T Cell Antigen-Specific Receptorextras.springer.com/2005/978-3-540-44172-4/data/... · dependent...

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T Cell

T cells, or T lymphocytes, develop in the thymus, andare a subfamily of circulating leucocytes that play animportant role in the adaptive immune response andfurthermore serve as crucial effector cells through an-tigen-specific cytotoxic activity and the production ofsoluble mediators (cytokines/chemokines). The char-acteristic feature of all T cells are clonal antigen-spe-cific heterodimeric receptor molecules on the surface(T cell receptor; TCR). The accessory molecules CD4and CD8 define the effector function and MHC restric-tion of T cells. Some T cells migrate to various loca-tions throughout the body and interact with antibodyproduction of B cells. Subsets of T cells (CD4+) havebeen classified as type 1 or type 2 T helper cells,depending on the cytokines they produce. Anothersubset is formed by the (CD8+) cytotoxic T cells.

3Cancer and the Immune System

3Streptococcus Infection and Immunity

3Mast Cells

T Cell Antigen Receptor (TCR)

The clonally distributed antigen receptor of T cells iscomposed of a heterodimer consisting of α and β or γand δ polypeptide chains, each containing one N-ter-minal variable (V) domain and one plasma membraneinserted constant (C) domain. Both heterodimers areexpressed in association with the signal transducingCD3 chains. Most T cells are CD4+ or CD8+, andexpress the αβ TCR. They recognize foreign peptidespresented by self -MHC molecules. A small numberof T cells express the γδ TCR, which recognizes dif-ferent types of mostly unknown ligands. Antigen rec-ognition by γδ bearing T cells is not MHC restricted.The antigen-binding domain of α or γ chains is en-coded by the V (variable) and J (joining) genes, that ofβ or δ chain by V, D (diversity) and J genes, which arerecombined during T cell development. The genenames are usually numbered, with a Greek letter as asuffix for the chain (e.g. Vβ 8.1 for the variable gene8.1 of the β chain). Alleles are designated by lower-

case letters (a, b). In the WHO nomenclature, humanlocus and gene are given a combination of capitalletters and numbers (e.g. (TCR)BV8S1). Roman fontsindicate gene products and italics the genes. The an-tigen-binding sites of the chains are formed by proteinloops called complementarity determining regions(CDR), which are connected by conserved frameworkregions. CDR1 and CDR2 and a fourth hypervariableloop, which is involved in superantigen binding, are Vgene encoded. The CDR3 are VDJ or VJ encoded. Thehigh variability of the CDR3 results from the combi-nation of the V and J , or the V, D, and J genes, as wellas additional events such as introduction of so-calledN- or P-nucleotides, and imprecision of recombina-tion. The number of different TCR which can be cre-ated by these mechanisms exceeds that of lymphocytesin the body.

3Superantigens3Chronic Beryllium Disease

3Mucosa-Associated Lymphoid Tissue

3Cell-Mediated Lysis

3Cytotoxic T Lymphocytes

3Helper T Lymphocytes

3Lymphocyte Proliferation

T Cell Antigen-Specific Receptor

The TCR is a molecule on surface of T cells composedof two polypeptides (α and β chains) of nearly equalmolecular weights. Similar to antibody molecules theTCR has N-terminal variable amino acid sequenceswhich combine to provide the individualized specific-ity (idiotype) shared by all TCR of a single cell of asingle clone; the C-terminal portion is common to allα and β chains of the TCR. The antigen-specific re-ceptor recognizes and binds peptides of thymus-de-pendent antigens (proteins) when presented byclass I and II molecules which are encoded in themajor histocompatibility complex (MHC). The pep-tides are generated by antigen-processing and anti-gen-presenting cells like macrophages, dendritic cellsand B cells.

3Metals and Autoimmune Disease

3Idiotype Network

T Cell-Dependent Antibody Response

3Assays for Antibody Production

T Cell-Dependent Antigen

An antigen that requires the presence of T cell help tostimulate the B cell to secrete antibody. Such antigensdo not elicit a productive antibody response by B cellsunless the B cell receives help from a CD4 T cell. Helpis generally supplied in the form of both a contact-dependent signal via CD40 plus specific T cell-se-creted cytokines. Generally, protein antigens are T-de-pendent antigens.

3Plaque-Forming Cell Assays

3Memory, Immunological

3Immunoassays

T Cell-Independent Antigen

An antigen that can stimulate B cells to secrete anti-body in the absence of T cell help.

3Plaque-Forming Cell Assays

T Cell Oligoclonality

This describes the restriction of an antigen-specificT cell response to one or several T cell receptor con-figurations. Following HLA–antigen–TCR interaction,this limited number of responder T cells undergo clon-al expansion and increase the size of their TCR spe-cific subpopulations.

3Chronic Beryllium Disease

T Cell Receptor (TCR)

3Antigen Presentation via MHC Class II Molecules

T Cell Receptor (TCR) Complex

The T cell receptor (TCR) complex consists of eighttransmembrane chains that are expressed on the sur-face of T cells. The TCRα and TCRβ chains bindantigenic peptide when presented to them in the con-tact of MHC (major histocompatability) molecules.

The other six chains form the CD3 complex and con-sist of CD3γ which forms a heterodimer with CD3ε,CD3δ which also forms a heterodimer with CD3ε anda homodimer of CD3ζ. The CD3 chains are the targetsfor kinase phosphorylation. It is the CD3 complex thatpropagates the signal from the TCR complex to down-stream signaling cascades.

3Signal Transduction During Lymphocyte Activation

T Cell Selection

3Thymus: A Mediator of T Cell Development andPotential Target of Toxicological Agents

γδT Cells

T cell receptors are composed of two different poly-peptide chains, α and β chains (αβT cells) or, in aminor population of T cells, of γ and δ chaisn (γδTcells). The maturation of T cells occurs either in thethymus or, under special circumstances, (γδT cells)extrathymically. The first critical stage in T cell matu-ration is the successful rearrangement of the TCR βchain (pre-TCR complex). A functional TCR β chaininitiates the arrangement of the TCR α locus and ex-pression of both the CD4 and CD8 molecules(CD4+CD8+ double positive thymocytes). In contrastto these subsets of αβ T cells only little is known ofthe physiological effector function or antigen specific-ity of γδT cells. The receptor of these T cells is muchmore homogenous compared to αβ T cells. They arefound in epithelia from where they do not recirculate.One hypothesis is that they participate in the pre-adap-tive immune response.


T-Dependent Antibody-Forming CellResponse

3Plaque Versus ELISA Assays. Evaluation of Humor-al Immune Responses to T-Dependent Antigens

T Helper 1 Cells

3Helper T lymphocytes

626 T Cell-Dependent Antibody Response

T Helper 1 Cytokines (Th1)

Cytokines such as IFN-α and IL-2, are autocrine andparacrine signaling molecules produced by CD4+

T cells in response to MHC class II antigen stimula-tion, and stimulate growth and activation of immuno-cytes and other inflammatory cells.


T Helper 1 (Th1) Cells

CD4 T cells that produce cytokines such as interferon(IFN)γ, and interleukin IL-2 but not IL-4 and IL-5. Bythis they direct cellular immune responses.


3Flow Cytometry

T Helper 1 (Th1) Response

A reaction mediated by CD4-positive T cells thatserves to activate macrophages and promote digestionof intracellular bacteria. Th1 cells secrete cytokinessuch as interferon-γ (which activates macrophages)and lymphotoxin-α (which activates macrophages, in-hibits B lymphocytes, and is directly cytotoxic to somecells).

3Lymphocytes


T Helper 1–T Helper 2 Balance

Balance in immune response after contact with anti-gen, especially important for response to allergenswhere T helper activity drives immune response eitherto cellular (delayed-type hypersensitivity, Th1) or an-tibody (IgE, Th2) mediated allergic reaction. Ratsprone to a T helper 1 reaction (Lewis rats) showedmore resistence to Salmonella infection compared torats prone to a T helper 2 reaction (Brown Norwayrats).

3Salmonella, Assessment of Infection Risk

T Helper 2 Cells


T Helper 2 (Th2) Cells

CD4 T cells that produce cytokines such as interleu-kins IL-4 and IL-5 but not IL-2 and interferon IFNγ.By this they direct humoral immune responses.


3Flow Cytometry

T Helper 2 (Th2) Response

A reaction mediated by CD4-positive T cells that killinfected cells and direct the destruction of extracellularpathogens by activating B cells. Th2 cells secrete cy-tokines such as the interleukins IL-4 and IL-5 (whichactivate B lymphocytes) and IL-10 (which inhibitsmacrophage activation).

3Lymphocytes

T Helper Cell

CD4+ helper cell subgroups that are defined by a dif-ferent pattern of cytokine release. The Th1 subgroupproduces a cytokine profile to induce inflammationand cell-mediated immunity. The Th2 subgroup pro-duces a cytokine profile to induce antibody synthesis.Both subgroups act antagonistically to each other tosecure an enhanced, but balanced immune response.

3Cytokines

3Maturation of the Immune Response

3Leukocyte Culture: Considerations for In Vitro Cul-ture of T cells in Immunotoxicological Studies

3Food Allergy

T Helper Cell Polarization

3Maturation of the Immune Response

T Helper Lymphocyte

3Trace Metals and the Immune System

T Lymphocyte

White blood cell with characteristic appearance, cell-surface markers, and function. They undergo differen-

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tiation in the thymus. T lymphocytes control mostaspects of the immune response, and are involved di-rectly in attack on virus-infected cells and aberrantcells, such as malignant cells and cells originatingfrom a different individual (as in a transplanted organ).

3CD Markers

3Canine Immune System

3Delayed Type Hypersensitivity

3T3 Neutral Red Uptake (NRU) Test

The in vitro 3T3 neutral red uptake (NRU) phototox-icity test was developed and validated in a joint EU/COLIPA project (1992–97). The aim was to establisha valid in vitro alternative to the available in vivo tests.The parameter for the detection of cell viability and formeasuring the total activity of a cell population isbased on the uptake of the vital dye neutral red intocellular lysosomes of living murine BALB/c 3T3 fi-broblasts.

3Three-Dimensional Human Skin/Epidermal Modelsand Organotypic Human and Murine Skin ExplantSystems

T Regulatory Cells (Tregs)

T regulatory or suppressor T cells play important rolesin the regulation of immune responses and mediate adominant immunologic tolerance. The mechanisms bywhich naturally occurring Tregs are able to suppressCD4+ and CD8+ T cell proliferation are not yetknown. The CD4+CD25+ Tregs represent a subset ofsuppressor T cells and have been shown to play acritical role in the prevention of organ-specific auto-immunity and allograft rejection.

3Transforming Growth Factor β1; Control of T cellResponses to Antigens

T Suppressor Lymphocyte


Tachycardia

Heart rate above 100 beats per minute.

3Septic Shock

Tachypnea

Increased number of breaths per minute.

3Septic Shock

TAPA-1 (Target of an AntiproliferativeAntibody-1)

TAPA-1 (CD81) is a 26 kDa surface protein expressedon the surface of B cells as well as T cells. It bindsseveral different integrins and is believed to be in-volved in activation, cell adhesion and migrations.


Taqman

A DNA probe (labeled with a fluorescent reporter dyeand a fluorescent quencher) used to detect specificsequences in PCR products. When amplification oc-curs the Taqman probe is degraded by the 5' exonucle-ase activity of Taq DNA polymerase, thus separatingthe quencher from the reporter. The increase of repor-ter dye fluorescence is used to determine the presenceof specific gene sequences.

3Polymerase Chain Reaction (PCR)

Target Cell

The cytotoxic activity of immune cells is targeted to-wards specific cell types, which vary depending on thecytotoxic cell type involved.

3Limiting Dilution Analysis

Target Cell Killing

3Cell-Mediated Lysis

Targeted Mutant Mouse

3Knockout, Genetic

628 3T3 Neutral Red Uptake (NRU) Test

TbAT1 Genes

Trypanosomes are unable to synthesize purines denovo and rely on nucleoside transporters. The Try-panosoma brucei adenosine transporter 1 (TbAT1),also described as the trypanosomal P2-transporter, en-ables adenosine uptake. In addition, it confers suscep-tibility to antitrypanosomal drugs such as arsenicals.Various point mutations have been identified in theTbAT1 gene of resistant trypanosomes.

3Trypanosomes, Infection and Immunity

TCDD

3Dioxins and the Immune System

Telomeres

Telomeres are the physical ends of chromosomes.They are specialized nucleoprotein complexes thathave important functions, primarily in the protection,replication, and stabilization of the chromosome ends.In most organisms telomeres contain repeated simpleDNA sequences composed of a G-rich strand and a C-rich strand (called terminal repeats). These terminalrepeats are highly conserved—in fact all vertebratesappear to have the same simple sequence repeat (upto 2000 times) in telomeres (TTAGGG)n. After eachcell division, telomere shortening takes place. Telo-mere length is therefore indicative for the numbersof divisions a cell has been through. Critically shorttelomeres trigger replicative senescence and cell cyclearrest. The innate immune system provides the firstline of defence against many microorganisms and isessential for the control of common bacterial infec-tions. It comprises macrophages, neutrophils, and nat-ural killer cells. These cells of the innate immune re-sponse play also a pivotal role in the initiation of asubsequent adaptive immune response.

3Aging and the Immune System

TEQ/TEF

The complex nature of polychlorinated dibenzo-p-di-oxins, dibenzofurans and biphenyls, which are usuallygenerated together in occupational or environmentalexposure complicates the risk evaluation for humans.This is a concept introduced to facilitate risk assess-ment and regulatroy control of exposure to these mix-

tures. 2,3,7,8-TCDD has been assigned a toxic equiv-alency factor (TEF) of 1.0. TEF values for individualcongeners of dioxins, furans, and biphenyls in combi-nation with their concentration can be used to calculatethe total TCDD toxic equivalents concentration(TEQs) contriubted by all dioxin-like congeners inthe mixture using appropriate equations. Compoundsare included in the scheme and assigned a TEF if theyshow structural relationships to PCDD or PCDF, bindto the aryl hydrocarbon receptor, elicit aryl hydrocar-bon receptor mediated biochemical and toxic re-sponses, and persist and accumulate in the food chain.


Teratogen

Any substance or exposure that causes birth defects.

3Birth Defects, Immune Protection Against

Testosterone

3Steroid Hormones and their Effect on the ImmuneSystem

Tests for Autoimmunity

Raymond Pieters

Head ImmunotoxicologyInstitute for Risk Assessment Sciences (IRAS)Yalelaan 2P.O. Box 80.1763508 TD UtrechtThe Netherlands

Short Description

A considerable number of chemicals, including manydrugs, are capable of inducing autoimmune-like dis-eases in man (1–3).Autoimmunogenic chemicals rarely induce similarclinical adverse effects in test animals and are hardlyever identified in general toxicity testing. Hence, au-toimmune-like symptoms often become apparent onlyafter introduction to the market. In combination withthe fact that these symptoms can induce very seriousor life-threatening conditions, the autoimmunogenicityof chemicals, and drugs in particular, poses a hugeproblem to certain sectors of society—patients, clini-cians, pharmaceutical companies, and governmental

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agencies. Conceivably, there is an urgent need forscreening tests to identify such chemicals.The main reason for the inability to assess a chemical'spotential to cause autoimmune-like diseases is that theunderlying mechanism is very complex, and can in-volve the interplay of many predisposing factors. Animportant factor is the genetic make-up, the majorhistocompatability complex (MHC) haplotype, non-MHC regulatory genes, metabolic polymorphisms,and gender; but many environmental factors (such asongoing infections and food ingredients) are alsoknown to co-influence autoimmune phenomena (Fig-ure 1).The complexity of the etiology may be the reason thatonly a few drug-using patients develop autoimmune-like derangements, but also explain why symptomssuddenly appear after a long period of symptom-freedrug usage.A set of rational criteria to establish autoimmune eti-ology of diseases in man was postulated in 1962 and isreviewed in (4). One of the criteria requires the pre-sence of circulating antibodies or cell-mediated auto-immunity. Others require that the corresponding auto-antigen should be identified, and—more importantly—that the disease can be reproduced by passive trans-fer of that antibody or the self-reacting cells or byimmunization with the self antigen.Today, it is realized that autoreactivity (both autoreac-tive B as well as T cells) is a normal and necessaryproperty of a healthy immune system and that onlyfew self-reactive autoantibodies or autoreactive lym-phocytes may be considered pathogenic (i.e. directedagainst a pathologically relevant autoantigen and cap-able of causing tissue damage and reproducing diseasein experimental animals). The frequency of thesepathogenic autoreactive antibodies or lymphocytesmay be significantly higher in diseased compared tocontrol population (2,3).

Although autoreactivity is a healthy phenomenon andchanges in autoimmune-linked parameters do not ne-cessarily result in an autoimmune disease, it is impor-tant to note that changes in such parameters may beused to flag a chemical as possibly autoimmunogenic.

CharacteristicsAt present no clearly defined screening tests for auto-immunity in animals exist. The popliteal lymph nodeassay (PLNA) is a simple straightforward local lymphnode assay that may be useful to screen for initialimmunostimulating capacity of chemicals. But thisassay can only be regarded as a first screening testfor immunosensitizing potential and to indicate that achemical might induce autoimmune-linked symptoms.It is preferable for screening tests for autoimmunity touse relevant exposure routes and demonstrate systemicchanges in parameters indicative of autoimmune-linked responses. Diagnosis of autoimmune-linkeddiseases in test animals, like rats or mice, may bebased on a combination of general well-being, routineclinical tests and (immuno)histology. Clinical investi-gations should include general hematology (e.g. tocheck for anemias) as well as tests for complementactivity, or acute-phase proteins, and for erythrocytesedimentation. Liver and renal impairment should bemonitored biochemically (3). Morphologically, a widerange of organs should be checked for indications ofinflammation, overt apoptosis (in the thymus in parti-cular). Peripheral immunologic organs should bechecked for indications of activation (e.g. hyperplasiaor formation of germinal centers) (5).Morphological indications of tissue inflammation, ac-tivated immune organs or immunomodulation (e.g.thymus atrophy) should be followed up by more thor-ough investigations into alterations of autoimmuneparameters (5). Because development of actual auto-immune disease depends on a complex interplay of(non)inherent factors (see Figure 1), relevant changesin any of the animals should be considered as an alertto pursue further investigations. This is certainly thecase in outbred animals which are used for evaluationof toxicity, but also in inbred animals which are alsonot always 100% responsive.The initial focus in follow-up studies should be ondetection of autoantibodies, which can be directedagainst a wide spectrum of autoantigens (2). In casethe target autoantigen is not yet known, and particu-larly for screening purposes, the indirect immunoflu-orescence (IF) technique may be useful. The immuno-fluorescence technique, which is also used in the clin-ic, has been used successfully in animal studies.Briefly, cryosections or isolated cells grown on micro-scopic slides (for instance HepG2 tumor cells for an-tinuclear antibodies (ANA) or freshly isolated granu-locytes for antineutrophil cytoplasmic antibodies

Tests for Autoimmunity. Figure 1 Representation ofrisk factors that are possibly involved in development ofautoimmune derangements. Adapted from (7).

630 Tests for Autoimmunity

(ANCA)) are an incubated with serum suspected tocontain autoantibodies followed by a incubation withfluorochrome-labeled second-step antibody. Interest-ingly, the immunofluorescence technique can be ap-plied to cryosections of a range of relevant organs(such as kidney, thyroid, liver, skin, adrenals, andsex organs), although false-positive staining (perhapsas a result of antibody binding to Fc receptors) parti-cularly in inflamed tissue has to be taken into account.When the specificity is known, autoantibodies can bedetected by various other techniques (most notablyenzyme-linked immunoassay, ELISA). In manycases, it may suffice to perform an ELISA for ANA.Compound-specific lymphocyte transformation tests(LTT) can be used in cases of drug allergy or chemicalexposure (see for instance Schnyder et al 2000) butdetection of autoreactive T cells in case of chemicals ismuch more difficult. This is mainly due to the fact thatthe relevant autoantigen (chemically altered or pre-viously cryptic epitopes) is hardly ever known, andalso because specific autoreactive T cells are relativelyscarce even in clinical situations. A solution would beto immortalize selected self-reactive T cell clones, butthis is not easy to incorporate in a general testingmodel for autoimmunity.

Pros and ConsAll of these methods may at best provide circumstan-tial evidence for autoimmune effects and/or etiology inanimals. The advantage of these methods is that theycan all be used in animal toxicity studies without in-terference with the study per se, and only ask for moreextensive analyses of samples (blood, serum and or-gans) that are already to be isolated at dissection. But,importantly, as the immunological effects dependgreatly on genetic make-up, autoimmune effects maybe easily missed when small groups of outbred testanimals are used. So animal tests for autoimmunity(including the parameters discussed here) should bedone with larger test groups and should be performedover relatively long periods of exposure (> 90 days).Importantly, adverse effects which are indicative ofautoimmunity—even if they occur in only one ani-mal—should already be taken as an alert to executefollow-up studies with inbred animal strains, such asthe frequently used high-IgE-responding Brown Nor-way (BN) rat. To date, only a limited number of com-pounds (e.g. HgCl2, gold salts, d-penicillamine, nevir-apine, hexachlorobenzene) have been shown to induceautoimmune-like phenomena in this rat strain.

Relevance to HumansChemical-induced autoimmune effects detected in an-imals can be predictive for the human situation. How-ever, as in humans the prevalence of autoimmune ef-fects will be low in (outbred) animals as well. Studies

with particularly sensitive rat strains, for instance, suchas the BN rat, may identify much better the hazard ofautoimmunogenic potential of a chemical. Notably,such a sensitive rat has to be regarded as a representa-tive of very susceptible humans.

Regulatory Environment

At present guidelines for detection of autoimmuno-genic capacity do not exist. It should be realized thatnone of the present animal models—including the BNrat model—is capable of detecting autoimmunogenicpotential of a wide range of different chemicals. Thepopliteal lymph node assay (PLNA) is an animalmodel that may indicate whether a chemical is immu-nostimulatory. Immunostimulation may result in sen-sitization of the immune system and is considered oneof the prerequisites for inducing autoimmunity.A number of the parameters proposed here, however,could be easily or are already incorporated in existingguidelines. For instance, the OECD guideline 407 in-cludes the hematology, clinical biochemistry and pa-thology of a series of organs. But without further ana-lyses of (auto)antibody levels, larger test groups ofinbred animals and long exposure periods(> 90 days) a chemical's potential to induce autoim-munity will hardly ever be detected in these toxicitystudies.So, future research to design predictive protocols andscreening models is greatly needed. This could be in-itiated by thorough research into the relevance of theabove-mentioned parameters in repeated-dose studiesover a relatively long period with inbred strains of rats(e.g. BN and Lewis strains) as well as mice (e.g. SJLand C3H/He strains), but also in outbred animals thatare normally used in toxicity studies. Such studiesshould first be performed in a limited number ofwell-equipped laboratories, and should be followedby more extensive ring studies.

References

1. D'Cruz D (2000) Autoimmune diseases associated withdrugs, chemicals and environmental factors. ToxicolLetters 112–113:421–432

2. Verdier F, Patriarca C, Descotes J (1997) Autoantibodiesin conventional toxicity testing. Toxicology 119:51–58

3. D'Cruz D (2002) Testing for autoimmunity in humans.Toxicol Letters, 127:93–100

4. Shoenfeld Y, Isenberg D (eds) (1990) The Mosaic ofAutoimmunity, Factors Associated with AutoimmuneDisease. Introduction. Research Monographs in Immu-nology, Volume 12. Elsevier, Amsterdam

5. Frieke Kuper C, Schuurman H-J, Bos-Kuijpers M,Bloksma N (2000), Predictive testing for pathogenicautoimmunity: the morphological approach. ToxicolLetters 112–113:433–442

6. Schnyder B, Burkhart C, Schnyder-Frutig K et al. (2000)Recognition of sulphamethoxazole and its reactive

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metabolites by drug-specific CD4+ T cells from allergicindividuals. J Immunol 164:6647–6654

7. Kammüller ME, Bloksma, N, Seinen W (1989) Immunedisregulation induced by drugs and chemicals. In:Kammüller ME, Bloksma N, Seinen W (eds) Autoim-munity and Toxicology. Elsevier, Amsterdam, pp 3–25

2,3,7,8-tetrachlorodibenzo-p-dioxin


Tetravalent Vanadium

Tetravalent vanadium is the ionic form of vanadiumwhen four outer shell electrons (that is, two from 4sand two from 3d orbitals) have been shed, therebygiving the atom an overall charge of +4.

