 300 000 newborns will have of autoimmune disorders. In the as controls without higher genetic been screened, and about 8000 rheumatic disorders, such as risk) and assembling them into a higher-risk infants enrolled. The systemic lupus erythematosis, they are an obvious part of the pathogenic Figure 13.5. The stages in the natural history and pathogenesis of childhood type process; in organ-specific disorders, 1 diabetes (T1D). Genetic susceptibility creates an immunological environment such as T1D, they are thought to be that predisposes to pancreatic islet cell autoimmunity. An environmental trigger is suspected in most if not all cases of T1D. Autoimmunity then becomes evident by the largely paraphenomena, but still presence of autoantibodies to islet cell antigens. The presence of autoantibodies to serve as useful markers. Originally two or more antigens suggests a progressive condition and a significant risk (> 50%) discovered by immunofluorescence of developing T1D. As islet cells are destroyed by immune-mediated inflammation, microscopy using pancreas tissue the pancreas loses the ability to produce insulin, ultimately resulting in type 1 (type 1) diabetes. Complications, largely related to inflammatory pathologies, cause to visualize antibody binding to islet morbidity and mortality. The progression from autoimmunity to frank diabetes is cells (173), most testing today is done highly variable, but the strongest genetic risk factors tend to be associated with the biochemically using purified islet cell earliest onset of disease antigens produced by cloned genes. Autoantibodies to three major islet cell antigens have been the important determinants of T1D risk in epidemiologic and natural history studies, but antibodies to other islet cell antigens have been reported on the basis of distinct tissue binding patterns (172). A series of prospective studies by research centres around the world has established a consensus

230 Table 13.3. Eligible MHC genotypes in the TEDDY study that confer higher risk for Several technical, operational and Type 1 diabetes ethical caveats attend TEDDY, and other long-term population-based

General Population Eligible Genotypes prospective studies. Technically, the high-throughput tests used for DR4-DQA1*0301-DQB1*0302 / DR3-DQA1*0501-DQB1*0201 genetic screening do not identify HLA haplotypes with certainty, and DR4-DQA1*0301-DQB1*0302 / DR4-DQA1*0301-DQB1*0302 indeed high resolution testing is DR4-DQA1*0301-DQB1*0302 / DR8-DQA1*0401-DQB1*0402 neither necessary nor cost-efficient DR3-DQA1*0501-DQB1*0201 / DR3-DQA1*0501-DQB1*0201 for this purpose (171). In TEDDY, screen-eligible haplotypes are First-Degree Relative Eligible Genotypes independently confirmed by higher resolution methods after recruitment. DR4-DQA1*0301-DQB1*0302 / DR4-DQA1*0301-DQB1*0201 In addition, the screening laboratories DR4-DQA1*0301-DQB1*0302 / DR1-DQA1*0101-DQB1*0501 annually undergo a proficiency testing survey, which has repeatedly DR4-DQA1*0301-DQB1*0302 / DR13-DQA1*0102-DQB1*0604 confirmed the validity of the different DR4-DQA1*0301-DQB1*0302 / DR4-DQA1*0301-DQB1*0304 screening methods in use (176). The

DR4-DQA1*0301-DQB1*0302 / DR9-DQA1*0301-DQB1*0303 autoantibody tests have undergone rigorous standardization, but insulin DR3-DQA1*0501-DQB1*0201 / DR9-DQA1*0301-DQB1*0303 autoantibody, the most important one for identifying the onset of participants will be followed with no intervention to prevent T1D onset autoimmunity, remains a technically blood sampling every three months currently exists, it is generally not challenging assay (179). Further for islet autoantibody measurements considered a candidate for routine concerns focus on the use of banked until age four, and then every six public health application. However, samples, since some of the tests to months until the age of 15. These some diabetologists believe genetic be done in the future may be affected Unit 3 cohorts are to be followed over a risk for T1D should be part of routine by storage. This is particularly true