3Vanadium and the Immune System

TGF-β13Transforming Growth Factor β1; Control of T cell

Responses to Antigens

Th1/Th2 Balance

An important mechanism in the immune regulationinvolves homeostasis between the T helper 1 (Th1)and T helper 2 (Th2) activity of CD4+ T helper cellsexpressing different cytokine patterns. T helper cellsshowing Th1 activity are more prone to induce a cell-mediated immunity whilst T helper cells obtainingTh2 activity are more prone to induce a humoral-type immune response. T helper cells showing eitherTh1-type or Th2-type reactivity are exclusively char-acterized by differences in cytokine expression.Briefly, Th1 reactivity is predominantly connected tointerferon (IFN)-γ, IL-2, and IL-12 secretion. In con-trast Th2 cells express mainly IL-4, but also IL-5, IL-6, IL-10 and IL-13. The Th1/Th2 balance is integratedin the immune regulation in a dynamic and reversiblemanner, depending also on kinetics and dose–responseof the immune response.


Three-Dimensional Human Skin/Epidermal Models and OrganotypicHuman and Murine Skin ExplantSystems

Hans-Werner Vohr . Eckhart Heisler

PH-PD, ToxikologyBayer HealthCare AGAprather Weg 18D-42096 WuppertalGermany

Synonyms

human skin recombinants, reconstructed human skin/epidermis, 3-D human skin/epidermal equivalents, invitro engineered skin/epidermal substitutes, artificialskin/epidermis, organotypic murine or human skin ex-plant system, MSE, HSE, hOSEC

Definition

Human full-thickness skin models and reconstitutedepidermal equivalents are in vitro-engineered tissuecultures that provide a three-dimensional architecturewhich is biochemically, morphologically and function-ally comparable to human epidermal tissue/skin invivo. Organotypic skin explant systems are based onex vivo skin removed from humans or mice and sub-sequently cultured in toto. All the models were shownto be useful in screening for topically applied irritat-ing, 3corrosive or photocytotoxic compounds. Re-sults from experiments with systemically applied com-pounds have already been published with such mod-els, too. Furthermore, in recent studies it was demon-strated that 3-D skin models also provide the capacityto further characterize and screen for substances with asensitizing potential.

Characteristics

Reconstructed Human Epidermal Models

Reconstructed human epidermal models are built upfrom proliferating, differentiating and cornifying ker-atinocytes which are airlift-cultured on a porous poly-meric membrane. The design of the cell culture con-ditions (air-liquid interphase and medium/ingredients)drives the cells to differentiate and form a three-di-mensional (3-D) epidermal multilayer with a function-al and stratified surface. Most of the key structuralelements of native epidermis like 3keratins, 3trans-glutaminase and lipid composition that characterizethe status of keratinocyte differentiation are presentin 3-D human epidermal equivalents.

632 2,3,7,8-tetrachlorodibenzo-p-dioxin

Full-Thickness Human Skin Substitutes

Full-thickness human skin substitutes additionally pro-vide a dermal layer that usually consists of a collagenmatrix which is populated by living fibroblasts. In anearly state of research the use of de-epidermizedhuman dermis as the backbone of full-thickness skinequivalents has been discussed as well. In comparisonto single-cell culture systems, the most predominantfeature of these artificial skin models is the existenceof a physiological and functional barrier (the stratumcorneum) that regulates percutaneous absorption/pen-etration of compounds as well as transepidermal waterloss. Although the barrier functions of artificial skinmodels are different from the situation in vivo, theresults from studies evaluating the penetration proper-ties of various reference test compounds have shown agood correlation to in vivo data.

Organotypic Skin Explant Systems

Organotypic skin explant systems from human, rats or—to a lesser extent—mice have also been establishedfor evaluating percutaneous absorption and penetra-tion. However, in comparison to reconstructed skinmodels the explant cultures naturally provide a phys-iological cell composition and micro-architecture in-cluding immunocompetent cells (e.g. Langerhanscells).For toxicological and immunotoxicologic research,both topical treatment (application of compounds tothe dry stratum corneum) as well as systemic-liketreatment (application of substances directly into thecell culture medium) are possible using 3-D skin mod-

els. Hazard identification is based on the measurementof decreased cell viability and changes in tissue mor-phology after treatment (histological examination; seebelow). In recent studies it was also shown that topicaland systemic-like treatment of 3-D skin models withhazardous compounds often results in induced expres-sion and/or release of immunomodulating proteins(cytokines, chemokines, matrix metalloproteinases,growth factors, and other parameters which are in-volved in a variety of biochemical pathways; seebelow). The determination of these parameters givesa detailed overview of the cell status which can addi-tionally confirm the results from viability testing andhistological examinations (multiple endpoint analysis;MEA).

Screening for Irreversible Cutaneous Toxicity

(Corrosion)

Screening for Acute Irritation

Both artificial skin models and organotypic skin ex-plant systems are suitable for screening for dermal ir-ritation induced by topically applied irritating orphoto-irritating compounds and formulations. Mostlikely in this situation is that in vivo substances witha strong 3irritant potential provoke severe destructionof the reconstructed or explanted tissues and affect theintegrity of residential cells. The use of these systemsto test chemicals, compounds, or formulations accord-ing to their irritant properties depends on the measure-ment of cell viability after topical treatment with com-pounds and additional time-related incubation. Cyto-toxic and photocytotoxic effects cause a significant

Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine Skin ExplantSystems. Figure 1 Two different reconstructed tissues: H&E stained sections of untreated reconstructed humanfull thickness skin model (Advanced Cell Systems, AST-2000) and epidermal model (Skinethic RHE ). Both modelsare comercially available. (Picture of AST-2000 by kind permission of Advanced Cell Systems, St. Katharinen,Germany).

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decrease in cell viability. For this reason, determina-tion of cell viability is essential for the assessment ofcompound biocompatibility using artificial skin mod-els or organotypic skin explant systems. However, inmost of the published test protocols MTT conversionis used as a single endpoint parameter for the determi-nation of cell viability and consequently the degree ofcytotoxicity caused by irritation and photoirritation.Recently the identification of more specific parametersallows a multiple endpoint analysis (cell viability, his-tological examination, release of IL-1α;).

Expression of Immunomodulating Proteins and

Screening for Dermal Sensitization

Both irritation and sensitization of the skin are relatedto the expression and release of immunomodulatingproteins such as cytokines, chemokines and cell sur-face proteins, especially within the epidermis. Thelocal immune system of the skin in vivo is based onthe interactions between epidermal keratinocytes, epi-dermal Langerhans cells, and dermal fibroblasts. Onceactivated by antigen uptake and processing, Langer-hans cells undergo morphological changes and start tomigrate to the local draining lymph nodes. ThereT cells become activated upon successful antigen pre-sentation. In cases of cutaneous irritation causing epi-dermal cell damage, keratinocytes release a cocktail ofproinflammatory proteins from their intracellular re-servoirs. This finally results in a non-specific activa-tion of the skin’s immune system (see also 3contacthypersensitivity section).Considerable efforts have been made to integrate Lan-gerhans cells into reconstructed human skin models.However, there is still no complex in vitro systemavailable that provides functional antigen-presentingcells in the epidermis or dermis. Nevertheless, kerati-nocytes are also thought to be involved in the initialsteps of irritation and sensitization. Topical treatmentof artificial skin models with irritating compoundsleads especially to the release of interleukins IL-1αand IL-8 by keratinocytes. Furthermore, the subse-quent analysis of cell culture supernatants by differentELISA techniques (enzyme-linked immunoassay) ad-ditionally show an induced release of different chemo-kines and cytokines as shown in Table 1.The profile of released proteins depends on the kind ofmodel used for the experiments. In comparison to re-constructed epidermal models, full-thickness skinmodels provide a set of parameters that are related tothe interaction between epidermal keratinocytes anddermal fibroblasts. In recent studies carried out withsensitizing substances the ratio between IL-1α and IL-8 release after topical treatment with the compoundsrevealed promising results that suggest that recon-structed human skin models are capable of discrimi-nating 3sensitizers from compounds with an exclu-

sively irritant potential. Other studies identified pro-mising parameters (increased release of the chemo-kines monocyte chemoattractant protein 1 (MCP-1)and interferon-inducible protein (IP-10) from ahuman full-thickness skin model AST-2000 after treat-ment with the standard sensitizer (oxazolone) that cer-tainly can contribute to a successful discriminationbetween sensitizers and irritants in vitro.From an immunological point of view, however, it isof prime importance for sensitization testing to analyzeparameters ( 3MIG, Langerin, TARC, etc.) that arecharacteristic for the cross-talk between keratinocytes,fibroblasts and antigen-presenting cells in their naturalsetting. For this reason, research on skin sensitization(screening, mechanistic) is particularly focussed on theuse of organotypic skin explant systems as well as thedevelopment of skin recombinants that incorporatefunctional antigen-presenting cells.

Pros and ConsExperimental Strategies

Methods used in in vitro dermal toxicology are oftenbased on single-cell culture systems, which in turn arebuilt up from either freshly isolated primary cells de-rived from cosmetic surgery, foreskins or well-estab-lished cell lines. Methods for cytotoxicity and photo-cytotoxicity testing, like the 3T3 neutral red uptake(NRU) test, have been successfully validated. Howev-er, test principles based on single-cell cultures are sub-ject to some limitations due to their lack of a physio-logical barrier. For this reason they are usually re-stricted to soluble substances and therefore fail whenit comes to testing hydrophobic compounds or formu-lations. Furthermore, the concentrations of compoundsinducing irritation in single-cell cultures are signifi-cantly lower than those determined in in vivo experi-ments. Due to the absence of a stratified surface, falsepositive results may also occur, because substancesmay be classified as (photo)cytotoxic by 3T3 NRUalthough they are physicochemically unable to passthrough the physiological barrier (the stratum corne-um).By using 3-D skin models it is possible to overcomethese problems, and they offer a promising test systemfor topical and systemic-like compound administra-tion. Furthermore, artificial skin models and organo-typic skin explant systems may be suitable for screen-ing for sensitizing properties of compounds in vitro.With respect to this last point research is still in pro-gress, but a convincing system may be available in thenear future.

Test Principles

As already mentioned, MTT testing is often used as asingle-endpoint parameter for predicting the irritantpotentials of substances, although cytotoxicity is not

634 Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine S

a sufficient stand-alone parameter for predicting cuta-neous irritation. In vitro testing associated with MTTconversion is always subject to some limitations, be-cause the test principle is based on a chemical redoxreaction which may also run without any participationof living cells. This may lead to false positive results.Another problem with MTT, especially concerning 3-D skin models, was observed when test results werecompared to histological examinations of recon-structed skin models after compound treatment. Dueto cellular activity, formazan crystals were found to beformed especially in the cells from the basal layer. For

this reason, it is not possible to detect undesired com-pound-related effects on cells from the stratum spino-sum or stratum granulosum by MTT (see Figure 2).Other test principles for the determination of cell via-bility are based on the quantitative analysis of en-zymes from the cytosol of cells. When cells losetheir integrity through damage to the plasma mem-branes, the leakage of these proteins can be recordedand quantified by bioluminometric or other optical en-zymatic test systems.In this context the measurement of lactate dehydroge-nase (LDH) and/or adenylate kinase leakage is oftendiscussed as a defined parameter for the analysis ofsubstance-related cytotoxic effects on in vitro cell sys-tems.Finally, the induced release of proinflammatory med-iators like IL-1α additionally serves as a good param-eter for the characterization of skin irritation, becauseIL-1α was found to be released from cells which areinfluenced by irritating chemicals. Although MTT is areliable and valid parameter for the analysis of cellviability, the results should be supplemented and ver-ified additionally by multiple endpoints, such as ex-pression and release of proinflammatory mediators,decrease of the barrier function determined by trans-epidermal water loss (TEWEL) and/or evaluation ofmorphological changes (histologic examination).

Comparison to In Vivo Test Principles

The replacement of in vivo methods for corrosivityand irritancy according to Draize by in vitro recon-structed skin models is often discussed, especiallyfrom an ethical point of view. In addition, the use of3-D skin models is less time-consuming than in vivo

Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine Skin ExplantSystems. Table 1 Expression/release of immunomodulating proteins from 3-D skin models

Parameter Expression/Release Release Inducible

Interleukin-1α ++ (a,b,c) Yes

Interleukin -1β + (b,c) Slightly

Interleukin -6 +++ (+a,b,c) Yes

Interleukin -8 +++ (+a,b,c) Yes

Tumor necrosis factor-α + ((a),b,c) Slightly

Monocyte chemoattractant protein MCP-1 +++ (+b,c) Yes

MIG + (c) Slightly

Interferon-inducible protein IP-10 + (b,c) Slightly

Macrophage inflammatory factor MIP-3α + (c) Slightly

Matrix metalloproteinase MMP-3 ++ (+b,c) Slightly

Matrix metalloproteinase MMP-9 ++ (b,c) Yes

a, epidermal model; b, full thickness skin model; c, organotypic skin explant system; +, low level; ++, medium level; +++, highlevel; (+), high background.

Three-Dimensional Human Skin/Epidermal Modelsand Organotypic Human and Murine Skin ExplantSystems. Figure 2 H&E stained section of SkinethicRHE after treatment with 0,4% SDS and 24 hours ofincubation (5% CO2, 37°C, max hum.) The areamarked with the red arrow shows massive destructionof cells in the upper epidermal layers. However, thebasal layer (blue arrow) is not affected. Here, MTT testgave false negative results. Although cell viability wascorrectly determined, the integrety of the cells in theupper epidermal layer was hardly affected. This effecthowever, was undetectable by MTT alone (By kindpermisson of SkinEthic Laboratories, Nice, France).

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testing, and if the costs for animal health and care aretaken together, reconstructed skin models are less cost-effective than animal testing, too. However, a rankingbetween strong irritation and weak or mild irritationbased on experimental results from testing with recon-structed skin/epidermis still seems to be questionable.The establishment of in vitro test methods for sensiti-zation is not that easy, although protein fingerprintingof cells from organotypic skin explant systems andreconstructed epidermal/skin models revealed promis-ing results that contribute to the in vivo situation. Inrecent studies it was shown that the expression andrelease of immunomodulating proteins (Table 1)serve as good parameters for the characterization ofcompounds with sensitizing properties. However, theuse of these parameters as criteria for predicting sen-sitization has not been validated so far. For this reason,guinea pig assays like those described by Buehler orMagnusson and Kligman are still the most reliablemethods for sensitization testing, even though theyare based on visible subjective parameters like the for-mation of erythema. In this context, another validmethod is LLNA/IMDS (local lymph node assay/inte-grated model for the differentiation of (chemical)-in-duced skin reactions) which characterizes sensitizingcompounds with the help of cellular parameters, but isstill based on animal treatment.

PredictivityIrritation of the skin caused by exposure of individualsto different kinds of hazardous compounds or formu-lations is the most common non-specific immune re-action observed in human skin. In vivo (animal) testprinciples according to the methods of Draize are fre-quently used for the identification of substances withirritant potential. For several reasons, however, thesetest methods are questionable. The analysis of sub-stances according to Draize testing is mainly basedon the evaluation and scoring of macroscopic para-meters such as overcasting of the rabbit eye corneaor redness of the skin after treatment with the com-pounds being tested. As far as this point is concerned,it has recently been shown that the choice of endpointsfor the assessment of acute skin irritation according tointernational standards (methods according to Draize)may lead to misclassification of substances. Further-more, the transfer of established data from animal test-ing to the human situation in vivo is still controver-sially discussed. For this reason the human patch testwas established. This ideally meets the requirements,but patch testing in human is restricted to weak ormoderate irritating compounds. These pragmatic dis-advantages of in vivo animal and human testing forskin corrosion or acute skin irritation are furthermoreaccompanied by the discussion of the ethical justifica-tion of animal testing in toxicological research. With

the use of reconstructed tissue models it is possible toovercome most of the problems described above.From multiple endpoint analysis (see Characteristics)reliable parameters are available that are simple todetermine, while the output is more stringent than vi-sual evaluation of results.Furthermore, artificial skin models were proven to bereproducible in intra- and interlaboratory multicenterstudies. As mentioned above, the predictivity of recon-structed tissue models is limited. In comparison tohuman in vivo skin the different physiological barrierfunction of the reconstructed stratified surface maycause problems because the risk of false positive re-sults cannot be totally excluded. In addition, distin-guishing between weak and moderate irritating com-pounds is sometimes not easy. However, research isfocussing on new parameters that could help to solvethese problems. Despite this early state of affairs, it ispossible to state that human reconstructed tissue mod-els exhibit acceptable predictivity in screening for cor-rosive compounds (sensitivity and specificity > 80%).Although the validation and catch-up validation stu-dies for acute irritancy of topical applied formulationand/or raw materials are still in progress, a high cor-relation of sensitivity has already been estimated bythe relevant ECVAM Task Force.Another main topic of interest concerns alternative invitro models for skin sensitization. At present, no re-constructed tissue model is available that meets theguideline criteria for adequate screening. However,considerable efforts have been made to search forparameters (cytokines, chemokines) which specificallycharacterize the complexity of the processes leading toskin sensitization (skin penetration, formation of pro-tein-hapten complexes, antigen uptake and processing,migration of LC to the local draining lymph nodes,presentation of antigen to T cell populations in thedraining lymph nodes). In the light of this complexity,the use of organotypic skin explant systems seems tobe very promising, because they provide the samemicro-architecture and the same cell composition asin vivo skin and are therefore potent tools for mechan-istic studies.

Relevance to Humans

The test results from animal testing for irritancy andcorrosion according to Draize are controversially dis-cussed among toxicologists. In cases of acute irritationthese test methods have never been validated and theyprincipally depend on a collection of cross-connectedempirical clinical and preclinical data. For this reason,the use of reconstructed human tissues is of particu-larly great value, because the cells used for these skinconstructs are of human origin. Although some differ-ences in the characteristic barrier function have been

636 Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine S

described, the experimental design closely matches thehuman situation in vivo.Unfortunately, screening for sensitization in vitro iseven more complex because artificial tissue structuresare necessary which must in addition provide immu-norelevant cross-talk activities. For hazard identifica-tion, on the other hand, fingerprinting of proteins re-leased from 3-D in vitro skin models has already beenevaluated and some of these parameters were shown tohold key positions in immunological pathways (IL-8,MCP-1, IL-1α, IL-6, etc.). These may therefore helpto screen for compounds with a sensitizing potential invitro. As mentioned earlier, human skin explant sys-tems in particular are believed to be very suitablemodels for further characterization of immunorelevantparameters. In the heat of discussion about testing forsensitization, one should keep in mind that in vivoanimal testing (guinea pig assays or the (modified)local lymph node assay) or human patch testing, aswell as all possible in vitro models which are goingto be established and validated in the future, are notcapable of taking all parameters influencing the induc-tion of skin sensitization into account (individual para-meters such as genotype, age, sex, side of contact/pen-etration and of course the overall condition of theskin).

Regulatory EnvironmentSkin Irritation/Corrosion

The international standards for skin irritation and cor-rosion are still based on in vivo test principles accord-ing to the methods of Draize et al. (1944). However,the considerable efforts of organizations like ECVAM,ICCVAM, COLIPA, the Steering Committee on Alter-natives to Animal Testing (SCAAT) have had a favor-able and lasting influence on the establishment of invitro test methods of international guidelines. The useof several in vitro human skin models for skin corro-sion was validated by ECVAM in 2000. For acute skinirritation, however, a first prevalidation study failedbut the process of improving the use of reconstructedtissue models especially in this field of toxicologicresearch is strictly ongoing.

Guidelines for Determination of Substance-Induced

Skin Corrosion* OECD Guideline 402: Acute Dermal Tox.* OECD Guideline 404/405: Acute Dermal/Occular

Tox Irritation and Corrosion* OECD Guideline 410: Repeated Dose Dermal Tox.* OECD Guideline 430: In Vitro Skin Corrosion—

Rat TER (Trans Epidermal Resistance) Test* OECD Guideline 431: In Vitro Skin Corrosion—

Human Skin Models* Annex V of Directive 67/548/EEC (1997)* US Code of Federal Regulations (1991)

Sensitization

Up to now no in vitro screening model has been avail-able to correctly predict exclusively sensitizing proper-ties of compounds. From an immunological point ofview this is not surprising because of the lack of an-tigen-presenting cells in most of the reconstructedhuman tissues. However, the induced release of im-munomodulating proteins indicates promising para-meters for successful discrimination between irritatingand sensitizing substances. As long as none of thereconstructed or organotypic models match the criteriafor a successful prevalidation study, immunotoxicolo-gic research must rely on guinea pig test principlesaccording to Bühler, Magnusson and Kligmann, oron a refined test assay like the LLNA or the integratedmodel for the differentiation of (chemical)-inducedskin reactions (IMDS).

Guidelines for Determination of Substance-Induced

Sensitization

* OECD Guideline 406: Skin Sensitization (1992)* OECD Guideline 429: LLNA (2002)* U.S. EPA-OPPTS Harmonized Test Guideline

870.2600 on Skin Sensitization (1998)* FDA (CDER) (Draft) Immunotoxicology Evalua-

tion of Investigational New Drugs (2001)* CPMP/SWP/398/01 (Draft) Note for Guidance on

Photosafety Testing (2001) (as modified LLNA)

References

1. Botham PA, Earl LK, Fentem JH, Roguet R, van de SandtJJM (1998) Alternative methods for skin irritation testing:the current status. ECVAM Skin Irritation Task ForceReport 1. ATLA 26:195–211

2. Zuang V et al. (2002) Follow-up to the ECVAMprevalidation study on in vitro tests for acute skinirritation. ECVAM Skin Irritation Task Force Report 2.ATLA 30:109–129

3. Spielmann H et al. (2003) Report of the SecondSkinEthic Workshop: In Vitro Reconstructed HumanTissue Models in Applied Pharmacology and ToxicologyTesting, Nice, France

4. Coquette A, Berna N, Vandenbosch A, Rosdy M, DeWever B, Poumay Y (2003) Analysis of interleukin-1alpha (IL-1 alpha) and interleukin-8 (IL-8) expressionand release in in vitro reconstructed human epidermis forthe prediction of in vivo skin irritation and/or sensitiza-tion. Toxicol In Vitro 17:311–321

5. Heisler E, Ahr HJ, Vohr HW (2001) Local immunereactions in vitro: Skin models for the discriminationbetween irritation and sensitization. Exp Clin Immuno-biol 204:1–2

Three Rs

Reduction (fewer animals), refinement (less severe

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procedures), and replacement (in-vitro alternatives) ofanimal experiments, first proposed by Russel andBurch in 1959.

3Canine Immune System

Thrombin

Thrombin is a multifunctional serine protease that hasprocoagulant activities when diffusable in the bloodstream. But it loses this ability and initiates a potentanticoagulant pathway when bound to its endothelialcell receptor thrombomodulin, thereby mediating gen-eration of the anticoagulant enzyme-activatedprotein C. The cellular activities of thrombin on plate-lets, endothelial or smooth muscle cells are mediatedthrough G protein-coupled protease-activated recep-tors (PAR) that are initially cleaved by thrombin be-fore a newly generated peptide motif of the receptorcan serve as an internal tethered ligand for initiation ofcell signaling.

3Blood Coagulation

Thrombocytopenia

Thrombocytopenia is a condition in which the normalconcentration of platelets (thrombocytes) in the bloodis decreased. A significant shortage of platelets canresult in bruising and easy bleeding.

3Leukemia

3Antiglobulin (Coombs) Test

Thrombocytopenic Purpura

A rare autoimmune disorder characterized by a short-age of platelets, leading to bruising and spontaneousbleeding. Approximately half of the cases are idiopath-ic (unknown cause). Other cases are caused by drugs,infections or autoimmune disorders such as lupus er-ythematosus.

3Interferon-γ

Thymic Hypoplasia

An immunodeficiency that selectively affects theT lymphocyte limb of the immune response. There islymphopenia with diminished T cell numbers.


Thymocyte Development


Thymocyte Education


Thymocyte Selection


Thymus

C Frieke Kuper

Toxicology and Applied PharmacologyTNO Food and Nutrition ResearchZeistThe Netherlands

Synonyms

Thymus, thymus gland, sweetbread (when used asfood)

Definition

The thymus is a primary lymphoid organ in verte-brates; in mammals it is located in the cranioventralmediastinum and lower part of the neck. The primefunctions of the thymus in mammals are the develop-ment of immunocompetent T lymphocytes from bone-marrow-derived stem cells, the proliferation of maturenaive T cells to supply the circulating lymphocyte pooland peripheral tissues and the development of immu-nological self-tolerance. The thymus elaborates a num-ber of soluble factors (thymic hormones) which regu-late several immune processes, including intrathymicand post-thymic T-cell maturation, and neuroendo-crine processes such as the synthesis of neuroendo-crine hormones by the central nervous system.

Characteristics

Anatomy and Histology

The thymus is located in the cranioventral mediasti-num and lower part of the neck, whereas small islandsof thymic tissue may be present near the thyroid and

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parathyroid glands. In young animals it is roughlypyramid-shaped with its base located ventrally. Thegland consists of two lobes, fused in the midline byconnective tissue. The two thymic lobes are enclosedby a fibrous capsule from which septa traverse into theorgan, dividing it into lobules. The lobules have basi-cally the same architecture, with a subcapsular area, a

3cortex, corticomedullary junction and a 3medulla.The cortex is easily recognizable in hematoxylin andeosin(H&E)-stained sections by its high density ofthymocytes (immature lymphocytes) and thereforedarker appearance when compared with the less den-sely populated medulla. The framework of the thymusis formed of epithelial reticular cells in which thebone-marrow-derived lymphoid (thymocytes/lympho-cytes) and non-lymphoid cells (macrophages, dendriticcells) are packed. The vast majority of lymphocytesare T cells, but accumulations of B cells do occur.Epithelial aggregates with centrally located cell debris,the so-called 3Hassall’s bodies, are a characteristicfeature in the medulla.The different thymic compartments are associated withdifferent T cell maturation processes, namely early(cortical) maturation and late (medullary) maturation,which in turn are associated with differences in themarker expression and cytology of epithelial cells,lymphocytes, macrophages and interdigitating cells(Figure 1).Moreover, the capacity of epithelial cells to synthesizethymic hormones differs, the major site of hormonesynthesis being the medullary epithelium (1). A char-acteristic and unexplained microenvironment isformed by the cortical and medullary areas whichare devoid of epithelial cells but full of thymocytes,the so-called epithelial-free areas or EFAs (2). Thefunction of these EFAs is unknown, although medul-lary EFAs may be associated with autoimmune diabe-tes. Foci of myelopoiesis are found in the connectivetissue septa, within the lymphoid tissue at the outerrim of the lobules, and at the corticomedullary zone.Hemoglobin-containing cells can be found among themyelocytic series in the interlobular septa, at the outerrim of the lobules. In the medulla no erythroid pre-cursors have been observed. Blood vessels enter thelobules via the interlobular trabeculae/septa andbranch at the corticomedullary area to supply the cor-tex and medulla. Postcapillary venules in the cortico-medullary region have a specialized cuboidal epitheli-um similar to that of the high-endothelial venules ofthe lymph node, which allows passage of lymphocytesinto and out of the thymus. Sheaths of connective tis-sue and an epithelial cell layer with its basement mem-brane are found around the blood vessels. The spacebetween the epithelial basement membrane and thevessel lining is often quite broad around the cortico-medullary vessels and is called the perivascular space.

This space may contain all kind of blood cells andmost often contain fine lymphatics. Nerves coursealong the blood vasculature.During ontogeny, hematopoietic progenitor cells mi-grate into the thymic epithelial primordium betweendays 11–13 of fetal life in mice. Small lymphocytescan be found in the thymic primordium at aboutday 14 (mouse) or day 15 (rat) of fetal life. The thy-mus is fully developed, meaning a cortex and medullacan be distinguished, at day 17 of fetal life in themouse and by days 19–21 in rats, and the organgrows considerably immediately after birth. Thisgrowth is caused by the immense postnatal antigenstimulation; at that time large numbers of matureT cells are demanded. The thymus starts to involuteafter adulthood is reached. With age, the two thymiclobes diverge caudally and in old animals are almostcompletely separated; the thymus is then restricted tothe area cranially to the aortic arch. The number oflymphocytes decrease, especially in the outer cortex.Although areas with different lymphocyte density,suggesting the presence of cortex and medulla, areoften present in advanced age, the general arrangementof the cortex enclosing the medulla is not strictlymaintained. This gives the thymus an irregular appear-ance. The expanding perivascular connective tissuemeshwork and increasing perivascular lymphocyte ac-cumulations may further disturb the normal pattern.The septa and capsule harbor increasing numbers ofadipose cells, which eventually invade the thymic pa-renchyma.In addition to the expansion of the connective tissuecomponent, epithelial cords and tubules are large andnumerous in the old thymus and the epithelial Has-sall’s bodies become relatively more prominent thoughin absolute numbers they decrease. Adrenergic inner-vation of the gland is maintained in old animals. Thy-mic involution may be related to changes in the hor-monal status of the individual; circulating thymic hor-mone is reduced to very low levels in adults. Theconsequences of age-related involution are obvious:the emigration of lymphocytes from the thymusshows a dramatic decrease. Apparently, the persistentgeneration of new antigen-recognition repertoire in theT cell population of adults is not needed. Instead, thebody can defend itself using the established repertoireand extra-thymic self renewal of the T cells. Pregnancyin rodents results in radical, but reversible changes.After an initial rise in thymic weight in early pregnan-cy, involution starts with lymphocyte cell death in thecortex. In wild populations, cyclical enlargement andregression is documented. For instance, most birdsshowed an involuted thymus at the time of matingand laying, whereas on subsequent egg incubationthe thymus size is increased.

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T-Cell Maturation

T cells reside in the thymus during their maturationfrom progenitor cells to immunocompetent T cells.The process of T-cell maturation includes a numberof steps which are associated with location in differentmicroenvironments (3). (See Figure 1).The immature cells, which enter the lobules by theblood vasculature at the corticomedullary junction,first move to the outer subcapsular cortex, wherethey appear as large lymphoblasts. They then passthrough the cortex where the cells become small lym-phocytes with scanty cytoplasm. Finally, the cellsmove to the medulla, where they appear as medium-sized lymphocytes. These translocational stages in de-velopment can be monitored by the immunologic phe-notype: cells change from CD4−CD8− (double nega-tive) at a very immature stage into a CD4+CD8+ (dou-ble positive) phenotype, which is characteristic for al-most all lymphocytes in the cortex. In the medulla,T cells have the phenotype of relatively mature cells,with distinct CD4+CD8− (about 70%) and CD4+CD8+

(about 30%) populations. This phenotypic change isaccompanied by a crucial aspect of intrathymic T-cellmaturation: the genesis of the T cell receptor (TCR)consisting of the alpha-beta heterodimer (4). The DNAgenomic organization encoding these chains is ingerm-line configuration, with a variety of gene seg-ments encoding the variable part of the receptor mol-ecule. Before transcription and translation into TCRbecomes possible, combinations have to be made ofgene segments encoding the variable and constantparts of the TCR.This process of gene rearrangement requires the thy-mus microenvironment. The cell can synthesize thereceptor after completion of this gene rearrangement.The receptor is then expressed on the cell membranewith the CD3 molecule, which acts as the transmem-brane signal-transducing molecule after TCR stimula-tion. Even when the TCR has not yet been synthe-sized, this CD3 molecule is already present in the cy-toplasm of the cell. T cells at this stage of maturation

Thymus. Figure 1 Schematic presentation of a thymus lobule with cortex, corticomedullary region, medulla andan epithelial-free area (EFA). In the lobule, a simple overview of thymocyte maturation is presented: round cellsrepresenting T lymphocytes (T) with their membrane markers CD4 and/or CD8

640 Thymus

can be recognized by cytoplasmic staining with CD3reagents.TCR gene rearrangement is similar to the rearrange-ment of genes encoding immunoglobulin heavy andlight chains that takes place in the bone marrow mi-croenvironment. However, after surface expression ofthe TCR, the cell undergoes a process unique toT cells, namely, specific selection on the basis of rec-ognition specificity. First, the cell is examined for itsaffinity for its own major histocompatibility complex(MHC; self restriction). T cells with an intermediateaffinity for self MHC peptides are allowed to expand(positive selection). Secondly, T cells with a high af-finity for self MHC are deleted (negative selection). Inthis way, the random pool of antigen-recognition spe-cificities of T cells is adapted to the host's situation.The T cell repertoire in germ-line configuration cannotbe fully expressed but is influenced by the individual'sown MHC haplotype.It is generally accepted that the epithelial microenvi-ronment of the thymic cortex plays a major role inpositive selection. This microenvironment expressesMHC class I and class II products and morphologi-cally (at electron microscopic level) shows close inter-actions with lymphocytes. This close interaction is re-flected by the complete inclusion of lymphocytes in-side the epithelial cytoplasm (thymic 3nurse cell).Negative selection has been ascribed to either the epi-thelial compartment or the medullary dendritic cells.The cortex can be considered a primary or centrallymphoid organ because of its antigen-free microenvi-ronment. In contrast, antigens can move relativelyfreely into the medulla and encounter antigen-present-ing dendritic cells as well as antigen-reactive T cells.Thus the medulla has properties of a secondary lym-phoid organ.

Preclinical RelevanceThe dynamics of the thymus with ongoing reactions ofcell proliferation and differentiation, and gene ampli-fication, transcription and translation makes it highlysusceptible to toxic insults. Compounds that interferewith these processes are often immunotoxic. There-fore, a decrease of thymus weight in preclinical studiesis often a first indicator of toxic action of a xenobioticagent on the immune system, although some com-pounds, like cyclosporine, profoundly alter thymic his-tophysiology, without apparent effect on thymusweight. The dynamic nature of the immune systemprovides it with great regenerative capacity: the origi-nal architecture of the thymus is restored rapidly fol-lowing involution induced, for example, by irradia-tion, or treatment with glucocorticosteroids or organo-tin compounds.Thymus in aged or immunocompromised animals mayhardly be visible. For histology adipose and connec-

tive tissues from the cranioventral mediastinum, whichcontains thymic tissue, should then be collected. Thethymus is also very susceptible to acute (glucocorti-coid-related) stress (5). It is conceivable that with agethe thymus becomes less sensitive to toxic insults andthat toxic effects on the thymus with age have lessfunctional importance, because of age-related thymicinvolution. However, the components that constitutethe various thymic compartments are still present inhealthy old animals, as was shown by reconstitutionstudies. Therefore, a decreased sensitivity to toxiccompounds may not be a general property of the in-voluted thymus in aged animals.

Relevance to HumansThe use of data obtained in laboratory animal speciesfor man presents difficulties when species differ inorgan anatomy and histophysiology and sensitivity.The thymus is present in all vertebrates, possiblywith few exceptions, and there are only a few struc-tural differences between the species (6). Anatomicaldifferences relate to thymus location and number ofthymic lobes, the prominence of epithelial aggregateswith centrally located cell debris, the so-called Has-sall’s bodies, and the presence of B cell follicles. Dur-ing the third month of gestation the thymic primordi-um becomes colonized by marrow-derived stem cells.When these stem cells are indeed thymocyte precursorcells, their migration into the thymic primordium atthat time is considerably earlier—relative to gestationtime—in humans than in mice or rats. Differences inimmunotoxicity between laboratory animals and manappear to depend predominantly on differences in tox-icokinetics and metabolism of substances. Moreover,the interindividual differences and the age-related in-traindividual variations are probably more markedthan interspecies differences. It should be emphasizedthat the “normal” architecture of the thymus, as knownfrom textbooks, can be expected only between the lategestational period and young adulthood, and beforepregnancy.The universality of the immune system observed inmammals and the data obtained so far indicate thatdata from laboratory animals can be extrapolatedquite well to humans.

Regulatory EnvironmentRegulatory toxicity testing, which uses immune para-meters, is still under development. This applies topharmaceuticals and industrial substances as well.Nevertheless, most guidelines recognize the impor-tance of the thymus. For instance, the EuropeanUnion guidelines on repeated-dose toxicity testingwith pharmaceuticals require the macroscopic and mi-croscopic examination of the spleen, thymus, andsome lymph nodes with respect to the immune system.

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Moreover, a multilaboratory, 28-day oral toxicitystudy (OECD guideline 407) with the model immuno-toxicants azathioprine and cyclosporine demonstratedthat the most consistent effects were observed in thethymus (7).

References

1. Dabrowski MP, Dabrowski-Bernstein BK (1990) Immu-noregulatory Role of the Thymus. CRC Press, BocaRaton

2. Bruijntjes JP, Kuper CF, Robinson J, Schuurman H-J(1993) Epithelium-free area in the thymic cortex of rats.Dev Immunol 3:113–122

3. Van Ewijk W (1991) T-cell differentiation is influencedby thymic microenvironments. Ann Rev Immunol 9:591–615

4. Werlen G, Hausmann B, Naeher D, Palmer E (2003)Signaling life and death in the thymus: Timing iseverything. Science 299:1859–1863

5. Godfrey DI, Purton JF, Boyd RL, Cole TJ (2000) Stress-free T-cell development: glucocorticoids are not obliga-tory. Immunol Today 21:606–611

6. Zapata AG, Cooper EL (1990) The immune system:comparative histophysiology. In: The Thymus. JohnWiley, Chichester, pp 104–150

7. International Collaborative Immunotoxicity Study (ICI-CIS) Group Investigators (1998) Report of validationstudy of assessment of direct immunotoxicity in the rat.Toxicology 125:183–210

Thymus: A Mediator of T CellDevelopment and Potential Target ofToxicological Agents

Michael Laiosa

NIAID/NIHBethesda, MO 20897USA

Allen Silverstone

Upstate Medical University166 Irving Ave.Syracuse, NY 13210USA

Synonyms

T-cell development, thymocyte development, T-cellselection, thymocyte selection, thymocyte education,positive selection, negative selection, thymus, thymusatrophy, thymus involution

Definition

T-cell development is the process by which hemato-poietic progenitor cells from the bone marrow home to

the thymus and undergo a complex process of differ-entiation, proliferation and selection to become matureT-cells that will emigrate from the thymus to periph-eral lymphoid organs such as the spleen and lymphnodes. Additional maturation and differentiation intoT-helper (Th) type 1 and Th type 2 subsets occur in theperiphery and are discussed elsewhere.

CharacteristicsThe 3thymus is the central organ for T-cell develop-ment in the body, and the principle function of thethymus is to regulate T-cell recognition of self antigenspresented by the body to insure that useless or self-reactive T-cells do not mature. T-cell development ischaracterized by progenitor cells that originate in thefetal liver or bone marrow and enter the thymusthrough the blood stream (1). The thymocytes thenundergo a highly regulated process of differentiation,proliferation, selection, and maturation to become T-cells. The stages of murine thymocyte differentiationcan be distinguished by differentially expressed sur-face molecules stained with fluorochrome-labeledantibodies and detected using flow cytometry. Thethymocyte subpopulation that appears earliest is iden-tified by expression of the lymphoid homing receptorCD44 and cKit, the receptor for the stem cell factor,(CD44+CD25−, DN1) (1). Subsequently, the high af-finity interleukin receptor IL-2α (CD25) and the heatstable antigen (HSA, CD24) are upregulated and theproliferation rate of this population also increases(DN2) (1). Following expression of CD25, CD44 isdown modulated leading to the next stage of differen-tiation, CD44−CD25hi (DN3) (1). In the DN3 popula-tion, the αβ and γδ T-cell antigen receptor (TCR)lineages begin to diverge as recombination activatinggene products 1 and 2 (RAG1, RAG2) begin somaticgene rearrangement of the TCR β locus (1). Success-ful rearrangement and surface expression of a func-tional TCR β chain in a complex with the pre-Tαprotein results in a burst of proliferation and the gra-dual reduction of CD25 expression on the cell surface(DN4) (1). Subsequent to successful expression ofTCR β, rearrangement of TCR α begins and theCD8 and CD4 molecules are expressed on the cellsurface(1). It has been calculated that it takes 3–4 days for a DN3 cell to differentiate into the DPstage of T-cell development (1).Once TCRα rearrangement is complete, theCD4+CD8+ double positive (DP) thymocytes begin arigorous selection process by engaging their αβ TCRwith complexes of self peptides bound to major histo-compatibility complex (MHC) class I and II proteins(1), expressed by epithelial, myeloid, and dendriticantigen presenting cells (APCs) in the cortex of thethymus (2). The TCR-MHC interaction leads to one ofthree possible outcomes depending on the nature of

642 Thymus: A Mediator of T Cell Development and Potential Target of Toxicological A

the interaction. TCRs with no or weak affinity forMHC will die by neglect. In comparison, potentiallyself-reactive TCRs with too high or strong affinity forthe peptide MHC complex undergo negative selection.Only TCRs with the appropriate affinity for peptideMHC complexes will undergo maturation, CD4(class II MHC) or CD8 (class I MHC) lineage com-mitment and 3positive selection (1).The signal transduction that results in positive selec-tion begins with phosphorylation of the intracellularportion of the TCRζ chain by the src kinase Lck.Phosphorylation of TCRζ results in the subsequentrecruitment of Zap70, which becomes activated andphosphorylates the linker of activated T-cells (LAT).The phosphorylated LAT acts as a docking complex,which recruits and activates a number of moleculesinvolved in TCR signal transduction and calcium ion(Ca2+) flux (3). The generation of a Ca2+ flux has beenshown to depend on phospholipase C γ (PLCγ),which generates inositol-3-phosphate (IP3) and diacyl-glycerol (DAG) (1). IP3 is responsible for the increasein intracellular Ca2+ and leads to the activation of thecalcineurin pathway and the NFAT family of transcrip-tion factors (1). In contrast DAG is involved in acti-vating protein kinase C (PKC) family members andcan be a mediator in activation of the Ras pathway. InDP thymocytes it is thought that DAG activates theguanine nucleotide exchange factor RasGRP1 leadingto activation of the extracellular signal-related kinase(ERK) (1). ERK activation in thymocytes undergoingpositive selection is thought to be involved in activat-ing the early growth response-1 (EgR-1) nuclear tran-scription factor (1). The positively selected DP thymo-cytes then upregulate Bcl-2 and mature to becomeeither class II restricted (CD4+; T helper) or class Irestricted (CD8+; T cytotoxic) single positive thymo-cytes. Additional selection occurs in the medulla of thethymus before final maturation and emigration of theSP T-cells into the periphery (1).Although 3negative selection results in a profoundlydifferent outcome (cell death rather than maturation)many of the signaling pathways utilized are the sameor similar. Most current data on thymocyte selectionfavor a model where the affinity between a TCR andself peptide-MHC complexes determines whether athymocyte will be positively selected or deleted.High affinity interactions with TCR and self peptide-MHC may activate additional signaling pathways suchas the Jnk pathway, which ultimately lead to apoptosis(1). In comparison, TCRs with weak or no affinity forself peptide-MHC complexes will die by neglect in thethymus within 1–3 days (1). Only thymocytes posses-sing the appropriate affinity and duration of bindingbetween a TCR and self peptide-MHC complexes canbe positively selected (1).A number of toxicological agents have been identified

which can interrupt or inhibit various stages of T-celldevelopment, which ultimately leads to atrophy of thethymus. Agents that have been shown to cause thymicatrophy in vivo include corticosteroids, estrogens andestrogen-like compounds, polychlorinated biphenyls(PCBs), and polychlorinated dibenzodioxins and di-benzofurans (PCDD and PCDF). Representativeagents that are known to induce thymic atrophy andpossible mechanisms by which they can induce atro-phy are listed in Table 1 (4,5).Evidence of thymic atrophy after toxicant exposurehas a relatively strong correlation to predicting if anagent will be immunotoxic as defined by classic im-munotoxicity assays such as delayed-type hypersensi-tivity (DTH), and the sheep red blood cell (SRBC)challenge assay (6). However, linking immunotoxi-cant-induced defects in thymic development to defi-ciencies in a functional response has been a majorobstacle in the field of immunotoxicology. Relatingthymic atrophy to alterations of functional responseshave suffered from a lack of data and agreement on thetype of assays, kinetics, and dosing protocols to beused.

Relevance to HumansThe thymus has been shown to be essential for devel-opment of T-dependent immune responses. Indeed,patients with the rare 3DiGeorge syndrome wholack a thymus present with a severe immunodeficiencyassociated with a complete lack of T-cells. The Di-George T-deficiency can be completely restored bythe transplantation of an allogeneic thymus graft (7).The essential role for the thymus in T-cell develop-ment has been further appreciated in recent clinicalstudies. These studies show that despite the longstand-ing observation of 3thymus atrophy with increasingage, the adult thymus is fully capable of producing andselecting new T-cells following periods of systemic T-cell depletion. Following chemotherapy, production ofnew thymic-derived naive T-cells has been observed(7). Additionally, infection with HIV has been shownto cause a dramatic thymic pathology characterized bythymic atrophy and a block in T-cell development atthe CD3−CD4−CD8− stage of development. However,thymopoieis can be restored in some HIV patientsundergoing highly active antiretroviral therapy( 3HAART) (7). Finally, evidence of TCR gene rear-rangement in recent thymus emigrants has been ob-served in normal adults of at least 60 years of age (7).These data strongly support an active and dynamicrole for the thymus organ in mediating new T-cell de-velopment throughout an individual’s life.The effect of 3immunotoxicants as mediators of thy-mic atrophy in humans has been controversial anddifficult to assess for some time. The lack of consensuson whether a particular toxicant can cause thymic at-

Thymus: A Mediator of T Cell Development and Potential Target of Toxicological A 643

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rophy is due in part to the obvious ethical considera-tions with human studies. Moreover, the vast majorityof immunotoxicity assays that have been developedare in rodent models that possess inherent flawswhen attempting to determine dose, pharmacokinetic,and risk assessment comparison models to humans.The challenges of relating risk assessment models tohumans should be overcome in the future as immuno-toxicologists begin to develop nonhuman primatemodels, novel in vitro models and comparative 3tox-icogenomic studies to fill in the gaps in knowledgeabout particular toxicants as related to T-cell develop-ment and immunotoxicity (6,8,9).


Regulatory agencies in the USA have recently startedto stress the importance of understanding how immu-notoxicants affect the developing immune system inchildren. The need to understand the effects of immu-notoxicants in children is particularly important be-cause of the possibility that during the period whenthe immune system is most actively developing, it maybe especially sensitive to the effects of an immunotox-icant. Moreover, immunotoxicant exposure in childrenmay lead to more severe effects and/or a higher riskfor long-term deleterious outcomes when compared todoses determined for adults (9). Although there arecurrently limited data comparing adult and child re-

sponses to immunotoxicants on the developing im-mune system, several possibilities for differencesexist. An immunotoxicant may affect the developingimmune system of a child but not an adult. Further-more, an immunotoxicant may affect the developingimmune system of a child at a lower dose than in anadult (9).In an attempt to get the full picture about childhoodexposure to immunotoxicants and the effect of expo-sure on the developing immune system of children,several EPA sponsored workshops have listed theneed for expanding exposure studies in very younganimals as a high priority. These workshops includethe EPA sponsored workshop on endocrine disruptorsheld in 1995, and the EPA sponsored workshop by theRisk Science Institute of the International LifeSciences Institute held in 1996. More recently, theEPA added a recommendation to the two-generationreproductive study (OPPTS 870-3800), stating: for F1and F2 weanlings that are examined macroscopically,the following organs should be weighed for one ran-domly selected pup per sex per litter: brain, spleen andthymus (9). The recommendation to use thymus andspleen weights was made because numerous studieshave concluded that thymic and splenic weight maybe immunotoxicant predictors (6).In 2001 the EPA created a developmental immunotox-

Thymus: A Mediator of T Cell Development and Potential Target of Toxicological Agents. Table 1 Agentsknown to cause thymic atrophy and mechanism of atrophy induction1

Agent Mechanism

Androgens Loss of DP thymocytes; mediated by androgen receptor

Cisplatin Apoptosis in proliferating thymocytes

Cyclosporin A Prevents Ca++ mobilization; inhibits positive selection; delayednegative selection

Dexamethasone (and other corticosteroids) Apoptosis in DP thymocytes

Dibutyl and tributyltin Possible apoptosis; inhibition of proliferation of DN thymocytes

Diethylstilbestrol (DES), estradiol, estro-gens and estrogen-like chemicals

No evidence of apoptosis, possible effects on progenitors and cellcycle; estrogen receptor-mediated

Ethylene glycol monomethyl ether Reduction in DP thymocytes, but no evidence of apoptosisReduction of lymphocyte progenitor capacity

Ethanol Apoptosis; increase in CD4+ mature cells, loss of CD25+ DN cells;evidence of Ca++ increase and protein kinase C activation

Malnutrition, vitamin deficiency Increase of glucocorticoid levels; apoptosis of DP thymocytes

2,3,7,8, tetrachlorodibenzo-p-dioxin No evidence of apoptosis in vivo; inhibition of bone marrowprogenitors; inhibition of cell proliferation in thymic DN cells; alleffects mediated by the aryl hydrocarbon receptor

T-2 toxin and other mycotoxins Elimination of putative lymphocyte progenitor cells in fetal liver; noevidence of apoptosis induction.

1 Adapted from Luster et al. (4) and Silverstone (5)

644 Thymus: A Mediator of T Cell Development and Potential Target of Toxicological A

icology working group. The mission of the this groupis to determine:* the state of science to support the creation of a

guideline for developmental immunotoxicology* what should be included in such a guideline* how this guideline would be validated* when a developmental immunotoxicology guide-

line would be used (9).