period of 15 years for the appearance newborn screening, since the for T-cell function tests (93) that could 13 Chapter of islet cell autoantibodies and knowledge can prevent morbidity, reveal the repertoire of lymphocyte diabetes, with documentation of especially in young children where specificities directed against islet cell early childhood diet, reported and the acute onset of T1D can go antigens or environmental triggers. measured infections, vaccinations, unrecognized, occasionally with fatal Operationally, long-term prospective and psychosocial stressors. The consequences. Genetic assessment studies are expensive and require TEDDY Consortium will allow for alone can increase the likelihood dedicated management with a coordinated, multidisciplinary ratio several-fold, but the positive committed field centres. Ethically, approach to this complex disease predictive value (PPV) is still quite low: risk communication with the recruited (see http://www.teddystudy.org). 1–2%, depending on the population. families must be approached Collection of information and The appearance of autoantibody carefully, and the requirements of samples in a standardized manner to one of the islet cell antigens participation must not be unduly will achieve greater statistical increases the risk substantially, and demanding to mitigate stress and power than smaller independent the presence of autoantibodies to foster retention in the long-term effort. investigations. Most importantly, two or more antigens raises the Such effort is justified by the promise the TEDDY study will establish PPV to around 50% (178). Children of primary prevention for T1D, a central repository of data and with such positive serologies should eliminating life-long dependence on biologic samples for subsequent be monitored for blood glucose insulin, and the disabling morbidities hypothesis-based research, levels, especially when they become that accompany it. applying immunologic and genetic acutely ill. This tiered approach biomarkers to samples collected in to using biomarkers (in this case, higher risk children. metabolic as well as immune) may Newborn screening for T1D risk well be a model for future public is currently a research activity; since health applications.

Unit 3 • Chapter 13. Evaluation of immune responses 231 Immune biomarkers of Figure 13.6. Factors influencing the risk that transplacental maternal IgG antibodies neurodevelopmental could cause developmental neuromental disorders, such as autism and schizophrenia. disorders The relevant parameters are: (1) the concentration of maternal antibodies that could cross-react with brain antigens, (2) the permeability of the blood-brain barrier, and (3) the threshold at which neurodevelopmental damage may occur, presumably Neuromental disorders (NMDs), related to the developmental state of the fetus. The clinical manifestations for any such as autism, schizophrenia, particular type of neuromental dysfunction may be detected during infancy, or attention deficit syndromes and may only be recognized many years postnatally. This will depend on the degree of initial involvement in the function of the developing nervous system, and when the epilepsy, represent a biological threshold for the neuropathological disorders to become clinically manifest, is lowered enigma and a public health by genetic and/or postnatal environmental influences. The IgG antibody levels will imperative. While specific genetic also be affected by immune factors in the mother or progeny: the IgG subclass of mutations have been identified in antibodies to antigens common between an infectious agent and the brain; whether the antigens involved are polysaccharides or proteins; genetic influences on the a small proportion of NMDs (180), immune responsiveness to these antigens of the mother; the time of her acquisition etiologic factors for the majority of the infectious agent, before, or during, pregnancy, as well as the agent’s natural remain unclear. A growing body of chronicity or reactivation potential, and the transfer by feto-fetal transfusion of IgG/ scientific evidence suggests that antibodies from one member of a monozygotic twin pair to the other, due to a common chorionic placentation. The blood-brain barriers may become more permissive in many NMDs have an early etiological case of trauma (e.g. a prolonged vaginal delivery), in which case lower antibody levels origin associated with aberrant may cross to the nervous system. Adapted from (181) with permission brain development during gestation (181), and neuroimmunomodulatory factors have been implicated in the prenatal pathogenic process (182,183). Studies in animal models, as well as limited human studies and epidemiological data (184), suggest an etiologic role for autoimmune or cross-reactive antibodies maternally transferred across the when the fetal blood-brain barrier is permeable to IgG. This concept has been developed into a full model termed the gestational neuroimmunopathology (GENIP) hypothesis (Figure 13.6) (181). old fetus can readily make IgG brain tissue could disrupt normal One line of evidence supporting antibodies to protein antigens neurodevelopmental processes. the GENIP hypothesis comes from of intrauterine infectious agents The mechanism for such disruption the consideration of the placental such as syphilis (185). However, could be directly upon neurons barrier and of the immune responses polysaccharides, structurally (187,188), or more subtly upon the in very young children (181). The identical to the bacterial α-2,8-linked regulation of axonal growth and placenta restricts passage of capsular polysaccharides, are also connectivity (189,190). For instance, maternal IgG2 antibodies, which synthesized by the mammalian antibodies to ganglioside GM1, are the subclass most enriched central nervous system, where identified in cases of paediatric for reactivity to encapsulated they regulate neuronal function autoimmune neuropsychiatric bacteria, such as meningococcus in association with the neural disorders associated with B and E. coli K1. Similarly, very cell adhesion molecule (186). If streptococci, act by stimulating the young children are unable to mount antibodies that react with these enzyme calcium/calmodulin kinase effective immune responses against polysaccharides (or with other II (191). In general, cross-reactive bacterial polysaccharide antigens. sialic acid epitopes, such as those or polyreactive antibodies that effect The withholding of protective of the many gangliosides in the neuronal development or function antibodies against common deadly developing nervous system) were are seen as an emerging theme in in highly susceptible present before the blood-brain neuroimmunology (192). infants is evolutionarily difficult barrier became fully impervious Another line of evidence comes to explain, since the six-month- to IgG transfer, their effects on from animal models that show