Lastly, in 2003, the National Institute of Environmen-tal Health Sciences (NIEHS) and National Institute forOccupational Safety and Health (NIOSH) cosponsoreda consensus workshop on methods to evaluate devel-opmental immunotoxicity. This workshop made sev-eral recommendations for immunotoxicant screeningassays as well as assays that needed further validationand assays for research development (4). The recom-mended screening assays for developmental immuno-toxicants were the primary antibody response (T-de-pendent), delayed-type hypersensitivity response,complete blood count (CBC), and weights of thymus,spleen and lymph nodes. Assays that require addi-tional validation include phenotypic analyses, macro-phage function and natural killer cell activity. Finally,stem cell functional assays were listed as assays thatrequire additional research and development (4).

References

1. Starr TK, Jameson SC, Hogquist KA (2003) Positive andnegative selection of T cells. Ann Rev Immunol 21:139–176

2. Anderson G, Jenkinson EJ (2001) Lymphostromalinteractions in thymic development and function. NatRev Immunol 1:31–40

3. Germain RN, Stefanova I (1999) The dynamics of T cellreceptor signaling: complex orchestration and the keyroles of tempo and cooperation. Ann Rev Immunol17:467–522

4. Luster MI, Dean JH, Germolec DR (2003) Consensusworkshop on methods to evaluate developmental im-munotoxicity. Environ Health Persp 111:579–583

5. Silverstone AE (1997) T cell development. In: Sipes G,McQueen CA, Gandolfi AJ (eds) Comprehensive Toxi-cology, 1st edn. Elsevier Science, New York, pp 39 ff

6. Holladay SD, Blaylock BL (2002) The mouse as a modelfor developmental immunotoxicology. Hum Exp Toxicol21:525–531

7. Spits H (2002) Development of alpha-beta T cells in thehuman thymus. Nat Rev Immunol 2:760–772

8. Buse E, Habermann G, Osterburg I, Korte R, WeinbauerGF (2003) Reproductive/developmental toxicity andimmunotoxicity assessment in the nonhuman primatemodel. Toxicology 185:221–227

9. Holsapple MP (2003) Developmental immunotoxicitytesting: a review. Toxicology 185:193–203

Thymus Atrophy

Loss of thymocyte weight and cellularity after expo-sure to an immunotoxicant.

3Thymus: A Mediator of T-Cell Development andPotential Target of Toxicological Agents

Thymus-Dependent Antigen

Thymus-dependent antigens (TD) are protein antigenswhich only can induced an antibody response with thehelp of thymus-derived T helper cells. This T cell helpis also essential for the class switch observed duringTD immune responses.

3Idiotype Network

Thymus Gland

The thymus is a primary lymphoid organ, the site of T-cell development. It is situated in the anterior superiormediastenum, behind the breastbone. The organ, inparticular its epithelial cells and connective tissue pro-vide the microenvironment wherein thymocytes pro-liferate, rearrange their T-cell receptor genes, and un-dergo positive and negative selection. The thymusslowly atrophies after puberty, but can become fullyfunctional again in clinical situations like radiationtherapy and stem cell transplantation.

3Thymus



3Systemic Autoimmunity

Thymus Involution

3Thymus: A Mediator of T-Cell Development andPotential Target of Toxicological Agents

Tight Junctions

An intercellular junctional structure, typically found inepithelia and endothelia. In the tight junction the twomembranes of neighboring cells are brought into closeproximity through binding of specific transmembraneproteins. This results in a selectivity barrier that sealsthe apical lumen from the basolateral intercellular

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space and also establishes cellular polarity by prevent-ing membrane-linked molecules from freely diffusingbetween the apical and the basolateral cell surface.

3Cell Adhesion Molecules

Time-Resolve Fluorometry

An instrumental design to collect emission at a certaintime interval after the pulsed excitation and to improvethe detection sensitivity by means of a temporal rejec-tion of background.

3Cytotoxicity Assays

Tissue Factor

This cellular receptor for factor VII/VIIa is constitu-tively expressed on cells of the media and adventitia ofthe vessel wall. When it is exposed to plasma clottingfactors at sites of vascular injury it serves as a potent(extrinsic) cofactor for the activation of factor X. Tis-sue factor is also associated with platelets and micro-particles and is responsible for intravascular activationof blood clotting in the absence of tissue damage.

3Blood Coagulation

Tm Mouse

3Knockout, Genetic

TNF-α

3Tumor Necrosis Factor-α

Tolerance

Anke Kretz-Rommel

Principal ScientistAlexion Antibody TechnologiesSuite A, 3958 Sorronto Valley RdSan Diego, CA 92121USA

Synonyms

Immunological unresponsiveness

DefinitionThe primary function of the immune system is to pro-tect the host from foreign materials while at the sametime ensuring that no attack against self proteins oc-curs. Immunological tolerance is the absence of immu-nological responsiveness to specific antigens, encom-passing unresponsiveness to self antigens, but also tol-erance to therapeutics such as antibodies, recombinantproteins and conventional drugs. Breakdown of im-mune tolerance is defined by the appearance of T-cells or antibodies to self antigen or the therapeuticentity. The result may be autoimmune disease or aller-gic or anaphylactic reactions. Furthermore, an immuneresponse to a drug may reduce its efficacy.Immune tolerance is an active process at both theB cell and T-cell level, involving processes takingplace in central lymphoid organs (thymus and bonemarrow) and peripheral lymphoid organs (blood,spleen, lymph node, mucosal immune system). Theunderlying mechanisms are subject to a continuousdebate involving clonal deletion, anergy, regulatoryT cells and regulatory dendritic cells. In this chapterthese concepts will be outlined with reference to drugsaffecting various tolerance mechanisms, and the inter-ested reader is referred to more in depths reviews.

Characteristics of T Cell ToleranceCentral Mechanisms

T cells develop in the thymus. Recombination of genesegments creates the two chains that make up theT cell receptor (TCR) resulting in a large repertoireof receptor specificities. To ensure the export to theperiphery of T cells that recognize peptides in thecontext of self major histocompatiblity complex(MHC), but do not strongly react to self antigens,the cells have to undergo positive and negative selec-tion processes as outlined in Figure 1.Selection is a rigorous process that results in the deathof approximately 95% of T cells. T cells first have toundergo positive selection on self peptide presented inthe context of self MHC. Successful signaling throughthe TCR has been suggested to raise the threshold ofactivation of these T cells possibly through the produc-tion of negative regulators (1). If the T cells still can beactivated in a subsequent encounter of self peptidepresented by MHC the T cell will undergo clonal de-letion by apoptosis, a process termed negative selec-tion. This leaves only T cells to be exported to theperiphery with a threshold of activation that can notnormally achieved by self peptides. Interference withnegative selection in the thymus has been proposed asa mechanism for the induction of autoimmunity.TCDD and cyclosporine have been evoked to affectboth positive and negative selection processes. Thereactive metabolite of the antiarrhythmic procainamidehydroxylamine (PAHA) has been shown to interfere

646 Time-Resolve Fluorometry

with positive selection in the thymus, resulting in theexport to the periphery of autoreactive T cells and au-toantibody production similar to that observed in pa-tients with drug-induced lupus.

Peripheral Mechanisms

T cells leaving the thymus still might respond to selfantigens if the antigens are present in such high con-centration that they can bind to “weak” receptors or ifthey did not encounter the self peptide in the thymuswhich might be the case for certain tissue-specificantigens. A number of peripheral mechanisms cancontrol these potentially self-reactive cells (2) as sum-marized in Figure 2.

Lack of Costimulation

Activation of T cells not only requires interaction ofthe TCR with peptide presented by MHC on antigen-presenting cells (APC), but also a second signal (cost-imulation). Among the most important of these costi-

mulatory molecules are members of the B7 family,interacting with CD28 on the T cell. Ligation ofCD28 by either B7-1 or B7-2 lowers the thresholdof TCR signaling needed to induce T-cell activationand increases the effect of that signal by promotingT cell expansion and proliferation. Recently, additionalmembers of the B7-CD28 family involved in the de-velopment or maintenance of immune tolerance havebeen identified such as ICOS which is expressed byactivated T cells. Ligation of ICOSL by ICOS pro-longs T cell activation. If a T cell receives a signalthrough the TCR in the absence of costimulation,cells are unresponsive to subsequent stimulation bythe peptide in context of MHC in the presence ofcostimulation—a process termed anergy. While thisphenomenon has only been demonstrated in vitro, itrecently has been recognized that naive T cells (T cellsthat have not been stimulated before) in the peripheryrequire frequent interaction with peptide presented byMHC in order to survive. This has been suggested to

Tolerance. Figure 1 Central tolerance mechanisms. After migration from the bone marrow to the thymus, T cellsfirst undergo selection on self peptides presented by thymic epithelial cells. Cells productively interacting with thepresented peptide proceed to negative selection resulting in deletion of cells with high affinity for self peptide.TEC=thymic epithelial cell; APC=antigen presenting cell.

Tolerance 647

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be an important mechanism of peripheral tolerance,maintaining a high activation threshold of T cellswhich can only be overcome by foreign antigen.Drugs could potentially provide a “danger” signal tothe immune system resulting in upregulation of costi-mulatory molecules and activation of self-reactiveT cells. However, clearly not all drugs inducing cellstress or cell death result in an activation of the im-

mune system. Evidence is emerging though that somecompounds can alter dendritic cells resulting in upre-gulation of the costimulatory molecule CD86 or pro-voking migration of dendritic cells by upregulating theCCR7 receptor.

Failure to Encounter Self Antigens (Immune privilege)

Under normal conditions, the cells in nonlymphoid

Tolerance. Figure 2 Peripheral tolerance mechanisms. A: Only T cells with high affinity for the antigen presentedby antigen presenting cells (APC) will proliferate. Since thymic emigrants have been tuned to have a threshold ofactivation generally above that achieved by most self peptides, T cell interaction with self peptide presented bymajor histocompatability complex (MHC) does not result in proliferation. B: If a T cell sees antigen in the context ofMHC in the absence of costimulatory signals, anergy can be induced. The T cell is subsequently unresponsive tochallenge with the cognate antigen by APCs, even in the presence of costimulatory molecules. C: Death moleculessuch as fatty acid synthetase (FAS) and tumor necrosis factor (TNF) get upregulated in the course of a T cellresponse to limit proliferation and cytokine production. T cells involved in the response to antigen will undergoactivation-induced cell death (AICD) by apoptosis. D: A number of immunoreceptors downregulate the T cellresponse. Some of them are upregulated during the T cell response to limit it, and some of them are constitutivelyexpressed on tissues to prevent damage by T cells. E: Tolerogenic dendritic cells can induce Treg which control theresponse of other T cells.

648 Tolerance

organs throughout the body are not in contact withT cells and are thus sequestered from the immunesystem. This lowers the probability of a low affinityself-reactive T cell encountering a specific self antigen.Only in the presence of “danger” signals such as pro-vided by bacteria can T cells enter non-lymphoid or-gans. Certain tissues are particularly protected fromthe entry of T cells, such as the interior of the eye,brain and testes. Constitutive expression of immuno-suppressive receptors and cytokines ensures protectionof these organs from immune-mediated damage.

Receipt of Death Signals

An important mechanism of maintaining immune ho-meostasis is the downregulation of the immune re-sponse after activation. Activation of antigen-present-ing cells by bacteria or viruses results in upregulationof costimulatory molecules and production of proin-flammatory cytokines such as tumor necrosis factor(TNF)-α. Persistence of an inflammatory environmentincreases the risk of activating T cells by self peptidesby providing costimulation to these cells. Also, there isa risk of cross-reactivity of T cells activated by patho-gens with self antigen, because activated T cells re-quire less costimulation. Therefore, most activatedT cells ultimately undergo a process of programmeddeath or apoptosis. Apoptosis of activated cells (acti-vation-induced cell death, AICD) occurs by cytokinewithdrawal and by induction through fatty acid syn-thetase (FAS) and TNF-α. FAS acts on FAS ligandexpressed on activated T cells. These cells thereforecan kill themselves as well as activated B cells andmacrophages. Also, expression of other receptorsmediating immune suppression play an importantrole in downregulating the immune response as dis-cussed in the following section.

Immunosuppressive Receptors

The immune response can be terminated by upregula-tion of the T cell surface molecule CTLA-4. WhileCTLA-4 is present at very low levels on restingT cells, it is markedly upregulated after T cell activa-tion. Similar to the positive costimulatory moleculeCD28, CTLA-4 binds to B7.1 and B7.2. Due to itssubstantially higher affinity for these molecules,CTLA-4 outcompetes CD28, thereby transducing in-hibitory signals to the activated T cell. More inhibitorymolecules have recently been identified. PD-1 is ex-pressed on activated T cells, B cells and myeloid cells,and engagement by its ligands PD-L1 and PDL-2 in-hibits T cell proliferation and cytokine production. Ex-pression of immunosuppressive receptors on nonlym-phoid organs is another safeguard mechanism againstself attack of T cells.

Regulatory Cells and Cytokine Milieu

A minor population of T cells known as regulatoryT cells (Treg) suppresses the proliferative responseand production of inflammatory cytokines of otherT cells. They may constitute a specialized T cell subsetto reduce the activity of autoreactive T cells. Treg con-stitutively express CTLA-4 and secrete transforminggrowth factor(TGF)-β and interleukin(IL)-10. Me-chanisms of action are still under debate, but theyseem to require direct cell-cell contact.In addition to regulatory T cells, dendritic cells andmacrophages play a major role in immune tolerance.The functional activities of dendritic cells are mainlydependent on their state of activation and differentia-tion. Terminally differentiated mature dendritic cellscan efficiently induce the development of T effectorcells, whereas immature dendritic cells are involved inmaintenance of peripheral tolerance. The means bywhich immature dendritic cells maintain peripheraltolerance are not entirely clear, however, their func-tions include the induction of anergic T cells, T cellswith regulatory properties as well as the generation ofT cells that secrete immunomodulatory cytokines. De-pending on the cytokines produced by the macro-phage/dendritic cell, the immune response can besteered towards a Th1 or Th2 response. Th1 cells pro-duce IFN-γ, IL-2 and TNF-α and regulate classicaldelayed (type IV) hypersensitivity. Th2 cells secreteIL-4, IL-5, IL-6 and IL-10 and participate in immedi-ate (type I) hypersensitivity reactions and B cell anti-body-mediated immunity. The effect of drugs on cy-tokine production and the importance of the cytokinemilieu resulting in drug-induced autoimmunity arebeing studied extensively.

Characteristics: B cell Tolerance

Similar to T cells, B cells are constantly being toler-ized to self antigens. For a thorough discussion ofB cell tolerance the reader might refer to Jacqueminet al. (3).

Central Mechanisms

B cells mature and undergo selection on self peptidesin the bone marrow. A large population of B cells withdifferent specificities is created by genetic recombina-tion within the immunoglobulin locus generating abroad range of heavy- and light-chain sequences thatrearrange to form a B cell receptor (BCR). If the im-mature B cell encounters extracellular antigen capableof crosslinking its BCR, a signal is created that willblock further development of this autoreactive cell.The B cell will initiate the receptor editing processto produce BCR with new antigen specificities. If itcannot alter its BCR effectively, the immature B cellwill be deleted by apoptosis. Some autoreactive

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B cells escape deletion and enter the peripheral circu-lation in an anergic state.

Peripheral Mechanisms

After recognition and uptake of antigen in the periph-ery, these partially activated B cells migrate throughthe lymphoid tissue. If an activated B cell encounters aT cell that has been activated by the same antigen,antibodies against that antigen are produced. B cellscannot respond to most antigens without receivinghelp from T helper cells. Therefore, ensuring self tol-erance of T cells is an important mechanism of keep-ing B cells from producing autoantibodies. However,drugs affecting B cell tolerance can ultimately result inautoimmunity when the individual has other predis-posing factors, as might be the case for pristane.

Additional Mechanism for Drugs to BreakImmune Tolerance

The most common hypothesis of how drugs result inimmune stimulation is the formation of drug-proteinconjugates by reactive drug metabolites with self anti-gens. The resulting haptens might be recognized asforeign by the immune system. Although formationof haptens has been demonstrated for a number ofdrugs associated with idiosyncratic immune adversereactions (e.g. phenytoin, carbamazepine, halothane,tielinic acid, procainamide and diclofenac) these ad-ducts are not a predictive factor for adverse immunereactions indicating that additional factors are requiredto induce the immune response. It has been demon-strated that binding of halothane to CF3CO proteinsmimics very closely the structure of the E2 subunitproteins of the 2-oxoacid dehydrogenase complexesand protein X—autoantigens associated with halo-thane hepatitis. Furthermore, binding of drugs to pro-tein can alter their cleavage and presentation after celldeath. Exposure of macrophages to mercuric chloridehas been show to alter fibrillarin processing, resultingin the appearance of self epitopes not normally en-countered by the immune system.In addition to covalent drug binding to proteins, non-covalent interactions of drugs such as sulfamethox-azole with MHC-peptide complexes have been impli-cated in immunological adverse reactions.While disruption of immune tolerance by classicalchemical drugs leaves many unanswered questions,immune responses after administration of bioengi-neered drugs is far more straightforward. The impor-tance of antibodies in therapeutics gains increasingrecognition. Often, these antibodies are of mouse ori-gin and certain residues are recognized as foreign bythe human immune system. Engineering methodsknown as “humanization” and pegylation decreasethe risk of an immune response against the therapeutic.

Preclinical Relevance

Adverse drug reactions affecting immune tolerance aredifficult to address in the preclinical setting. However,a number of assays have been developed to address thepotential of drugs to sensitize the immune system,such as the popliteal lymph node assay that assessesthe effects of drugs on macrophages, or assays lookingfor altered cytokine profiles. Few animal models de-monstrating chemically induced autoimmunity areavailable, but are specific for the compound used.As far as immunogenicity of biotherapeutics is con-cerned, some animal models have proved to be useful.For example, transgenic mice were developed to pro-duce and secrete human tissue plasminogen activatorto which they developed immune tolerance. Thesemice were capable of producing antibodies to a formof human tissue plasminogen activator that had beenmodified by a single amino acid substitution. Further-more, nonhuman primates have been used successfullyin predicting the relative immunogenicity of differentforms of human growth hormone. Also, computermodeling methods are used in predicting the immuno-genicity of proteins.

Relevance to Humans

Adverse drug reactions account for 2%–5% of all hos-pital admissions, a portion of which is based on im-mune-mediated reactions. With more than 80 recom-binant proteins in clinical use and more than 400 ther-apeutic antibodies in clinical trials, immune toleranceto these proteins is a major issue and predicting im-munogenicity is crucial (4).


Regulatory issues for drug-induced autoimmunity andallergy are covered in their respective chapters. Forclinical trials of recombinant proteins, patients arescreened for the development of antidrug antibodies.

References

1. Grossman Z, Singer A (1996) Tuning of activationthresholds explains flexibility in the selection and devel-opment of T cells in the thymus. Proc Natl Acad Sci USA93:14747–14752

2. Sharpe AH, Freeman GJ (2002) The B7-CD28 super-family. Nat Rev Immunol 2:116–126

3. Jacquemin MG, Vanzieleghem B, Saint-Remy JM (2001)Mechanisms of B-cell tolerance. Adv Exp Med Biol489:99–108

4. Pendley C, Schantz A, Wagner C (2003) Immunogenicityof therapeutic monoclonal antibodies. Curr Opin MolTher 2:172–179

650 Tolerance

Tolerance and the Immune System

Unresponsiveness to antigenic stimulation that iseither mediated by genetics, or is acquired by specialconditions of antigenic exposure. The immune systemhas established several mechanisms that prevent im-mune reactions against self antigens. Of central impor-tance is the tolerance of the immune regulatory helperT cells. Activation of helper T cells can be controlledby tolerance induction in the thymus, by sequestrationof antigens in immune privileged sites (brain, testis,cornea) and by active suppression of immune re-sponses by regulatory T cells.

3Antigen Presentation via MHC Class II Molecules

3Graft-Versus-Host Reaction

3Autoantigens

3Autoimmune Disease, Animal Models

3Antinuclear Antibodies

3Lymphocytes


Toll-Like Receptors

A family of receptors expressed by cells of the innateimmune system and directed against conserved struc-tures present on many micro-organisms. Ten membersof this receptor family are present in humans (e.g.TLR4 specific for lipopolysaccharide; TLR2 for pep-tidoglycan; TLR5 for flagellin). They are named afterthe Drosophila protein Toll which is involved in theantibacterial defense of the fruit fly.

3B Cell Maturation and Immunological Memory

Toxic Epidermal Necrolysis (TEN)

Toxic epidermal necrolysis (TEN) represents the mostserious extreme of the febrile mucocutaneous syn-drome in which there is a full-thickness sloughing ofthe epidermis. According to the criteria, TEN is de-fined as detachment affecting about 30% of the bodysurface area. Stevens-Johnson syndrome is similar toTEN in terms of the histopathology and the responsi-ble drugs, indicating that these two conditions are partof the same spectrum. Fas-Fas L interactions appear tobe involved in the epidermal necrolysis.

3Drugs, Allergy to

Toxic Oil Syndrome (TOS)

An illness associated with the ingestion of adulteratedrapeseed oil in Spain in 1981. The most distinctivelesion is a non-necrotizing vasculitis involving differ-ent types and sizes of blood vessels in every organ.


Toxicogenetics

The genetic basis for individual differences in suscep-tibility to toxicity, with single nucleotide polymorph-isms (SNPs) being the prime source of variability inthe genome.

3Toxicogenomics (Microarray Technology)

Toxicogenomic Studies

Studies in toxicology which screen for global changesin gene expression following exposure to a toxicolo-gical agent.


Toxicogenomics

The measurement of altered gene expression upon ex-posure to a compound or drug, thereby identifying thetoxicant and characterising its mechanism of action.

3Toxicogenomics (Microarray Technology)

Toxicogenomics (MicroarrayTechnology)

Rob J Vandebriel

Laboratory for Toxicology, Pathology and GeneticsNational Institute for Public Health and theEnvironment3720 BA BilthovenThe Netherlands

Synonyms

Gene profiling, expression profiling, global gene ex-pression analysis

Toxicogenomics (Microarray Technology) 651

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Definition

3Microarray technology is the simultaneous indivi-dual measurement of the mRNA expression level ofthousands of genes in a given sample by means ofhybridization. 3Toxicogenomics is the measurementof altered gene expression upon exposure to a com-pound or drug, thereby identifying the toxicant andcharacterizing its mechanism of action.

Characteristics

Although individual differences exist, the basic prin-ciple of microarray technology is the same for differ-ent platforms (1). The term platforms means types ofarrays or array suppliers, in the latter case combinedwith dedicated hardware and software. First, per gene,a single probe or a few different probes are generated,using either polymerase chain reaction(PCR)-ampli-fied complementary DNA (cDNA), or syntheticDNA segments (oligonucleotides or oligos) devisedon the basis of these cDNA sequences. Usually theyare spotted onto a glass surface in a regular array. Thisprocess is called spotting or arraying, and requiresdedicated machinery. Some companies manufactureoligos in situ, either using photolithography (Affyme-trix) or chemical coupling (Agilent).Several options exist to obtain arrays:* ready-made arrays (e.g. Affymetrix, Agilent)* custom-made arrays (e.g. Affymetrix, Agilent)* in-house spotting of a PCR-amplified clone collec-

tion (e.g. Invitrogen) or of an oligo collection(MWG, Operon, Sigma).

Other manufacturers of ready-made arrays include Op-eron, MWG, and Phase-1, but this list is by no meansexhaustive.A clone collection is a collection of bacteria, eachcontaining a plasmid consisting of a different cDNAinsert. Care has to be taken that the individual clonesindeed contain the correct insert; verifying clone setsby sequencing the inserts is not uncommon. Second,RNA or mRNA is isolated from cells or tissues andcDNA is synthesized. This cDNA is labeled using afluorescent label, either during or after synthesis. Thelabeled cDNAs are then hybridized to the array. TheAffymetrix platform uses a single labeled cDNA (Cy3)per hybridization, whereas other platforms rely on twolabeled cDNAs (Cy3 and Cy5; most often test andcontrol). The array is then read using a scanner (withfitted laser (s)) that measures for each spot the fluores-cence intensity. These data are then transferred to apersonal computer. This process is outlined in Figure 1.