232 antibodies can influence behaviour if United States are routinely collected clinical research to epidemiologic they reach the brain. Mice that were as DBS for newborn screening, the field studies and public health given autoantibodies associated samples required to test the GENIP applications, such as newborn with systemic lupus erythematosis hypothesis are readily available. screening and early cancer developed behavioural anomalies, Highly-multiplexed suspension detection. if, and only if, they also were arrays have been developed The translational process will given pharmacologic agents that for detecting a wide variety of have to confront challenges in opened the blood-brain barrier potentially relevant antibodies in the standardization of laboratory (193). Infant monkeys gestationally DBS, and they are currently being measurements, the establishment exposed to IgG antibodies purified applied to epidemiologic studies of biologic validity and the from the serum of mothers with of autism, epilepsy, and other meaningful interpretation of results. autistic children demonstrated the neuromental disorders. Perhaps A multidisciplinary approach that stereotypies characteristic of autistic the major concern in such studies engages epidemiologists, laboratory behaviour (194). lies not in the arcane realm of scientists and clinicians offers the A third line of evidence for GENIP immune biomarkers, but rather best chance for useful field studies is the identification of antibodies that with the classic epidemiologic and public health applications. react with brain tissue antigens in dilemma of establishing a consistent All the scientists involved in such children with neurodevelopmental case definition for these complex efforts should have some sense disorders (195,196). These studies neurodevelopmental conditions. of the way immune markers are have used research assays that are measured, of their functional role not widely employed or validated Future opportunities in health and disease, and of the by inter-laboratory studies, but the and challenges statistical methods that will be used methods are generally sound, and to analyse the data. While these the differences with control samples The continual advances of aphorisms can be applied to any are often striking. Although these biomedical research in immunology type of biomarker, the self-referential studies cannot prove causality, they offer an ever-widening opportunity sensory nature of the immune suggest an association that could to employ immune biomarkers system, its wide range of effector Unit 3 at least provide useful markers for in epidemiologic studies. In the functions, and the narrow margin

stratification within the complex foreseeable future biomarkers will between protection and pathology 13 Chapter spectrum of neurobehavioural lead to an increased understanding make them especially pertinent diseases. of the relationship between innate to the use of immune biomarkers. The key to providing direct and acquired immunity; the When properly met, the challenges evidence for the GENIP hypothesis influences of microRNAs on immune will open new routes to scientific is the newborn baby, whose cell differentiation and function; the discovery, disease prevention and blood contains large amounts of role of epigenetic mechanisms; the public health practice. placentally-transferred IgG from regulation of immune responses the mother, as well as the low levels by the T 1-T 2-T 17 network; the Disclaimer: The findings and conclusions H H H in this chapter are those of the author of antibodies made in utero by the environmental factors that trigger and do not necessarily represent the fetus. Samples from newborns allergy, autoimmunity and immune views of the Centers for Disease Control can therefore reveal the spectrum malignancy; and the interaction of and Prevention. of antibody reactivities to which the neurobehavioural and immune the developing brain has been systems in neuromental disorders. exposed. Since blood samples Also envisioned is a more rapid from virtually every newborn in the pace of translation from basic and

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