During and after this process a number of controlshave to be performed to assure that the results obtainedare correct. For the arrays these controls include theshape of the spots and the amount of DNA spotted (e.

g. by hybridization of labeled random hexamers).After hybridization these controls include a similaraverage staining intensity over the entire array, andplotting the intensity ratio of both labels against theintensity of the label for the control sample. This ratioshould be independent of the intensity for most of thegenes interrogated. To exclude artifacts caused by dif-ferential incorporation of the two labels into thecDNAs a dye swab is useful. If replicate samples aretested, statistics can be performed. Ratios of test vscontrol of > 2 are generally considered significant. Ifseveral time points, dose groups, or organs are ana-lyzed, more advanced statistics can be done, such ascluster analysis and/or principal component analysis(2). To this end several algorithms have been written,most of them being freely available on the internet.Commercial software packages have the advantageof easier data handling, compared to the tedious pro-cess of uploading data-sets to algorithms on the web(see Baxevanis and Francis Ouelette for a primer onthe subject) (3).The number of genes to be analyzed is of interest.Obviously, for mechanistic studies as well as for seed-ing databases that are ultimately aimed at identifyingtoxic profiles of compounds, the number of genesshould be maximal, nowadays meaning virtually allgenes. With statistics aiding in the process of geneselection, signatures of toxicity (such as peroxisomeproliferators) or pathology (such as liver necrosis) mayeventually be addressed by interrogating a small num-ber of genes.

Preclinical RelevanceA first important issue of toxicogenomics is to estab-lish specific types of toxicity, or even compounds onthe basis of signature expression profiles. A proof-of-principle approach to obtain such signature profilesproved to be successful (4,5). A first step towardspreclinical relevance is to obtain a database consistingof gene profiles for a range of model compounds.Since studies aimed at seeding such a database areusually divided between different laboratories andthe outcome has to be useful also for laboratories out-side the study group, care has to be taken that resultsfrom these laboratories can be compared, or used backand forth. With the current state of technology, variousmethodologies and platforms exist for assessing geneexpression, making it difficult to compare and compiledata across laboratories.An important initiative in this respect is the “minimuminformation about a microarray experiment”(MIAME) document (6), produced by the microarraygene expression database (MGED) society (http://www.mged.org). This set of guidelines is in the pro-cess of extension for toxicogenomics (MIAME/Tox),aiming to define the core that is common to most

652 Toxicogenomics (Microarray Technology)

toxicogenomic experiments. The major objective ofMIAME/Tox is to guide the development of toxicoge-nomics databases and data management software. Thedraft document can be found at http://hesi.ilsi.org. Ef-forts to build international public toxicogenomicsdatabases are underway at the National Center forToxicogenomics, National Institute of EnvironmentalHealth Sciences, USA (http://www.niehs.nih.gov/nct)and at the EMBL European Bioinformatics Institute(http://www.ebi.ac.uk/microarray/index.html) in con-junction with the International Life Sciences InstituteHealth and Environmental Sciences Institute (http://hesi.ilsi.org). This database will be made public inlate 2003 or early 2004.A provisional conclusion from experiments conductedso far is that multiple sources of variability exist, in-cluding expected sources of biological variability, iso-lation and labeling of mRNA samples, hardware andsoftware settings, microarray lot numbers and genecoverage, and annotation. Nevertheless, the gene ex-pression profiles relating to biological pathways arerobust enough to allow insight into mechanism, stronginformation on topographic specificity is provided,dose-dependent changes are observed, and concernsof over sensitivity may be unfounded (http://hesi.ilsi.org).

Relevance to Humans

A second important issue of toxicogenomics is thegenetic basis for individual differences in susceptibil-

ity to toxicity. Much of the variability in the genomestems from single nucleotide polymorphisms or SNPs,that occur roughly every 1000 nucleotides. A mapdescribing over 1.4 million SNPs (7) is available(http://snp.cshl.org). The next step is then to find anassociation of a particular SNP and a disease trait.Generally, two approaches can be taken to find suchassociations: one is a candidate gene approach, wheregenes in key biochemical pathways are investigatedfor SNPs, and in the second approach SNPs and there-by target genes are identified by whole genome ap-proaches. Mixed approaches can of course also betaken. An example of a successful candidate gene ap-proach is the SNP mapping of the hypersensitivity re-sponse (HSR) to the drug abacavir. Over 100 SNPswere tested on the basis of candidate genes. Poly-morphisms from two of the candidate genes (tumornecrosis factor(TNF)-α and human leukocyte antigen(HLA)-B57) were found to be highly associated withthe hypersensitivity response to abacavir (8).Similar to gene profiling, creating a database that de-scribes associations between SNPs and disease is animportant goal. Using high-density SNP mapping itshould be feasible to study the genetic basis for severalcommon diseases simultaneously. For drug adverseeffects this will surely be more difficult since onlyfew patients with a certain drug prescribed will showadverse effects.A recent development comes from the finding that thehuman genome can be parsed into haplotype blocks,

Toxicogenomics (Microarray Technology). Figure 1 Schematic illustration of microarray analysis. In thisparticular example PCR amplified cDNAs are dotted.

Toxicogenomics (Microarray Technology) 653

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being regions over which there is little evidence forhistorical recombination and within which only a fewhaplotypes are observed (9). Markers for these haplo-type blocks are now available, which makes it possibleto identify the genetic control of responses to toxicantswithout the necessity to identify the specific SNP re-sponsible.


Regulations that rely on genomics are not yet in placebut there is little doubt that within the next 5–10 yearsgene expression profiles will be used for safety as wellas efficacy assessment. This requires a firm databaseof expression profiles that can be directly related towell characterized toxicological and pathological end-points.Second, risk assessment has traditionally been per-formed across whole populations with widely varyingresponses. The goal is that by genetically identifyingsensitive subpopulations, the accuracy of risk assess-ment can be improved. Possibly, this may eventuallylead to personalized risk profiles.

References

1. Duggan DJ, Bittner M, Chen Y, Meltzer P, Trent JM(1999) Expression profiling using cDNA microarrays.Nature Genet 21S:10–14

2. Eisen MB, Spellman PT, Brown PO, Botstein D (1998)Cluster analysis and display of genome-wide expressionanalysis. Proc Natl Acad Sci USA 95:14863–14868

3. Baxevanis AD, Francis Ouelette BF (eds) (2001)Bioinformatics. John Wiley & Sons, New York

4. Hamadeh HK, Bushel PB, Jayadev S et al. (2002) Geneexpression analysis reveals chemical-specific profiles.Tox Sci 67:219–231

5. Hamadeh HK, Bushel PB, Jayadev S et al. (2002)Prediction of compound signature using high densitygene expression profiling. Tox Sci 67:232–240

6. Brazma A, Hingamp P, Quackenbush J et al. (2001)Minimum information about a microarray experiment(MIAME)—toward standards for microarray data. NatureGenet 29:365–371

7. Sachidanandam R, Weissman D, Schmidt SC et al. (2001)A map of human genome sequence variation containing1.42 million single nucleotide polymorphisms. Nature409:928–933

8. Roses AD (2002) Genome-based pharmacogenetics andthe pharmaceutical industry. Nature Rev Drug Disc1:541–549

9. Gabriel SB, Schaffner SF, Nguyen H et al. (2002) Thestructure of haplotype blocks in the human genome.Science 296:2225–2229

TR1 Cells

3Suppressor Cells

Trace Metals

Those metals commonly found in minute amounts inthe organism.


Trace Metals and the Immune System

Judith T Zelikoff

Depart. of Environmental MedicineNew York University School of Medicine57 Old Forge RoadTuxedo, NY 10987-5007USA

Synonyms

CD4+, T helper lymphocyte, CD4+/CD8−, T helperlymphocyte, CD8+, T suppressor lymphocyte,COPD, chronic obstructive pulmonary disease, asth-ma, bronchitis, emphysema.

Definition

Trace metals are normally present in minute quantitiesin the body. Many of them are also transition elements,essential for life due to their ability to control meta-bolic and signaling functions, such as zinc (Zn), man-ganese (Mn), and copper (copper). However, thesesame essential metals can also be toxic because oftheir ability to evade established controls for cellularuptake, transport, and compartmentalization. Alumi-num (Al) is a toxic trace element, unavoidable bythe general population because of its widespread en-vironmental distribution. The immunotoxicity of tracemetals other than Al, copper , Mn, and zinc can befound in a number of review articles (1–3).

Molecular Characteristics

Aluminum

Aluminum is the third most prevalent element in theEarth's crust. It is an A-type metal, or hard acid, thatstrongly prefers oxygen-donor ligands; hydroxide, ci-trate, phosphate, and nucleoside phosphate groups areprobably the most important low-molecular-mass bio-ligands for the predominant trivalent cation (Al3+). Italso binds readily to the two high-affinity iron-bindingsites of the serum transport protein, 3transferrin (TF).There is a wide variation in the ability of differentligands to solubilize and transport the Al3+ ion to crit-ical target sites.

Copper

Copper is a Group II (or IB) element, the third most

654 TR1 Cells

abundant transition metal found in living things. Itexists in one of two stable oxidation states: as cuprous(Cu1+) and cupric (Cu2+) ions. Consequently, its bio-logical chemistry is dominated by participation inredox reactions. Copper is necessary in the diet foriron utilization and as a cofactor in enzymes associatedwith oxidative metabolism. It is transported in serumbound initially to albumin and later more firmly to α-ceruloplasmin where it is exchanged in the cupricform; normal copper serum level is 120–145 μg/l.At elevated levels, copper is toxic to cells, presumablyby binding indiscriminately to thiol moieties or bycatalyzing a Fenton-type reaction to produce reactivehydroxyl radicals. Binding of copper by biologicalligands such as small peptides, large proteins, and en-zymes is required to minimize potential deleteriouseffects. Most stored copper is usually bound to metal-lotheinein (MT), a ubiquitous class of proteins that iswell suited to the role of metal sequestration.

Manganese

Manganese is the only Group VIIB element com-monly found in biological environments. Althoughthe inorganic chemistry of manganese displays arange of stable oxidation states, its biological chemis-try is dominated by the divalent form (Mn2+). BecauseMn2+ is very similar in size and charge density tomagnesium (Mg2+) and Zn2+ and also prefers to as-sume thetrahedryl and octahedral geometric structures,Mn2+ can replace Mg2+ in the enzyme pyruvate car-boxylase and Zn2+ in superoxide dismutase (SOD)with only negligible effects on enzyme activities.

Zinc

Zinc is found in large quantities in the vertebrate body(second only to iron); it is the first member ofGroup IIB elements and forms stable complexeswith sulfur, phosphate, and carbon atoms. Biologicalcomplexes contain zinc only in the divalent oxidationstate (Zn2+). Since Zn2+ is the only stable oxidationstate of the metal, it does not play a redox-active rolein biological processes. However, Zn2+ can activelyparticipate in enzymatic reactions as a Lewis acid oras a structural cofactor. Zinc is part of, or a cofactorfor,such enzymes as carbonic anhydrase, carboxypepti-dase, SOD, lactate dehydrogenase, phosphatase, andglutamate dehydrogenase. Zinc also displays a struc-tural role in biological systems, as exemplified by itsrole in maintaining the integrity of zinc finger tran-scription factors that bind to DNA and regulate thetranscription of genetic information.

Relevance to HumansAluminum

While some daily exposure to aluminum is unavoid-

able, inhalation by the general population is usuallyconsidered negligible (i.e. 0.14 mg aluminum dust perday). However, smelters, miners, welders and otherworkers involved in various metal industries areoften acutely exposed to localized atmospheres con-taining 2–4 mg/m3 of aluminum, resulting in time-weighted-average (TWA) intakes of > 23 mg per 8-hour shift. Increases in pneumonia, bronchitis, asthma,pneumoconiosis, lung cancers, and pulmonary fibrosishave been described in occupationally exposed work-ers. In addition, there is little doubt that aluminum cancause encephalopathy, osteopathies, and anemia inkidney dialysis patients. Although early studies set100 μg/l plasma as the level of aluminum belowwhich neurotoxicity failed to occur, recent studieshave demonstrated subtle neurocognitive and/ or psy-chomotor effects, as well as EEG abnormalities in di-alysis patients expressed at levels well below thislimit. Infants are a particularly susceptible subgroupfor aluminum toxicity partly due to their rapidly grow-ing and immature brain and skeleton and their devel-oping blood-brain barrier; preterm infants are gener-ally recognized to be at risk for aluminum loading dueto their immature kidney function. While the referencerange for blood aluminum levels in healthy individualsis < 10 µg/l, studies in infants have demonstrated plas-ma aluminum levels > 50 μg/l after oral intake ofaluminum-containing antacids.

Copper

As an 3essential element, copper promotes iron ab-sorption from the gastrointestinal system, it is involvedin the transport of iron from tissues into plasma, ithelps maintain myelin in the nervous system, it isnecessary for hemoglobin synthesis, and it is impor-tant in the formation of bone and brain tissue. Apartfrom occupational exposure, daily copper intakeaverages ∼ 0.02 mg. The fine balance required forcopper in humans is evident in genetically inheritedinborn errors of copper metabolism. For example, inWilson's disease there is failure to excrete copper fromthe liver to the bile, resulting in copper overload in theliver, brain, kidneys, and cornea; and in Menkes dis-ease, which is characterized by severe copper deficien-cy due to an error in copper transport from the intes-tines. Copper, usually in the form of cuprous oxideand cupric hydroxide (which converts to cupricoxide), is generally encountered in high concentrationsin the air of metallurgical processing plants, iron andsteel mills, and around coal-burning power plants. Incontrast to airborne copper concentrations in rural/sub-urban areas that average 0.01–0.26 μg/m3, particulatecopper levels in workplace sites be 50–900 μg/m3.Inhalation of such levels can result in an immunolog-ically-based condition called “copper fever”.

Trace Metals and the Immune System 655

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Manganese

Manganese, an essential trace element for all livingorganisms, is necessary for bone formation, cholester-ol and fatty acids synthesis, and as a dissociable co-factor for several enzymes including SOD. Despite itsessentialness, the toxic effects of manganese are wellknown, particularly those associated with the nervoussystem (4). Manganese is widely employed in manyindustries: in alloy steel manufacture for deoxidationand to promote hardenability; in the electric industryfor production of dry cells; in the chemical industry,where they are used as oxidants, for the manufactureof fertilizers, paints and varnishes, and in the produc-tion of glass and glazes (3). Apart from the directrelease of manganese into the air by several types ofmining industries and alloy and steel production facil-ities, manganese is introduced into the ambient envi-ronment by the combustion of manganese-containingfossil fuels (used as anti-knock additives and combus-tion improvers). Manganese (whose toxicity in manycases depends upon compound solubility) has beenfound at measurable levels in the majority of sus-pended particulate matter (including coal flyash) inurban environments. While air levels of manganesein many metropolitan areas containing steel or alloyplants can range from 0.5–3.3 μg/m3, the majorityhave levels ≤ 0.1 μg/m3; average air levels in the ab-sence of any contributing point sources are in therange 0.03–0.07 μg/m3. Alternatively, occupationalairborne levels of manganese are usually in the range1–≥ 100 μg/m3 (although levels as high as 1 mg/m3

have been measured); workplace permissible exposurelimits (PEL) of 300 (TWA) and 500 μg/m3 have beenrecommended by the World Health Organization andOSHA (Occupation Safety and Health Association),respectively.

Zinc

Zinc is ubiquitous in the environment and present inmost foodstuffs, water, and air. It is a nutritionallyessential element that serves as a cofactor for morethan 70 metalloenzymes. Daily dietary intake of zincis usually 12–15 mg/day and ~ 20%–30% of ingestedzinc is absorbed; zinc deficiency results in a widespectrum of clinical effects depending upon age,stage of development and deficiencies of related me-tals (i.e. zinc deficiency can exacerbate impaired cop-per nutrition and exacerbate cadmium and lead toxic-ity). Airborne concentrations of zinc are usually < 1μg/m3, with the majority of zinc being derived fromautomobile exhaust, soil erosion, and local commer-cial, industrial or construction activities. In urbanareas, atmospheric zinc concentrations are in therange 0.02–0.50 μg/m3; rural air contains 0.01–0.06μg/m3. Because zinc also contaminates certain work-place environments, national guidelines of 1.0 mg, 5–

10 mg, and 0.1 mg/m3 have been established for sol-uble zinc, insoluble zinc oxide, and carcinogenic zincchromate, respectively.

Putative Interactions with the Immune SystemAluminum

Although limited in number, immunotoxicologic stu-dies using a variety of animal models have demonstra-ted that injection of soluble aluminum compounds in-creases mononuclear cell mitotic index; injection ofthe metal or insoluble aluminum agents alter mono-cyte/macrophage numbers and immune function (1).Dietary exposure of rodents to soluble aluminum re-duces cytokine production, T helper (Th) andT suppressor (Ts) cell numbers, and host resistanceto Listeria monocytogenes infection. Repeated inhala-tion exposure of rabbits and hamsters to soluble alu-minum increases lung immune cell numbers; similareffects were not seen in aluminum-exposed workers.Effects of inhaled aluminum on host resistance areinconsistent, showing decreased resistance to subse-quent bacterial challenge in some studies and no effectin others. Differences between the studies are thoughtto be due to intratracheal versus inhalation exposureroutes. In vitro studies employing soluble aluminumsalts demonstrate a range of effects on immune cellsderived from a variety of animal species includinghumans. For example, aluminum chloride treatmentof rat alveolar macrophage reduced reactive oxygenintermediate production.

Copper

Much the same as for manganese and zinc, studies ofcopper immunotoxicity are complicated by the factthat copper is essential to maintenance of immuno-competence and, thus, most immunotoxicity occursas a result of copper insufficiency. Splenomegalyand thymic atrophy are consistent findings in cop-per-deficient mice. Alterations in antibody responseand B-lymphocyte function are also well documentedwith experimentally induced copper deficiency. Incontrast, serum antibody levels in humans with nutri-tional or genetic copper-deficiency are reported to benormal. B-lymphocytes are increased in number incopper-deficient animals, but they respond poorly tomitogen stimulation. Although the effects of copperdeficiency on T-lymphocyte populations are well char-acterized, the overall effect on cell-mediated immunityis unclear. Except in female rats, 3CD4+ and 3CD8+

subsets are decreased in the peripheral blood andspleen of copper-deficient rodents. Though no gendereffect has been observed in mice, the immune systemof male rats appears more susceptible to copper defi-ciency than that of females. Clinical studies involvinghealthy men on low copper diets fail to support theanimal studies with respect to circulating T-lympho-

656 Trace Metals and the Immune System

cytes and CD4+ and CD8+ subsets. While effects ofcopper-deficiency on innate immunity are inconclu-sive in animal studies, reduced neutrophil numbersand functionality are well defined in clinical studies.The most consistent immune defect associated withcopper deficiency in epidemiologic, clinical, and tox-icological studies is impaired host resistance due pri-marily to suppressed antibody-mediated responses andphagocyte antimicrobicidal activities (2).

Manganese

While relatively few studies have investigated the ef-fects of manganese on the immune system, immuno-toxicity appears dependent (like many of the othermetals discussed herein) upon compound solubility.Immune responses of the lung appear particularly sen-sitive to the immunomodulating effects of manganese.Inhalation of insoluble manganese compounds reducesthe ability of the lungs to resist and clear subsequentbacterial/viral infections and exacerbates ongoing viralinfections (3). Inhalation studies examining the effectsof soluble manganese reveal little effect on lung im-mune cell-related functionality. In contrast, studieswherein rabbit alveolar 3macrophages were exposedin vitro to manganese chloride demonstrated decreasedcell viability and number, increased incidence of celllysis, and reduced phagocytic activity.

Zinc

Zinc deficiency (like copper) can impair humoral andcell-mediated host immunocompetence. Zinc-deficientchildren and laboratory animals consistently presentwith 3thymic hypoplasia; oral administration of zincsupplements appear to reverse this effect. In zinc-defi-cient animals, secondary antibody responses to T-de-pendent antigens are suppressed in conjunction withaccelerated thymic hypoplasia and a decreased numberof CD4+ cells. Administration of zinc to immunosup-pressed human populations appears to increase num-bers of CD4+ and CD8+ thymocytes which, in turn,give rise to increased numbers of Th-cells importantfor the activation of cytotoxic T- and B-lymphocytes.In contrast, zinc suppresses concanavalin A-inducedT lymphocyte proliferation by in vitro-exposedhuman immune cells (2) and compromises pulmonaryhost resistance against bacterial infection (5); suppres-sive effects of inhaled zinc on pulmonary antimicro-bial activity are most likely due to zinc-induced reduc-tions in macrophage phagocytic activity. While themain immunological effect of occupational zinc expo-sure is metal fume fever, inhalation of particulate zincby occupationally exposed workers also alters pyro-genic, chemotactic, and anti-inflammatory cytokines.While zinc appears to play an important regulatoryrole in membrane-associated events of certain nonspe-

cific immune cell types, effects of zinc on innate im-munity are conflicting.

References1. Zelikoff JT, Cohen MD (1997) Metal immunotoxicology.

In: Massaro EJ (ed) Handbook of Human Toxicology.CRC Press, New York, pp 811–852

2. Omara FO, Brousseau P, Blakley BR, Fournier M (1998)Iron, zinc, and copper. In: Zelikoff JT, Thomas PT (eds)Immunotoxicology of Environmental and OccupationalMetals. Taylor and Francis, London, pp 231–262

3. Cohen MD (2000) Other metals: aluminum, copper,manganese, selenium, vanadium, and zinc. In: Cohen M,Zelikoff JT, Schlesinger RB (eds) Pulmonary Immuno-toxicology. Kluwer, Boston, pp 267–299

4. Inoue N, Makita Y (1996) Neurological aspects of humanexposures to manganese. In: Chang LW (ed) Toxicologyof Metals. CRC Lewis, New York, pp 415–421

5. Zelikoff JT, Chen LC, Cohen MD et al. (2003) Effects ofinhaled ambient particulate matter (PM) on pulmonaryanti-microbial immune defense. Inhal Toxicol 15:101–120

Trans-Signaling

The soluble Interleukin-6 receptor α-chain binds inter-leukin-6 and can then interact with the transmembranereceptor subunit glycoprotein 130 (gp130) and inducesignal transduction. Thus a cell lacking an endogenousbinding subunit of the interleukin-6 receptor can re-spond to Interleukin-6 in the presence of the solublereceptor-derived from distant producer cells, hencetrans-signaling.

3Cytokine Receptors

Transcription Factors

Proteins (enzymes) that bind to regulatory sequences(response elements) in the promoter region of a gene,forming a complex to which RNA polymerase binds.The process of transcription converts the genetic in-formation contained in DNA into an RNA message forsynthesis of a specific protein.

3Glucocorticoids


Transendothelial Migration

This is the exit of circulating leukocytes from bloodinto tissue by means of traversing the microvascularendothelium. This process involves loose interactionsof blood leukocytes with the luminal side of blood

Transendothelial Migration 657

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vessels (mostly mediated by selectins), and this is fol-lowed by firm adhesion and leukocyte transmigration.This latter step critically depends on the rapid andtransient modulation of integrin function, which itselfis controlled by chemokine receptor signaling. Onlythose leukocytes firmly arrest that bear the appropriateset of chemokine receptors and, therefore, the chemo-kines present on the luminal side of microvessels areviewed as key controllers of leukocyte extravasation.

3Immune Cells, Recruitment and Localization of

Transferrin

A protein that combines with and competes for ironwith bacteria.


Transferrin Receptor

These are cell membrane receptors for transferrin.They play a role in iron uptake by the cell, and arehighly expressed in proliferating cells.

3Interferon-γ

Transforming Growth Factor β1;Control of T Cell Responses toAntigens

Susan C McKarns

Laboratory of Cellular and Molecular ImmunologyNIAID/NIHBuilding 4, Room 111, MSC 0420, 4 Center DriveBethesda, MD 20892USA

Synonyms

TGF-β1(the nomenclature is used worldwide with thenumber designating the isoform)

Definition

The transforming growth factor-β (TGF-β) superfam-ily consists of more than 40 structurally related se-creted proteins (1). Three members (TGF-β1, 2, 3)are expressed in mammals; despite a 70%–76% se-quence homology, these isoforms have expression pat-tern and functional differences. Whereas, TGF-β2 andTGF-β3 are important for cellular differentiation, de-velopment, and embryogenesis, the effects of TGF-β1

are predominantly—albeit not exclusively—immuno-logic. Lymphoid cells selectively produce TGF-β1.The name 'transforming' is something of a misnomerbecause this factor is not always associated with on-cogenesis. TGF-β1 is possibly the most pleotropic ofall the 3cytokines and growth factors, and its activityis cell-type- and context-dependent. The ability ofTGF-β1 to suppress cell growth distinguishes itfrom most other cytokines/growth factors. Mechanis-tically, it converts receptor ligation at the cell surfaceinto an enzymatic signaling cascade within the cell tochange the level of expression of target genes. In thismanner, it is able to target a vast array of immune celllineages to modulate their ability to proliferate, differ-entiate, survive, perform effector functions, and mi-grate to sites of antigen presentation and/or inflamma-tion. These events are vital to the initiation, progres-sion, and resolution of inflammatory responses. Dys-regulated expression or function of TGF-β1 is impli-cated in autoimmune disease, chronic inflammation,and tumor progression. The driving force behindTGF-β1 seems to be maintaining homeostasis of con-trolled immune responses, and it achieves its goal byorchestrating a network of intracellular signalingcrosstalk that enables cells to rapidly respond tochanges in their environment.

Characteristics

Cellular sources

TGF-β1 expression is present at the four-cell embryostage and persists, in most tissues, during morphogen-esis and into adulthood. Most all mature cells havebeen shown to produce this factor. Likewise, nearlyall cell types have functional TGF-β1 receptors.Although controversial, it has been postulated thatthe primary effector function of a small cohort of reg-ulatory T cells (e.g. Th3 and CD4+CD25+) is to secreteTGF-β1.

Regulation of activity

It is well established that the bioavailability and activ-ity of TGF-β1 are influenced by the environment(Table 2), and it generally is accepted that some of

Transforming Growth Factor β1; Control of T CellResponses to Antigens. Table 1 GenBankaccession numbers for transforming growth factor-β1(TGF-β1)

Accession numbers(partial listing only)

Species Gene Protein

Human (Homo sapiens) J04431, J05114 PO1137

Mouse (Mus musculus) AH003562 P04202

658 Transferrin

these changes increase human susceptibility to immu-nologic-related diseases. TGF-β1 predominantly is se-creted as a biologically inert complex consisting ofmature TGF-β1, latency associate protein (LAP),and latent TGF-β1-binding protein (LTBP). Prior tobinding to TGF-β receptors, the latent complex mustbe cleaved into the 25 kDa active TGF-β1 homodimer.

TGF-β1 signaling

The predominant mechanism by which TGF-β1 elicitsits activity is through modulation of gene transcription.TGF-β1 mediates the association of transmembranetype II (TβRII) and type I (TβRI) receptors. Ligandbinding propagates signaling through phosphorylationof multiple effector proteins. The only known directTGF-β1 signaling effectors are a class of structurallysimilar 3Smad proteins (2). Once the ligand hasbound to the serine-threonine kinase receptor a signal-ing complex is formed, leading to the phosphorylation

of Smad 2 and Smad 3 and their subsequent traffickingto the nucleus, where they bind well-defined Smadresponse elements and function as transcriptionalmodulators to regulate transcription of TGF-β1 targetgenes (Fig. 1).Smads can also positively regulate gene expression byrecruiting coactivators such as CBP/p300 or nega-tively by forming complexes with histone deacetylases(HDACs) or corepressors (such as c-ski and SnoN)which themselves associate with HDACs. One keynegative regulation of Smad signaling is the expres-sion of the inhibitory Smad, Smad7. Smad7 blocksTGF-β signaling by competing with R-Smads for as-sociation with TβRI, or by targeting receptors for ubi-quitin-mediated degradation. Potent inducers ofSmad7 are interferon-γ (IFN-γ), tumor necrosis factor(TNF)-α, and interleukins IL-1β, and IL-7. Inductionof Smad7 represents an important regulatory interplaybetween TGF-β1 and cytokines in immune cell func-tion. It is noteworthy that type TβRI and TβRII re-ceptors distinguish themselves from other cytokine/growth factor receptors by their specificity for serine/threonine, rather than tyrosine kinase, activity.Smad-independent signaling pathways also regulateTGF-β1 signaling. For instance, TGF-β1 activates mi-togen-activated protein (MAP) kinases including theextracellular regulated kinases (ERKs), c-Jun N-termi-nal kinases (JNKs) and p38 kinases. TGF-β1 has alsobeen shown to activate Rho-like GTPases and phos-phatidulinostiol-3-kinase (P13K) and signal throughprotein phosphatase 2A (PP2A). One key point ofcross-talk among signaling intermediates is MAP ki-nase activation that occurs downstream of growth fac-tors, integrins, and chemokine receptors. In mostcases, activated 3MAP kinases promote the actionsof TGF-β1 to enhance cell migration. Thus, activationof growth factor receptor and the pattern of cytokine/chemokine signaling have a tremendous impact on theresponse of cells to TGF-β1 (Fig. 1). Finally, TGF-β1

Transforming Growth Factor β1; Control of T CellResponses to Antigens. Table 2 Factors thatmodulate bioactivity of transforming growth factor-β1(TGF-β1)

Enhance TGF-β synthesis and secretion

Liver hepatotoxicants (carbon tetrachloride, acet-aminophen, alcohol)

Tissue injury (liver, renal, and lung)

Hypoxia

Stress

Viral infection

Parasitic infection

Steroid hormones (retinoids, vitamin D, andtamoxifen)

Activate extracellular latent TGF-β1

Mannose 6-phosphate/insulin-like growth factor 2receptor (M6P/IGF2R)

Transglutaminase

Plasmin/plasminogen activator

Apoptotic T cells

Reactive oxygen species

αvβ6 Integrin receptor

Suppress activation of extracellular latent TGF-β1

α2-Macroglobulin

Decorin

Endoglobin

Mucosal mast cell protease (MMCP)

Antagonize TGF-β1 signaling

Cytokines: tumor necrosis factor-α, interferon-γ, in-terleukins IL-1β, IL-6, IL-2

Transforming Growth Factor β1; Control of T CellResponses to Antigens. Figure 1

Transforming Growth Factor β1; Control of T Cell Responses to Antigens 659

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signaling can also modulate protein stability. For ex-ample, it enhances degradation of TβRI.

Immunological activities

A loss-of-function mutation in TGF-β1 results in therapid onset of lethal multiorgan inflammation and au-toimmune phenotype. These transgenic mouse modelsclearly establish the critical role of this factor in main-taining immune homeostasis for the prevention of dis-ease and chronic inflammation (Fig. 2).A role for TGF-β1 has been implicated, in severaldifferent mouse models, including 3tolerance, andparticularly in mucosal immunity. While these studiesclearly demonstrate the onset of inflammation in theabsence of TGF-β1 signaling, more recent data sug-gest that the role for TGF-β1 in controlling T cellhomeostasis may be restricted to preventing inap-propriate responses to self- or environmental antigens,rather than regulating T cell responses to low-avidityself-ligands (3). In addition to its immunosuppressiveand anti-inflammatory properties, TGF-β1 is capableof promoting inflammation (4). For example, at theearly stages of inflammation, it enhances lymphoid,neutrophil, monocyte, and macrophage migration, pre-sumably to enhance the localization of these cells atthe site inflammation. Probably TGF3 also prolongsthe inflammation associated with numerous autoim-mune disorders by actively sequestering activatedT cells at the site of inflammation. TGF-β1 also exertsnumerous suppressive effects on T and B lymphoideffector and antigen-presenting cells and many ofthese effects are summarized in Table 3.


Implications for disease

Dysregulated expression of TGF-β1 or response ofimmune cells to TGF-β1 signaling have been impli-cated in the pathogenesis of many human diseases,including hypersensitivity reactions such as asthmaand food allergies, as well as autoimmune disorders,including encephalomyelitis, arthritis, systemic lupuserythematosus, and allograft rejection. While it is com-

monly accepted that both environmental and geneticfactors contribute to the incidence of these immuneresponses, it remains unclear why some individualsare susceptible to these disorders while others arenot. Appropriate levels of TGF-β1 have been shownto be essential for maintaining immunologic balance,to prevent the pathogenesis of hypersensitivity reac-tions/chronic inflammation and autoimmune disorders.Perhaps a better mechanistic understanding of how itmodulates cellular and molecular pathways will pro-vide important insights that will enhance our under-standing of susceptibility to these diseases. Outlinedbelow are three prevalent immune disorders that occurin response to common environmental exposure;which, TGF-β1 is key to regulation of the ensuingpathological immune responses.

Asthma

The development of asthma in response to environ-mental antigens affects up to 20% of the populationin developed countries. Asthma is a chronic inflam-matory disease of the airways that is characterized bymononuclear infiltration, eosinophil degranulation,and bronchoconstriction. TGF-β1 is constitutively ex-pressed by airway epithelial cells, eosinophils,T lymphocytes, macrophages, and fibroblasts, andstored in the extracellular matrix of the airways. Ro-dent models of asthma suggest that it mediates bothanti-inflammatory and profibrotic effects (5). Prior toallergen exposure, it is thought to play a critical pro-tective role against the onset of asthma by suppressingairway inflammation and hyper-responsivenessthrough the suppression of T lymphocytes, dendriticcells, eosinophils, mast cells, and IgE production. No-tably, mononuclear cell infiltration into the lungs isprevalent in TGF-β1 null mice. Additionally,TGF-β1 may further suppress airway CD4+ T cell al-lergen exposure by enhancing activity of T regulatorycells. However, repeated long injury is accompaniedby a profound TGF-β1-mediated recruitment of fibro-blasts into the airways, a progressive deposition ofextracellular matrix, and subsequent fibrosis and bron-choconstriction.

Food allergy

Food allergy is characterized by an adverse hypersen-sitive response to food consumption. A normal healthygastrointestinal immune response must discriminatebetween harmful pathogens and harmless dietary anti-gens and commensal bacterial flora. The mucosal im-mune system has generated two adaptive immune re-sponses to meet this challenge: induction of a localsecretory IgA response, which is propagated in theabsence of a measurable systemic immune response,to clear potentially dangerous antigens; and inductionof oral tolerance, a state of non-responsiveness or

Transforming Growth Factor β1; Control of T CellResponses to Antigens. Figure 2

660 Transforming Growth Factor β1; Control of T Cell Responses to Antigens

hypo-responsiveness which minimizes unnecessaryimmune reactions against harmless antigens. A failureto induce or an inability to maintain oral tolerance mayleads to a food allergy. TGF-β1 is abundant through-out the mucosa and has been shown in several experi-mental models to play a profound role in maintainingoral tolerance (5). It is well documented that a popu-lation of TGF-β1-secreting T helper cells is generatedwhen low doses of antigen are consumed. TheseTGF-β1-secreting regulatory T cells also produce var-ious amounts of IL-4 and/or IL-10. Secreted TGF-β1suppresses T cell proliferation and promotes classswitching of B cell IgA isotypes to modulate the adap-tive immune response. Moreover, it also enhances the

preservation of the epithelial barrier between environ-mental antigens in the gut flora and T lymphocytes inthe mucosa to add yet another level of regulatory con-trol over adaptive T cell responses.Childhood food allergies have been associated with areduction in the number of mucosal TGF-β1-produ-cing lymphocytes. Aberrant levels of mucosal TGF-β1and associated dysregulated responses to the normalgut flora have also been implicated in the pathogenesisof inflammatory bowel disease. Collectively, thesedata implicate a role for TGF-β1 to maintain oral tol-erance in humans.

Transforming Growth Factor β1; Control of T Cell Responses to Antigens. Table 3 Biological activities oftransforming growth factor-β1 on the immune system

Parameter TGF-β1-mediated effect

T lymphocytes

TCR-induced CD4+ and CD8+ T cellproliferation

Suppress; memory CD4+ resistant to G1 cell cycle arrest

IL-2-induced CD4+ and CD8+ T cellproliferation

Suppress, dependent upon IL-2 concentration

Th1 differentiation Suppress, but dependent upon strength of T cell stimulation; inhibits T-betand INF-γ expression

Th2 differentiation Suppress; inhibits GATA-3 and IL-4 expression

Th1 effector function Suppress; inhibits INF-γ and IL-2 production; inhibits IL-12 signaling

Th2 effector function Suppress; inhibits IL-4 and IL-5 production

CD8+ cytotoxic T cell effector func-tion

Suppress

CD4+ and CD8+ migration/adhesion Enhance; increases CXCR4 and α4β7 expression

CD4+CD25+ regulatory T cell func-tion

Enhance; increases Foxp3, GITR, CD103, CTLA-4 expression

IL-12 signaling Suppress; downregulates IL-12 receptor β2 chain

T cell apoptosis Suppresse or enhance, dependent on microenvironment

B lymphocytes

Proliferation Suppress

Effector function Enhance IgA and IgG2b and suppressed most other isotopes

Antigen-presenting cells

MHC class I and class II molecules Suppress

Monocytes and macrophages

Monocyte chemotaxis Enhance or suppress

Macrophage chemotaxis Suppress

Neutrophils

Neutrophil chemotaxis Enhance or suppress

Chemokine and receptor expression Suppress or enhance: chemokine, receptor, and cell type-dependent

CTLA=cytotoxic T-lymphocyte-associated protein 4; GITR= glucocorticoid induced TNF receptor; Ig=immunoglobulin;IL=interleukin; INF=interferon; MHC=major histocompatibility complex; TCR=T cell receptor; TGF=transforming growth factor;Th=T helper cell.

Transforming Growth Factor β1; Control of T Cell Responses to Antigens 661

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Autoimmunity

Discordance in incidence of autoimmune disease inmonozygotic twins demonstrates a role for environ-mental exposure in regulating immune homeostasis.Although numerous environmental factors have beenimplicated, the underlying mechanisms remain rela-tively undefined. The autoimmune phenotype of theTGF-β1 knockout mouse, characterized by circulatingantinuclear antibodies and glomerular deposit s of im-mune complexes, probably best defines the role of thisfactor in the disease process. 100% of TGF-β1 knock-out mice succumb to a massive multiorgan inflamma-tion involving the heart, lung, liver, gut, salivaryglands, eyes, brains, and other tissues. The inflamma-tory infiltrates are predominantly perivascular andvary from neutrophilic in the stomach to lymphocyticin the brain. In agreement, systemic administration ofexogenous TGF-β1 or adoptive transfer of TGF-β1−producing T cells protect against autoimmune dis-eases in several experimental models, including diabe-tes, encephalomyelitis, inflammatory bowel disease,arthritis, systemic lupus erythematosus, and allograftrejection. Targeted deletion of TGF-β signaling inT cells alone has been demonstrated to be sufficientto induce an autoimmune phenotype. It remains to bedetermined whether other non-T cell TGF-β1-produ-cing cells (e.g. macrophages) contribute to the diseaseprocess as well. The precise mechanisms of actionsunderlying the ability of this factor to regulate auto-immune disorders remains speculative. Recent evi-dence implicates a significant role for regulatoryT cells. CD4+ CD25+ regulatory T cells, also calledsuppressor T cells, can be delineated into two subsetsof CD4+ CD25+ T cells with inherent activity to sup-press autoreactive T cells: these are ‘natural’ regula-tory CD4+ CD25+ T cells that emerge from the thy-mus, and adaptive regulatory CD4+ CD25+ T cells thatare induced in the periphery. TGF-β1 has been shownto positively regulate both subsets, and TGF-β1-mediated expansion of CD4+ CD25+ T cells protectsagainst autoimmune diabetes (7). However, in view ofthe diversity of pathology associated with autoimmunedisorders, it is highly likely that TGF-β1 also utilizesother critical mechanisms of action. For instance,modulation of Th2/Th1 cytokine balance, cell survi-val, migration, effector function, and Th3-mediatedtolerance represent likely alternative mechanisticroutes (8).

Relevance to HumansThe phenotype of the TGF-β1 mouse resembleshuman SLE, Sjögren syndrome, graft-versus-host dis-ease, and polymyositis, suggesting that TGF-β1 mayplay a similar regulatory role in human immunologicdisorders. The levels of TGF-β1in serum and of itsmRNA in tissue can be measured and have been used

as diagnostic or prognostic markers for other humandiseases. For example, high levels of the factor inRNA in tissues are associated with gastric cancer.High serum levels also correlate with the developmentof fibrosis in patients with breast cancer who havereceived radiation therapy. Understanding the mechan-isms of action of environment-induced immune disor-ders in experimental models will potentiate the devel-opment of better predictive risk assessment assays toprevent disease as well as more specific therapeuticregimens aimed at increasing effectiveness and dimin-ishing deleterious side effects.


Although interaction of chemicals with cytokines orchemokines may have an important impact on thefunction and regulation of the immune system theyare not regulated by any specific immunotoxicityguideline. The cytokine network is mentioned in dif-ferent guidelines or guideline drafts but exclusively inconnection with extended 'case-by-case' investiga-tions.The regulation of the immune system is complex, andidentification of the mechanism, of action of chemical-induced immune toxicity is critical for the understand-ing of the disease process. The regulatory cytokineTGF-β1 may be of special interest for such investiga-tions. A better understanding of the disease processwill provide the basis for the development of moresensitive and predictable assays for risk assessment.Chemical-induced immunotoxicity may be indirectlymediated via the soluble potent immune modulator,TGF-β1. TGF-β1 may be a useful biomarker of chem-ical-induced and/or environmental-induced immuno-toxicity. A critical challenge is to determine the appro-priate therapeutic level of active TGF-β1 or signalingpathways that positively influence cell responsivenessto ameliorate disease while minimizing deleteriousside effects.

References

1. Flanders CF, Roberts AB (2000) TGF-β. In: OppenheimJJ, Feldman M, Durum SK, Hirano T, Vilcek J, NicolaNA (eds) Cytokine reference: A compendium ofcytokines and other mediators of host defense. AcademicPress, New York, pp 719–746

2. Shi Y, Massague J (2003) Mechanisms of TGF-βsignaling from cell membrane to the nucleus. Cell13;113:685–700

3. Gorelik L, Flavell RA (2002) Transforming growthfactor-β in T-cell biology. Nat Rev Immunol 2:46–53

4. McCartney-Francis NL, Frazier-Jessen M, Wahl SM(1998) TGF-β: A balancing act. Int Rev Immunol16:553–580

5. Duvernelle C, Freund V, Frossard N (2003) Transforminggrowth factor-β and its role in asthma. Pulmon Pharma-col Ther 16:181–196

662 Transforming Growth Factor β1; Control of T Cell Responses to Antigens

6. Weiner HL (2001) Oral tolerance: immune mechanismsand the generation of Th3-type TGF-β-secreting regula-tory cells. Microbes Infect 11:947–954

7. Peng Y, Laouar Y, Li MO, Green EA, Flavell RA (2004)TGF-β regulates in vivo expansion of Foxp3-expressingCD4+CD25+ regulatory T cells responsible for protectionagainst diabetes. Proc Natl Acad Sci USA 101:4572–4577

8. Prud'homme GJ, Piccirillo CA (2000) The inhibitoryeffects of transforming growth factor-beta-1 (TGF-β1) inautoimmune diseases. J Autoimmun 1:23–24

Transforming Growth Factor β1(TGF-β1)

TGF-β1 is the prototype for a superfamily of secretedproteins that control many aspects of growth and de-velopment. It was named transforming growth factorbecause upon its discovery it was shown to induce atransformed or tumor cell phenotype in normal cells.TGF-β1 is now known to regulate a diverse array ofcellular functions unrelated to cell transformation.Within the immune system, TGF-β1 is critical forcell growth, differentiation, effector cell function, sur-vival, and migration.


3Mucosa-Associated Lymphoid Tissue

Transgenic Animals

Peter J Bugelski

Experimental PathologyCentocor, Inc.R-4-2, 200 Great Valley, ParkwayMalvern, PA 19355USA

Synonyms

Knock-out, knock-in, genetically modified, recombi-nant

Definition

Animals whose genome has been modified using re-combinant DNA technology so as to have a foreigngene expressed (knock-in) or a native gene suppressed(knock-out) in a heritable fashion. A number of trans-genic species have been created: mice, rats, pigs,goats, cattle, sheep and fish. Currently, the vast major-ity of transgenic animals are mice. Transgenic micehave applications in numerous areas of biomedicalresearch (e.g. neurologic, inflammatory, autoimmune,

neoplastic and cardiovascular disease), in immunolo-gy, mutagenesis and carcinogenesis research, and innovel target evaluation and drug discovery. In immu-notoxicology, although the potential usefulness oftransgenic mice is widely recognized, practical appli-cation has been limited.

CharacteristicsGeneration of Transgenic Animals (1)

There are two principal methods by which transgenicmice are created: 3microinjection of genetic materialinto the 3pronucleus of a fertilized ova; or gene trans-fection of embryonic stem cells (ES cells) cells fol-lowed by injection of the transgenic cells into a blas-tocyst. In either case, the resulting transgenic embryois implanted into a recipient female prepared for preg-nancy. The principal difference between the techni-ques is that, when successful, microinjection of thepronucleus results in homozygous offspring, whiletransfection of ES cells results in chimerae (see 3chi-mera) that must be selectively bred to yield homozy-gous animals.There are two principal types of transgenic events,those with one or more random insertions of the trans-gene and those where 3homologous recombinationresults in targeted insertion. Either type of transgenicevent can result in a knock-in (KI) that will express thecoding sequences of the transgene. Although randominsertion will by definition result in a mutation in therecipient genome, as most DNA is noncoding thesemutations are generally silent. Homologous recombi-nation is used to selectively disrupt expression of thehomologous gene, resulting in a knock-out (KO). Thisrequires design of the inserted DNA so as to containsequences homologous to the desired host species’gene.A third type of transgenic animal—the knock-in-knock-out (KI-KO) mouse—has also been created. Ifthe inserted DNA has a sequence homologous to amurine gene and also codes for a foreign protein, aKI-KO mouse can be created in a single step. Alter-natively, these two types of transgenic events can becombined in a two-step process to result in a KI-KOstrain.

Transgene Expression

In some cases, expression of the transgene by the hostis not important. For example in mutagenesis researchthe endpoint can be a mutation in the transgene thatwill be expressed and detected ex vivo. In most caseshowever, expression of the transgene is desired. Inser-tion of multiple copies of the gene and linkage to apotent promoter (e.g. simian virus 40 promoter, willlikely ensure widespread and high-level 3gene ex-pression. Depending on the experiments to be con-ducted, however, it may be important that the site (s)

Transgenic Animals 663

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of expression, the magnitude of expression and timingof expression be controlled. This can be accomplished,but is no means guaranteed by selecting the gene pro-moter sequences included in the transgene. Techniquesare now reasonably well established for controlling thesites and magnitude of expression and 3“conditional”gene expression in transgenic animals is an active fieldof research.

Preclinical RelevanceTransgenes

Transgenic mice have been created which express awide and ever increasing range of genes (as of June2003 the database maintained by BioMed Net lists2300 transgenic mice (2)). These genes include repor-ter constructs (e.g. β-galactosidase or green fluores-cent protein (GFP)) and viruses (e.g. hepatitis C),and a wide variety of human proteins.Of greatest relevance to immunotoxicology is the ex-pression of human cytokines, cell surface markers andimmunoglobulins. Mice transgenic for mutant reportergenes have application in genotoxicity, and mice trans-genic for mutant oncogenes have application as short-term replacements for 2-year cancer bioassays.In efforts to facilitate xenotransplantation, transgenicpigs have been created that express a small number ofhuman genes. There have also been reports on a modelof colitis in rats expressing a human major histocom-patibility antigen (HLA)-B27.

Function

Expression, however, is not sufficient for the transgen-ic strain to have preclinical relevance. The transgenegene product (i.e. the protein) must be functionallyactive in the transgenic animal. In the case of immu-notoxicology, this will generally require that thehuman protein binds, and will result in signal trans-duction in its respective murine receptor (e.g. CCRand FcR or binding proteins such as major histocom-patability complex (MHC) class II).

Genetic Background

Two strains of inbred mice are widely used for creat-ing transgenic mice; 129 and C57 black. Once a trans-genic strain has been created however, it may be pos-sible to “move” the transgene into an alternate geneticbackground (e.g. BALB/c, by selective breeding. Thiscan be of critical importance in application of trans-genic mice in immuntoxicology where the geneticbackground of the mice can have a significant impacton the experiment (e.g. delayed-type hypersensitivity,transplantation, immunogenicity and host defenseagainst infection or neoplasia).

Fecundity

Fecundity can also be an important factor in determin-

ing the success of application of transgenic mice intoxicology. Many strains of transgenic mice showlow fecundity as determined by fertility and numberof offspring. As we must have sufficient numbers ofanimals for study, low fecundity can have a seriousimpact on our ability to conduct a given experiment.Simple animal husbandry (e.g. selection of provenbreeders) may be sufficient to solve this issue. Main-taining the breeding colony as heterozygotes may berequired. However, as the offspring of heterozygoteswill be a mix of transgenic and nontransgenic, theoffspring must be genotyped or phenotyped prior toenrolment in studies.

Application of Transgenic Rodents in Immunotoxicol-

ogy

Transgenic mice have been used extensively for study-ing the immune system. As of June 2003 the NationalInstitutes of Health Medline lists over 2800 papersdescribing the use of transgenic mice to study immu-nology. Obviously, there are far too many examples tolist here. However, applications of direct relevance toimmunotoxicology are much rarer. Some selected ex-amples are listed in Table 1.

Relevance to Humans

As with any animal system, the relevance of transgen-ic animals to humans is somewhat limited. Some ofthe factors which lead to this limited relevance arelisted in Table 2. One must also keep in mind that inmost cases while the transgenic animal may be trans-genic for one human protein (and therefore immuno-tolerant to that human protein) it will likely not beinherently tolerant to any administered human thera-peutic protein. With these caveats in mind, a priori,transgenic mice should have a much relevance to hu-mans as any murine system.


Use of transgenic animals for demonstrating pharma-cologic activity and safety are gaining increasing ac-ceptance by regulatory authorities. They are specifi-cally dealt with in the following guidance documents:* FDA Guidance for Industry. Clinical Development

Programs for Drugs, Devices, and Biological Pro-ducts for the Treatment of Rheumatoid Arthritis(RA) http://www.fda.gov/cder/guidance/1208fnl.pdf

* FDA Guidance for Industry. ImmunotoxicologyEvaluation of Investigational New Drugs. http://www.fda.gov/cder/guidance/4945fnl.doc

* ICH Guidance for Industry. S1B Testing for Carci-nogenicity of Pharmaceuticals http://www.fda.gov/cder/guidance/1854fnl.pdf

3Animal Models of Immunodeficiency

664 Transgenic Animals

References

1. Hofker MH, Van Deursen J (eds) (2002) TransgenicMouse: Methods and Protocols. Methods MolecularBiology, Vol. 209. Humana Press, Clifton NJ

2. BioMed Net. http://www.biomednet.com/db/mkmd (ac-cessed June 2003)

3. Moser R, Quesniaux V, Ryffel B (2001) Use of transgenicanimals to investigate drug hypersensitivity. Toxicology158:75–83

4. Matheson JM, Lemus R, Lange RW, Karol MH, LusterMI (2002) Role of tumor necrosis factor in toluenediisocyanate asthma. Am J Respir Cell Mol Biol 27:396–405

5. Bugelski PJ, Herzyk DJ, Rehm S et al. (2000) Preclinicaldevelopment of keliximab, a Primatized anti-CD4 mono-clonal antibody, in human CD4 transgenic mice:characterization of the model and safety studies. HumExp Toxicol 19:230–243

6. Herzyk DJ, Bugelski PJ, Hart TK, Wier PJ (2002)Practical aspects of including functional endpoints indevelopmental toxicity studies. Case study: immunefunction in HuCD4 transgenic mice exposed to anti-CD4 MAb in utero. Hum Exp Toxicol 21:507–512

7. Takahashi R, Ueda M (2001) The milk protein promoteris a useful tool for developing a rat with tolerance to ahuman protein. Transgenic Res 10:571–575

8. Braun A, Kwee L, Labow MA, Alsenz J (1997) Proteinaggregates seem to play a key role among the parametersinfluencing the antigenicity of interferon alpha (IFN-alpha) in normal and transgenic mice. Pharm Res14:1472–1478

9. Francini G, Scardino A, Kosmatopoulos K et al. (2002)High-affinity HLA-A(*)02.01 peptides from parathyroidhormone-related protein generate in vitro and in vivoantitumor CTL response without autoimmune sideeffects. J Immunol 169:4840–4849

Transgenic Animals. Table 1 Examples of application of transgenic rodents in immunotoxicology (KI, knock-in;KO, knock-out)

Transgenic system Application Reference

Various cytokine KI andKO mice

Drug hypersensitivity 3

TNF-α receptor KO Mechanism of toluene diisocyanate asthma 4

Human CD4 KI-murine CDKO

General and immunotoxicity of a chimeric antihuman CD4 monoclonalantibody

5

Human CD4 KI-murine CDKO

Embryo–fetal and immunotoxicologic development study of a chimericantihuman CD4 monoclonal antibody

6

Human growth hormoneKI rats

Immunogenicity 7

Human interferon-α KI Breaking immune tolerance to interferon-α 8

Human carcinoembryonicantigen KI mice

Safety of human carcinoembryonic antigen tumor vaccine 9

Transgenic Animals. Table 2 Examples of sources of limitation of the relevance of transgenic mice to humanimmunotoxicity

Physiology

Kinetics Generally more rapid clearance of xenobiotics and therapeutic proteins in mice

Metabolism Differences between murine and human P450 usage and inducibility, substratespecificity and metabolite profile

Immunology

Ontogeny Differences in timing of cytogenesis, histogenesis and organogenesis of the immunesystem

Receptors Differences in binding affinity and signal transduction of human proteins for murinereceptors and binding proteins

Immunogenicity andtolerance

Differences between human and murine antigen processing and MHC restrictions

T cells Differences in T helper 1 and 2 usage and switching

B cells Differences in immunoglobulin class switching

Macrophages Differences in Fc receptor utilization

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Transgenic Mouse

Transgenic mice are genetically engineered mice thatover-express foreign DNA and are typically referred toas transgenic, while those in which foreign DNA hasreplaced an endogenous gene are termed gene targeted(or knockout). In the strict sense, however, both theseprocedures yield a transgenic mouse (i.e. one withadded genetic material).

3Knockout, Genetic

Transglutaminase

The epidermal keratinocyte transglutaminase I is a cal-cium-dependent enzyme that plays a central role inkeratinocyte cornification. It catalyzes the cross-link-ing between glutamine and lysine residues of isopep-tides at the inner surface of keratinocyte cell mem-branes, which is an essential step for the stabilizationof their cornified cell envelope (CCE).

3Three-Dimensional Human Skin/Epidermal Modelsand Organotypic Human and Murine Skin ExplantSystems

Transition Element

Elements that occupy the middle portions (the d-block) of the periodic table, have valence electronsin two or more shells instead of only one, and arecharacterized in most cases by variable oxidationstates and magnetic properties.

3Chromium and the Immune System

3Vanadium and the Immune System

Transporter Associated with AntigenProcessing (TAP)

TAP is composed of two subunits, TAP1 and TAP2.This heterodimer, which belongs to the ABC (ATP-binding cassette) transporter family is responsible forthe shuttling of peptides from the cytosol into thelumen of the endoplasmic reticulum.

3MHC Class I Antigen Presentation

Trichinella spiralis

A helminthic parasite, invading the gut mucosa andresiding as larvae in striated muscle tissues.

3Host Resistance Assays

Triglycerides

Tricglycerides are molecules that consist of a glycerolbackbone esterified to three fatty acids.

3Fatty Acids and the Immune System

Trivalent Chromium

The ionic form of chromium when three outer shellelectrons (one from 4s and two from 3d orbitals) havebeen shed, thereby giving the atom an overall chargeof +3.

3Chromium and the Immune System

Trypanosomes, Infection andImmunity

Ronald Kaminsky

Centre de Recherche Santé AnimaleNovartisCH-1566 St-AubinSwitzerland

Synonyms

hemoflagellates

Definition

Trypanosomes are protozoan parasites of the family ofTrypanosomatidae, belonging to the order of Kineto-plastida of the class of Zoomastigopohora.Three species are pathogenic to man—Trypanosomabrucei gambiense and T brucei rhodesiense causeAfrican human sleeping sickness in sub-Saharan Afri-ca, while T. cruzi causes Chagas disease in SouthAmerica (Table 1).

Characteristics

Characteristics of the parasites

The prominent morphological feature of the unicellu-lar protozoan parasites is the kinetoplast, an organellewhich contains about 15% of the cells DNA. The ki-netoplast can be visualized by Giemsa staining or flu-

666 Transgenic Mouse

orescent dies like DAPI. Movement of trypanosomesis via a flagellum which originates at the basal bodynear the kinetoplast and which is attached to the bodyof the parasite by an undulating membrane.The African trypanosomes are extracellular parasites(16–30 μm long) which move within the blood (hencetheir designation as hemoflagellates) or within the ce-rebral spinal fluid. T. cruzi occurs in man in both asextracellular and intracellular form. After introductioninto the blood T. cruzi invades various cell types in-cluding macrophages and muscle cells. The intracellu-lar form (3 μm in diameter) is much smaller than theextracellular form and does not posses a flagellum, butstill contains the kinetoplast.All bloodstream forms of African trypanosomes arecoated with variable surface glycoproteins (VSGs).The VSGs are anchored through a glycosyl phospha-tidyl inositol lipid to the body of the parasite. Thesehighly immunogenic VSGs have, at any one point oftime, the same structure resulting in a specific variantantigen type (VAT). However, the VSGs are periodi-cally removed and replaced with the result that theparasite population bearing one VSG are killed byan antibody response and are replaced by a new pop-ulation with another variant antigen type. This 3anti-genic variation is a mechanism that plays a key role inthe escape of trypanosomes from total destruction bythe immune response of their mammalian hosts (1).

Cyclical transmission

African trypanosomes are cyclically transmitted bytsetse flies (various Glossina species) (Table 1).After a fly has taken a blood meal from an infectedhost, the trypanosomes undergo various changes andmultiplication within the fly. They finally mature inthe salivary glands of the 3tsetse fly, to infectiousmetacyclic forms which are transmitted to a naivehost.The American T. cruzi is transmitted cyclically by

'kissing' bugs, the family of Reduviidae (Table 1),not by direct inoculation when the vector is feedingbut by contamination through parasites in feces. Tri-

Trypanosomes, Infection and Immunity.Figure 1 Trypanosoma brucei brucei bloodstreamforms.

Trypanosomes, Infection and Immunity.Figure 2 Trypanosoma cruzi: in vitro cultured amasti-gote forms in mammalian feeder cells.

Trypanosomes, Infection and Immunity. Table 1 Characteristics of human pathogenic trypanosomes

Trypanosomaspecies

Disease Transmission

vector

Mode oftransmission

Animal reservoirs Geographicdistribution

T brucei gam-biense

Sleepingsickness

Tsetse flies(Glossina spp.)

Bite Mainly dogs, pigs, andcertain game animals

West and Cen-tral Africa

T. brucei rhode-siense

Sleepingsickness

Tsetse flies(Glossina spp.)

Bite All major domestic ani-mals and various gameanimals

East Africa

T. cruzi Chagasdisease

Reduviid bugs(Triatoma spp.,Rhodnius spp.,Panstrongylusspp.)

Contaminationby bug feces

Domestic (dogs, cats,guinea-pigs), rodents andwild animals (opossumsetc.)

Southern andCentralAmerica

Trypanosomes, Infection and Immunity 667

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atoma infestans is the major transmitting species, butvarious others species including Triatoma spp., Rhod-nius spp., and Panstrongylus spp. are capable of trans-mitting T. cruzi.

Characteristics of the diseases

3Sleeping sickness is 100% fatal if left untreated.There are two disease stages for human African try-panosomiasis. A chancre, a primary lesion at the siteof the bite, it is not observed frequently. The firststage, when trypanosomes are in the blood, is charac-terized by non-specific symptoms, such as fever, se-vere headache, joint or muscle aches. The second stageof the disease (also called late stage) starts with theinvasion of the central nervous system by trypano-somes, which cross the blood-brain barrier 3–6 month post infectionem. It is in the late stage ofthe disease that the characteristic symptoms of sleep-ing sickness occur, such as sleep disturbances, altera-tion of mental state, muscle tone disorders, abnormalmovements, and sensory and coordination disorders,up to a final general apathy.The acute form of Chagas disease is characterized bygeneral malaise with a variety of clinical manifesta-tions. Symptoms can be very mild and atypical. At thesite of entry of T. cruzi a local inflammation called achagoma may develop; this is known as a Romanasign if it occurs at the eyelid. The acute form is fol-lowed by a period of an indeterminate form withoutany clinical symptoms. It is estimated that 20%–50%of persons with the indeterminate form of the infec-tions will suffer from cardiac, digestive, or neurologi-cal damage 10–20 years after infection (2).

Preclinical RelevanceAfrican and American trypanosomes can be manipu-lated in vitro and in various animal models. T. bruceigambiense appears to be the most difficult species forlaboratory work. In vitro assays and in vivo models arebeing used to identify new active compounds, non-variant vaccine targets, and to monitor drug resistance.Some forms play an important role in host resistancemodels for immunotoxicity screenings.

Relevance to HumansInfection

Sixty Million people in 36 countries of sub-SaharanAfrica live at risk of acquiring sleeping sickness. In1999 around 45 000 cases were reported, but the num-ber of people thought to have the disease at any onetime is between 300 000 and 500 000. Chagas diseaseaffects 16–18 million people, and about 100 million(25% of the population of Latin America) are at risk ofacquiring Chagas disease. Due to the chronic character(indeterminate stage) of Chagas disease, transmissionoccurs not only via insect vectors, but also by congen-

ital transmission, and from transfusions with contami-nated blood, and organ transplantations.

Immunity

Due to antigenic variation of the African trypano-somes, which can express approximately 1000 differ-ent variant antigen types (1), immunity to the parasitesdevelops only to specific VATs but does not provideprotection against infection.

Treatment

Two drugs, pentamidine and suramin, are used in thefirst stage of sleeping sickness prior to CNS involve-ment. The first-line treatment for late-stage cases,when trypanosomes are established in the CNS, isthe arsenic-based drug melarsoprol (3). The drug hasbeen in use since 1949. However, up to 5% of treatedpatients may die because of lethal encephalopathy dueto the drug. Recently a new treatment schedule (4) wasdesigned, but the number of patients with encephalop-athy syndromes was the same as before. Nevertheless,the new 10-day schedule is a useful alternative to thepresent standard 26-day treatment schedule. Eflor-nithine (DFMO) is used mainly as a back-up in in-stances of melarsoprol-refractory T. brucei gambiense.Its efficacy against East African sleeping sickness islimited due to an innate lack of susceptibility ofT. brucei rhodesiense based on higher ornithine decar-boxylase turnover.The unsatisfactory treatment situation for sleepingsickness is hampered further by the occurrence of mel-arsoprol-resistant trypanosomes (5) in several regionsof sub-Saharan Africa. A molecular mechanism in theresistant isolates was identified: the majority of indi-vidual resistant isolates from geographically distantlocalities contained the same set of point mutationsin their 3TbAT1 genes (6), which codes for an aden-osine transporter (7).The drug of choice for treatment of Chagas disease isnifurtimox, with benznidazole as a back-up. However,these drugs are associated with side effects (2). Nifur-timox and benznidazoles were introduced at the begin-ning of the 1970s. Treatment success varies accordingto the phase of Chagas disease, the period of treatment,and the dose, the age, and geographical origin of thepatients. Good results have been achieved in the acutephase, in recent chronic infection, and congenital in-fections. However, there is still controversy about theiruse in chronic cases (2).

Regulatory EnvironmentAt present there is only one new antitrypanosomaldrug on clinical trial in Africa—the diamidine deriva-tive DB289. Identification of novel compounds andtheir development to drugs is pursued by various pri-vate-public initiatives. Registration of new drugs

668 Trypanosomes, Infection and Immunity

might be facilitated when these drugs are classified asorphan drugs. As mentioned above, trypanosomes areindirectly regulated by different immunotoxicologyguidelines by the recommendation for infection mod-els using these parasites in host-resistance assays.More detailed information is given in the relevant en-tries in this book.

References

1. Borst P (2002) Antigenic variation and allelic exclusion.Cell 109:5–8

2. Coura JR, de Castro SL (2002) A critical review onChagas disease chemotherapy. Mem Inst Oswaldo Cruz97:3–24

3. Legros D, Ollivier G, Gastellu-Etchegorry M et al. (2002)Treatment of human African trypanosomiasis—presentsituation and needs for research and development. LancetInfect Dis 2:437–440

4. Burri C, Nkunku S, Merolle A, Smith T, Blum J, Brun R(2000) Efficacy of new, concise schedule for melarsoprolin treatment of sleeping sickness caused by Trypanono-soma brucei gambiense: a randomized trial. Lancet355:1419–1425

5. Kaminsky R, Mäser P (2000) Drug resistance in Africantrypanosomes. Curr Opin Anti-infect Invest Drugs 2:76–82

6. Matovu E, Geiser F, Schneider V et al. (2001) Geneticvariants of the TbAT1 adenosine transporter from Africantrypanosomes in relapse infections following melarsoproltherapy. Molec Biochem Parasitol 117:71–81

7. Mäser P, Sütterlin C, Kralli A, Kaminsky R (1999) Anucleoside transporter from Trypansoma brucei involvedin drug resistance. Science 285:242–244

Tryptophan

α-Amino-β-indole-propionic acid; a component ofproteins; it is chromogenic, producing a violet colorwith chlorine or bromine solution.

3Serotonin

Tsetse Fly

Tsetse flies (Glossinidae; more than 30 species) aresub-Saharan bloodsucking flies (Diptera). The femalesdo not lay eggs but give birth to living larvae. Bothsexes feed on the blood of humans, livestock, and wildanimals. Tsetse flies transmit human and animal patho-genic trypanosomes. Ingested trypanosomes of an in-fested host undergo a development cycle in the tsetsefly to mature to metacyclic forms which are infectivefor the next host.

3Trypanosomes, Infection and Immunity

TSK

An acronym for tight skin which is associated withthickened skin and fibrosis due to mutations in thefibrillin gene.


Tuberculin

Mixture of antigens obtained from the culture of My-cobacterium tuberculosis.

3Mitogen-Stimulated Lymphocyte Response

Tuberculin-Type Reaction

A classical example of a delayed-type hypersensitivity(DTH) is the tuberculin-type reaction. In sensitizedindividuals, it is induced by an intradermal injectionof tuberculin, an extract of Mycobacterium tubercolo-sis. This particular example of DTH was first de-scribed by R. Koch. He who observed that patientswith tubercolosis reacted with fever and shock afterthe subcutaneous injection of tuberculin. Typically, theT cell mediated local immune reaction appears one ortwo days after the application.The tuberculin test, however, is not an allergic reac-tion. It is a diagnostic proof for the previous infectionwith M. tubercolosis and also other pathogens such asM. leprae or Leishmania tropica.

3Delayed-Type Hypersensitivity

Tumor Antigen

Any molecule leading to immune recognition oftumor. A generic term that encompasses tumor-specif-ic antigens, antigens shared by normal and neoplasticcells, and specificities recognized by xenogeneic anti-bodies (e.g. human molecules bound by mouse mono-clonal antibodies) that can be non-antigenic in the spe-cies of origin.

3Tumor, Immune Response to

Tumor-Associated Antigens

Tumor-associated antigens (TAA) are tumor-specificproteins that can be recognized by immune effectorcells of the host. To date, a variety of TAA are

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known. These are derivatives of either (i) physiologi-cal self-antigens or tissue specific differentiation anti-gens that are dramatically overexpressed by tumorcells in comparison to other cells, (ii) mutated self-proteins or specific oncogenic antigens inappropriatelyexpressed by tumor cells, or (iii) those derived fromvirally encoded antigens. The recognition pattern in-duced by TAA allows the immune system to distin-guish the transformed neoplastic cells from surround-ing normal tissue cells and triggers the immune cas-cade against them.


Tumor, Immune Response to

Pier-Luigi Lollini

Cancer Research SectionDepartment of Experimental Pathalogy, University ofBolognaViale Filopanti 22I-40126 BolognaItaly

Synonyms

Immune response to cancer, anti-tumor immunity.

Definition

The immune system of the host responds to tumorgrowth as it does to infectious agents, with specific(e.g. T cells and antibodies) and non-specific (e.g. nat-ural killer cells and cytokines) effector and regulatorymechanisms. The immune response reduces the num-ber of tumors arising in the host, but is no longereffective against established tumors. Tumor immuno-therapy is the attempt to elicit a therapeutic immuneresponse in cancer patients.

Characteristics

The immune response against tumors was formallydemonstrated in the late 1940s and early 1950s usingtransplantable tumors induced with chemical carcino-gens or retroviruses in inbred mice (1). The experi-ments showed that mice vaccinated with a giventumor reject a subsequent challenge with the sametumor (immune memory), but fail to reject an unre-lated tumor (specificity).The immunization-challenge system was extensivelyused to characterize the effector and regulatory me-chanisms of the immune response against tumorsusing two strategies:* cellular and molecular analysis of local and system-

ic components elicited by immunization and/or in-volved in rejection

* use of mice with selective immune deficiencies ofgenetic origin (spontaneous mutation or geneticallymodified mice) or induced by exogenous treatmentslike monoclonal antibodies or drugs.

Specific immune responses against tumors are mainlydue to T cells. Cytotoxic T cells (CTL) expressing theCD8 surface molecule are the final effectors capable oftumor cell lysis. Helper T cells (Th) expressing CD4play a fundamental positive or negative regulatoryrole. Tumor immunologists tend to downplay the im-portance of B cells, antibodies and complement be-cause solid tumors are resistant to complement-mediated cytotoxicity (tumor cells express comple-ment inhibitors like CD55 and CD59) and in immuni-zation-challenge systems B cells can even favor tumorgrowth (“enhancement”).Most cells of the innate (also called natural or non-adaptive) immune system directly affect tumor growth,and are required for the generation of T cell immunity.Professional and non-professional phagocytes destroytumor cells and generate antigenic material that is sub-sequently picked up by antigen-presenting cells (APC)like dendritic cells, that are indispensable to activateT cell responses. Natural killer (NK) cells can killtumor cells in tissues and in the bloodstream, thusare important in the control of systemic metastaticspread. In the course of the immune response manycytokines released by various cell types have regula-tory and effector activities. Interferons IFN-α, IFN-β,and IFN-γ and tumor necrosis factors TNF-α andTNF-β, in addition to their roles as internal mediatorsof the immune system, directly inhibit tumor cell pro-liferation, trigger apoptosis, and induce the secretionof anti-angiogenic chemokines like MIG and IP-10(2).

Immune Surveillance

The 3immune surveillance hypothesis, originally pro-posed in the late 1950s, postulates that the immunesystem protects the host not only from infectiousagents, but also from tumor onset (1). Two predictionscan be derived from the theory:* tumors that grow despite the immune system have

found a way to escape surveillance, thus must bepoorly immunogenic

* tumor incidence should be higher in immunode-pressed than in immunocompetent individuals.

The low immunogenicity of spontaneous (as opposedto carcinogen-induced or viral-induced) tumors inmice was easily verified, and is also a property ofhuman tumors. Demonstration of the second predic-tion has been more controversial, because the degreeand duration of immunodepression in experimentalsystems and in human conditions is highly variable

670 Tumor, Immune Response to

and rarely complete. Only recently, with the advent ofknockout mice, has it been clearly demonstrated thataging immunodepressed mice develop significantlymore tumors than immunocompetent mice (1). Tumorsarising in such immunodepressed mice are more im-munogenic than tumors of immunocompetent mice,thus providing a further demonstration of the hypoth-esis. In long-term immunodepressed adult humans (e.g. transplant recipients or HIV-infected patients) theincidence of virus-induced tumors (such as Kaposisarcoma or cervical carcinoma) is increased, butmany other tumor types display an incidence similarto that of the immunocompetent population.

Tumor Antigens

The search for 3tumor antigens in human tumors wasconducted for many years by means of antisera andmonoclonal antibodies obtained after immunization ofrodents with human cells or tissues. This endeavor ledto the discovery of a wealth of molecules expressed byhuman tumors that are recognized by xenogeneic anti-bodies. However some molecules detected by rodentantibodies display little or no antigenicity in thehuman species, or data on recognition by the humanimmune system are not available. Application of theterm “tumor antigens” to molecules that are not recog-nized as such in the species of origin is inappropriate,whereas “tumor markers” is more appropriate. Eventhough the immunological import of tumor markers isdubious, they have a great clinical relevance in tumordiagnosis, prognosis, and follow-up. Some examplesare lactate dehydrogenase (used to monitor treatmentof testicular cancer, Ewing’s sarcoma and other humantumors), neuron-specific enolase (neuroblastoma andsmall cell lung cancer), and DU-PAN-2 (pancreaticcarcinoma).To distinguish “true” tumor antigens, that can induce aspecific immune response in the species of origin,leading to tumor rejection, the terms “tumor rejectionantigens” or “tumor specific transplantation antigens”are sometimes used. Having clearly established thedistinction between tumor markers and tumor anti-gens, here we will simply use the latter term. Molec-ular cloning of tumor antigens became possible in the1980s thanks to technologies based on T cell recogni-tion under syngeneic or autologous conditions. Identi-fication of tumor antigens and measure of specificresponses are currently based on T cell clones withhelper or cytotoxic activity in vitro, identification ofpeptides bound to major histocompatibility complex(MHC) molecules on the surface of tumor cells, mo-lecular cloning of T cell receptor (TCR) genes from

3tumor-infiltrating lymphocytes (TIL), soluble

3MHC tetramers produced in vitro and bound to syn-thetic peptides, and screening of DNA libraries withpatient’s sera ( 3SEREX) (2). It can be noted that

SEREX is antibody-based, however it makes use ofhigh affinity human IgGs that derive from a Th-in-duced immunoglobulin class switch, thus SEREXcan be viewed as a T-B hybrid technology.The main groups of tumor antigens are shown inTable 1 (3). One major fact is that most tumor antigensare not tumor specific. The protein expressed by tumorcells, and the antigenic peptides derived from it areidentical to those of normal cells, thus leading to theconclusion that the immune response to tumors is ac-tually an autoimmune response. Experimental andclinical proofs of the autoimmune nature of anti-tumor immune responses were obtained in melano-ma-bearing individuals, who develop autoimmune vi-tiligo as a consequence of vaccination with tumor anti-gens (2). The autologous nature of many tumor anti-gens is one of the reasons why tumors are poorly im-munogenic, suggesting that a break of immune toler-ance is a prerequisite to an effective anti-tumor im-mune response. In a few cases immune tolerancedoes not operate, either because normal cells expres-sing the antigen are in immunologically privilegedsites (e.g. 3cancer-testis antigens), or because the an-tigen is involved in a physiological network of im-mune responses (idiotypes of T and B cell neoplasms).The only truly 3tumor-specific antigens are those thatderive from mutations of oncogenes (RAS, CDK4) ortumor suppressor genes (p53), from chimeric proteinsencoded by chromosomal translocations (BCR-ABL),or from tumor-specific alternative splicing (MUC-1,possibly HER-2). Experimental evidence shows that,even when tumor antigens are not shared by normalcells and are tumor-specific, spontaneous immune re-sponses are quite low and ineffective in the tumor-bearing host.

Low Immunogenicity of Tumors

A complete understanding of the reasons why tumorsare poorly immunogenic is of paramount importanceto devise immunotherapeutic strategies to induce aprotective response (1). Basically tumors are toleratedby the immune system because their antigenic profileis almost identical to that of normal cells. In addition,genetic instability of tumor cells generates a largearray of phenotypes that can escape immune recogni-tion using a variety of passive and active strategies.Down-regulation of antigen expression is an obviousalternative that has been incompletely investigated.The most common defect in human tumors (80%–90% of all solid tumors) is a partial down-regulationof MHC class I molecules required for peptide bindingand T cell recognition (4). Active strategies may in-clude the induction of regulatory (i.e. suppressive)cells of myeloid (CD11b+/Gr1+) or lymphoid (CD4+/CD25+) origin, the secretion of suppressive cytokineslike TGF-β or IL-10, or the expression of pro-apopto-

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tic surface molecules (1). On top of all immune reg-ulations, an expanding tumor could overcome the im-mune response by sheer cell kinetics.

From Tumor Immunology to Immunotherapy

Spontaneous immune responses are incapable of era-dicating established tumors (spontaneous regressionhas been rarely described in human melanoma andrenal cell carcinoma). Preclinical evidence demon-strates that the immune response, if properly activated,can cure tumors. Analogous conclusions can be drawnfrom some successful clinical approaches. A convin-cing clinical example is the ability of allogeneic T celltransplants to reduce the risk of leukemic relapse by30%–40%, a phenomenon known as “graft versus leu-kemia” (GvL). The main strategies to induce a thera-peutic immune response in human patients are basedon the administration of preformed immunologic“drugs” ( 3passive immunotherapy) or of therapeuticvaccines ( 3active immunotherapy).Passive immunotherapy is currently the most success-ful way to target human tumors (2). A small number ofmonoclonal antibodies with significant activity againsthuman tumors emerged from clinical trials and is ap-proved for clinical use. The best known examples aretrastuzumab (Herceptin), a humanized monoclonal an-

tibody against HER-2 for breast cancer, and rituximab,a monoclonal antibody against CD20 for non-Hodg-kin’s lymphoma (NHL). Several other monoclonalantibodies against similar, or different, target antigensare being developed. It is interesting to note that thetherapeutic activity of monoclonal antibodies is onlypartly mediated by classical immune functions such ascomplement-mediated cytotoxicity and 3antibody-de-pendent cell-mediated cytotoxicity 3(ADCC). Thera-peutic effect is also attributable to the activity ofmonoclonal antibodies as “receptor antagonists”,through the inhibition of receptor dimerization andsignaling, and the induction of receptor internalizationand degradation.Immunotherapy with cytokines (2) received a consid-erable attention throughout the 1980s and 1990s. Themajor clinical drawback has been the high toxicity ofcytokines. Immune cytokines physiologically reachhigh local concentrations, but high systemic dosagesare usually associated with severe toxicity. Toxicityhampered the clinical development of promising mo-lecules such as IL-2, TNF-α, and IL-12. IFN-α is agood example of a cytokine that can be administeredsystemically at active dosages to cancer patients withtolerable toxicity. IFN-α initially showed therapeuticactivity against hairy cell leukemia, and has signifi-

Tumor, Immune Response to. Table 1 Examples of tumor antigens

Tumor antigen group Examples*

Cancer-testis antigens MAGE-A1–A12, B1–B4, C1, C2BAGEGAGE-1–8NY-ESO-1

Differentiation or lineage-specific tumor antigens gp100Melan-A (MART-1)Prostate specific antigen (PSA)TyrosinaseTyrosinase-related proteins (TRP)

Shared tumor antigens Carcinoembryonic antigen (CEA)HER-2/neuMUC-1Telomerase catalytic unit (TERT)

Mutated antigens RASβ-cateninCyclin-dependent kinase 4 (CDK4)MUM-1p53

Fusion proteins BCR-ABLPML-RARαPAX3-FKHRSYT-SSX1/2EWS-WT1, EWS-FLI1

* Complete listing is given in reference 3.

672 Tumor, Immune Response to

cantly prolonged survival in chronic myeloid leukemia(CML). It is also used for some solid tumors, such asmelanoma, with a significantly lower activity thanagainst hematologic malignancies. As previouslynoted for therapeutic monoclonal antibodies, IFN-αowes its anti-tumor activity to a combination of im-mune and non-immune effects. The latter include in-hibition of tumor cell proliferation, induction of celldifferentiation, and inhibition of neo-angiogenesis.Molecular definition of tumor antigens prompted alarge number of vaccination trials based on a varietyof immunological approaches to 3cancer vaccines (2).One possibility is to vaccinate with the DNA encodinga tumor antigen that is picked up and translated byhost cells (DNA vaccination). Alternatively, vaccinesare made of whole cells, recombinant proteins or syn-thetic peptides admixed with adjuvants. Dendritic cell-based vaccines exploit the pivotal role of antigen pre-sentation in the generation of T cell responses. Den-dritic cells cultured in vitro are fed (“pulsed”) withtumor antigens and then injected in vivo. Promisingresults were obtained in small phase I/II clinical trials,but definitive evidence of a marked clinical benefitfrom therapeutic cancer vaccines is still lacking.


Study of the immune response to tumors was largelyconducted in preclinical model systems. The resultswere mostly confirmed by human studies, when pos-sible, thus it is generally assumed that preclinical ev-idence and features of the immune response to tumorsapply to human and clinical situations.One area that requires caution is the toxicity of cyto-kines endowed with anti-tumor activity. Because ofspecies specificity, human cytokines are inactive, orpartially active in rodents, therefore mouse cytokinesmust be used for mouse studies. This situation is quitedifferent from the development and testing of conven-tional anticancer drugs, in which the same molecule isused in preclinical and in clinical studies. Some cyto-kines like TNF-α that display a potent anti-tumor ac-tivity in mice are clinically useless because in humansthe maximum tolerated dose is much lower than theeffective dose (2).A major stumbling block for cancer vaccines, as wellas for other forms of tumor immunotherapy, is the factthat most clinical trials recruit advanced patients thatare heavily immunosuppressed and poorly responsiveto vaccines, whereas preclinical data clearly show thatthe ideal use of vaccines would be for cancer preven-tion in healthy individuals at risk, or for adjuvant ther-apy against micrometastatic foci, rather than for ther-apy of bulky and advanced lesions (5). In conclusion itis conceivable that active immunotherapy will demon-strate its anti-tumor potential only when clinical stu-

dies will follow the path clearly marked by preclinicaldata.

Relevance to HumansCancer patients treated with monoclonal antibodiesrespond to therapy only if the tumor expresses highlevels of the target antigen, thus demonstration of highantigen levels in tumor lesions is a prerequisite fortherapy. Some indications are available for specificantigens (e.g. HER-2), for which clinical benefit ofantibody therapy at intermediate antigen levels is du-bious.Assessment of the anti-tumor immune response in can-cer patients is not routinely performed outside clinicaltrials of immunotherapy. Most immune tests applied inpatients receiving immunotherapy are not standar-dized, and in some instances are of questionablevalue. For example it is not clear if tests performedon peripheral blood lymphocytes (the easiest samplingroute) correlate with the immune response at tumorsites. Correlation of positive or negative clinical re-sults with the immune status of patients and with mod-ifications of the immune response induced by immu-notherapy is a major open issue (2).

Regulatory EnvironmentPreclinical data are required to design clinical trials,but analysis of human immune responses to tumors isconfined to in vitro systems, thus there is no need forguidelines concerning animal testing. Within clinicaltrials, study of the immune response is highly depen-dent on treatment (e.g. type of vaccine or cytokinesused) and in most instances there are no gold standardsor guidelines pertinent to immune testing of cancerpatients for what concerns antibody responses, cyto-kine release or T cell cytotoxicity against autologousor non-autologous tumor cells. Skin tests (cf. delayed-type hypersensitivity) are used to detect responses eli-cited by anti-tumor vaccines.Immunotherapy trials are being conducted with a widerange of approaches that span practically all classes oftherapeutic agents and therapies. The range of adverseor unwanted effects that can affect patients is corre-spondingly wide. In addition to general toxicity of thetherapeutic agent, and to the presence of contaminantsin the preparation, a specific type of potential adverseeffect of cancer immunotherapy is the induction ofautoimmunity. In practice all treatments aimed at in-ducing an immune response against tumor antigensshared by normal cells involve an autoimmune re-sponse. It must be underlined that regulatory require-ments for prophylactic vaccines to be administered tohealthy individuals in the general population are quitedifferent from those applying to cancer patients. In factsome relatively mild forms of autoimmunity inducedby immunotherapy in cancer patients, such as vitiligo

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in melanoma, are regarded as surrogate markers ofanti-tumor immune response (2).In various instances a single therapeutic rationale canbe implemented with different treatment modalitiesthat depend on different regulatory environments.For example, to enhance tumor antigen recognitioncytokines can be administered systemically to the pa-tient (drug therapy), or tumor cells can be geneticallymodified to secrete the cytokine (gene therapy), or befused with autologous or allogeneic dendritic cells(adoptive cell therapy). A complete listing of all theguidances, guidelines, and regulations pertaining toeach and every immunotherapeutic approach goes be-yond the scope of this article. The reader is referred tothe web sites of the regulatory bodies for comprehen-sive listings and full text of documents.

Regulatory Bodies and Agencies

* European Agency for the Evaluation of MedicinalProducts (EMEA) http://www.emea.eu.int

* European portal to the pharmaceutical regulatorysector (EudraPORTAL) http://www.eudra.org

* US Food and Drug Administration (FDA) http://www.fda.gov

* FDA Center for Biologics Evaluation and Research(CBER) http://www.fda.gov/cber

* FDA Center for Drug Evaluation and Research(CDER) http://www.fda.gov/cder

* Japanese Ministry for Health, Labour and Welfarehttp://www.mhlw.go.jp

* Organization for Economic Co-operation and De-velopment (OECD) http://www.oecd.org

* International Conference on Harmonisation ofTechnical Requirements for Registration of Phar-maceuticals for Human Use (ICH) http://www.ich.org

References

1. Pardoll D (2003) Does the immune system see tumors asforeign or self? Ann Rev Immunol 21:807–839

2. Rosenberg SA (2000) Principles and practice of thebiologic therapy of cancer. Lippincott Williams &Wilkins, Philadelphia

3. Renkvist N, Castelli C, Robbins PF, Parmiani G (2001) Alisting of human tumor antigens recognized by T cells.Cancer Immunology Immunotherapy 50:3–15

4. Garrido F, Algarra I (2001) MHC antigens and tumorescape from immune surveillance. Adv Cancer Res83:117–158

5. Lollini PL, Forni G (2002) Antitumor vaccines: is itpossible to prevent a tumor? Cancer Immunol Immun-other 51:409–416

Tumor Immunology


Tumor-Infiltrating Lymphocytes

Lymphocytes (usually T cells) isolated from tumorspecimens. Tumor-infiltrating lymphocytes (TILs)can be cultured in vitro to analyze their functionaland molecular features, and can be also injected invivo for therapeutic purposes.


Tumor Necrosis Factor (TNF)

TNF-α and TNF-β lymphotoxin are produced bymacrophages and T lymphocytes. First described ascytotoxins for tumor cells, later as important cytokinesfor the inflammatory response, cooperation with otherleukocytes, induction of fever and interference with fatmetabolism; therefore TNF is also named cachectin.

3Cytokines

Tumor Necrosis Factor-α

Victor J Johnson

Toxicology and Molecular Biology BranchNational Institute for Occupational Safety and Health1095 Willowdale RoadMorgantown, WV 26505USA

Synonyms

tumor necrosis factor-α, lymphotoxin, cachectin,TNF-α

Definition

Tumor necrosis factor-α (TNF-α) is a pleiotropicproinflammatory cytokine that mediates key roles inhomeostasis, cell growth and proliferation, tissue da-mage, repair and chronic diseases. TNF-α productionis induced by a plethora of stimuli including bacterialproducts, oxidative stress, other cytokines, and generaltissue damage. As such, this cytokine has a central rolein orchestrating many injury and disease states includ-ing immunotoxicity.

674 Tumor Immunology

Molecular Characteristics and Mediators ofSignaling

Human TNF-α is synthesized as a 26-kDa pro cyto-kine destined for expression on the plasma membrane.Proteolytic processing by members of the matrix me-talloproteinase family of enzymes results in the extra-cellular release of the mature soluble 17-kDa form ofTNF-α. Both the membrane-bound and soluble formsare biologically active and play important roles inoverlapping and distinct signaling processes. Signal-ing is achieved through ligation of two structurallydistinct receptor subtypes, TNF-receptor 1 (TNF-R1)and TNF-R2. TNF-R1 is constitutively expressed onmost nucleated cells whereas TNF-R2 has a more re-stricted expression, mainly on cells of the immunesystem and is inducible. A schematic of the major

signaling pathways and molecular mediators isshown in Figure 1.Membrane and soluble TNF-α form trimers that in-duce the trimerization of the TNF- receptor upon bind-ing. Activation of the receptor initiates the formationof unique signaling complexes that are distinct foreach receptor subtype. A 3death-inducing signalingcomplex is formed at the intracellular domain ofTNF-R1 involving the recruitment and binding of anumber of accessory proteins, including TNF-R1-as-sociated death-domain-containing factor (TRADD),Fas-associated death-domain-containing protein(FADD) and TNF-R-associated factor-2 (TRAF2).Binding is achieved through mutual death domains(DD) present on TNF-R1 and the accessory proteins,and the DD sequence is unique to the intracellularportion of TNF-R1. The resulting complex recruits

Tumor Necrosis Factor-α. Figure 1 Death and survival pathways in tumor necrosis factor (TNF)-α signaling.TNF-α exerts its biological effects through ligation of two distinct receptors, TNF-R1 and TNF-R2. Activation ofTNF-R1 results in the formation of a death-inducing signaling complex, consisting of TNF-R1 intracellular DD,TRADD, FADD and TRAF2. This complex recruits other intracellular signaling molecules that activate pathwaysculminating in cell death (caspase and JNK pathways) and cell survival (NFκB activation pathway). Additionally,activation of MAP kinases and NFκB can result in upregulation of genes involved in inflammation, including TNF-αitself. On the other hand, activation of TNF-R2 leads to the formation of a signaling complex via mutual TRAFdomains. This complex is known to lead to NFκB activation and anti-apoptotic signaling. Increasing evidencesuggests a role for TNF-R2 in apoptosis, possibly through potentiation of TNF-R1 pro-apoptotic signaling. Overall,these signaling pathways can contribute to immunotoxicity by directly inducing cell death and tissue damage,initiating and contributing to inflammation, and/or altering the proliferative capacity of cells and tissues.

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other proteins with enzymatic activity culminating inthe induction of several major signaling pathways, in-cluding the caspase pathway, mitogen-activated pro-tein (MAP) kinase pathways and pathways that leadto nuclear factor κB (NFκB) activation. The cell deathand tumor regression properties of TNF-α are attrib-uted to TNF-R1-mediated activation of caspases andc-Jun NH2-terminal kinase (JNK). JNK activation hasbeen shown to cleave the Bcl-2 interacting domain(Bid) resulting in jBid translocation to the mitochon-dria. This induces the release of Smac/DIABLO fromthe mitochondria, which then sequesters inhibitor ofapoptosis (cIAP) proteins leading to FADD-inducedactivation of caspase 8. Caspase 8 activates effectorcaspases 3 and 7 leading to the cleavage of severalintracellular proteins and apoptotic cell death. Apop-tosis is not the predominant outcome in most cell typesin vivo and can be prevented through the activation ofthe NFκB survival pathway. Activation of NFκB re-sults from the coordinated action of receptor interact-ing protein (RIP), NFκB inducing kinase (NIK) andinhibitor of κB (IκB) kinases. The outcome is phos-phorylation-dependent ubiquitination and degradationof IκB, which results in the release of NFκB into thecytoplasm which then translocates to the nucleus via anuclear localization sequence. Heterodimers andhomodimers of the NFκB/Rel family drive the tran-scription of many survival and inflammation genescontaining NFκB response elements. Therefore, thispathway functions to prevent cell death in normalhealthy cells and upregulates the production of pro-teins involved in inflammatory processes.The intracellular domain of TNF-R2 lacks the DD andinstead has TRAF domains responsible for the recruit-ment of TRAF1, TRAF2, and TRAF3. This complexrecruits cIAP and NIK both responsible for NFκB-mediated anti-apoptotic signaling. However, severalinvestigators have reported that TNF-R2 may be im-portant in the regulation and potentiation of TNF-R1-induced apoptosis. Several mechanisms have beenproposed, including TNF-R2, acting as a high-affinitytrap/ 3ligand passer and TNF-R2-induced upregula-tion of endogenous TNF-α production, both leadingto autocrine and paracrine activation of TNF-R1-mediated apoptosis. A dual role in cell survival anddeath has been shown such that in the absence of TNF-R1 signaling, TNF-R2 promotes not only proliferationof naïve T lymphocytes but also apoptosis in activatedCD8+ T lymphocytes. Additionally, the affinity ofTNF-R2 for membrane-bound TNF-α is much greaterthan that for the soluble form (affinities are equal forTNF-R1), suggesting that TNF-R2 is important in di-rect cell to cell regulation and localized immune re-sponses. TNF-R2 can also be shed from the plasmamembrane resulting in soluble TNF-R and downregu-lation of TNF-α signaling.

Relevance to ImmunotoxicityTNF-α is produced by many cell types including im-mune cells (macrophages, monocytes, dendritic cells,T lymphocytes, B lymphocytes), endothelial cells, epi-thelial cells, and fibroblasts following activation byappropriate stimuli. Therefore, this cytokine can beenvisioned to play a role in immunotoxicity in manytarget organs. Indeed, TNF-α, through its ability toinfluence inflammatory processes, has been demon-strated in response to immuntoxicants targeting theliver, kidney, lung, muscle, eye, skin and brain, toname a few sites. Gene knockout of TNF-α, its recep-tors, or neutralizing-antibody studies have been usedto investigate the role of TNF-α in immunotoxicity.For example, mice deficient in TNF-R1 and TNF-R2show reduced lung inflammation and cytokine chang-es in response to toluene diisocyanante, an occupation-al asthmogen. Removal of TNF- signaling almostcompletely abrogated inflammation and fibrosis inthe liver following treatment with the known immu-notoxicant, carbon tertrachloride. TNF-α plays an im-portant role in the immunotoxicity of many mycotox-ins such as vomitoxin and fumonisin B1. KidneyTNF-α levels increase in adriamycin-induced neph-ropathy and may play a direct role in the associatedproteinuria. Immediate production of TNF-α in theskin is evident following exposure to agents thatcause allergic and irritant contact dermatitis and ispositively correlated with the inflammatory response.Significantly, mice deficient in TNF-Rs show reduceddevelopment of contact dermatitis. In addition to itsrole in chemical-mediated immunotoxicity, TNF-αalso mediates critical events in the pathogenesis ofiodiopathic diseases involving the immune system, in-cluding bacterial infection and sepsis, chronic inflam-matory lung diseases, cancer, and autoimmunity.The vastness of TNF-α involvement in chemical andidiopathic immunotoxicity stems from its central rolein many cytokine/chemokine networks. TNF-α signal-ing (see Figure 1) can modulate the expression of otherimportant mediators of inflammation required for therecruitment and activation of effector cells (macro-phages, lymphocytes, neutrophils, eosinophils) thatcan contribute to tissue injury. Therefore, enhancedsynthesis and release of TNF-α following immunotox-icant exposure or during disease can initiate and ex-acerbate acute and chronic inflammation—knowncontributors to tissue injury, repair and remodeling (fi-brosis).

Relevance to HumansExamining the association between genetics and dis-ease prevalence and disease severity provides valuableinsight into the mechanisms of disease. As such, great-er than 1000 scientific studies have been conductedinvestigating the association between polymorphisms

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in the TNF family and diverse human diseases. Genet-ic variation in TNF-α has been associated with chem-ical toxicities and idiopathic diseases of the immunesystem and diseases with immune involvement. Exam-ples include occupational lung diseases, such as sili-cosis and coal workers' pneumoconiosis, chemothera-py-induced pulmonary fibrosis, adverse drug reac-tions, response to hepatitis B vaccination, asthma, di-abetes, and rheumatoid arthritis. The strong associa-tion with human disease has prompted research intopotential therapies related to inhibition of TNF-α sig-naling. To date, several US FDA approved protein-based injectable inhibitors that block TNF-TNF-R in-teractions have been used to successfully treat humandisease, including rheumatoid arthritis and juvenilechronic arthritis. Ongoing clinical trials show promisefor these therapeutics in other diseases like psoriasis,psoriatic arthritis, ankylosing spondylitis, and Crohn’sdisease. Second-generation small-molecule inhibitorsof TNF are now undergoing clinical trials and functionthrough blocking specific mediators in the TNF signal-ing cascade. Caution must be exercised in the use ofthese treatments as side effects including potential ex-acerbation of congestive heart failure, activation oflatent tuberculosis infection, development of antinu-clear antibodies, and systemic lupus erythematosishave been reported. Nevertheless, TNF-α is a pinnaclecytokine in acute and chronic inflammatory diseaseand toxicity and represents a promising therapeutictarget.

References

1. Luster MI, Simeonova PP, Gallucci R, Matheson J (1999)Tumor necrosis factor alpha and toxicology. Crit RevToxicol 29:491–511

2. Palladino MA, Bahjat FR, Theodorakis EA, MoldawerLL (2003) Anti-TNF-α therapies: the next generation.Nature Rev Drug Discov 2:736–746

3. Gupta S (2002) A decision between life and death duringTNF-α-induced signaling. J Clin Immunol 22:185–194

4. Chen G, Goeddel DV (2002) TNF-R1 signaling: abeautiful pathway. Science 31:1634–1635

5. Lui Z-G (2004) Adding facets to TNF signaling: the JNKangle. Mol Cell 12:795–796

6. Schook L, Laskin D (eds) (1994) Xenobiotics andInflammation. Academic Press, San Diego CA

Tumor Necrosis Factor Receptor-Associated Factor-6

TRAF-6 induces multiple signals from TOLL-like re-ceptors that sense infection.

3Interleukin-1β (IL-1β)

Tumor-Specific Antigen

Antigen expressed by tumor cells, but not by normalcells. Truly specific tumor antigens are generated byoncogenic genetic lesions, such as mutations in onco-genes and tumor suppressor genes, or chromosomalrearrangements leading to the synthesis of fusion pro-teins. The idiotype of T and B cell receptor in lym-phoid malignancies is considered a tumor-specific an-tigen (but there might be non-neoplastic clones sharingthe same idiotype). Cancer-testis antigens are also con-sidered tumor specific because male germ line cells(the only normal cell type sharing such antigens)lack MHC expression, thus cannot present the antigento the immune system.


Type I Error

A decision error in which a true null hypothesis isincorrectly rejected.

3Statistics in Immunotoxicology

Type I Reactions According to Gelland Coombs

3IgE-Mediated Allergies

Type I–IV Reactions

Gell and Coombs described antibody and T cell-mediated reactions with distinct clinical pathologyand underlying pathomechanism. The type IV reac-tions can be subdivided in type IVa–IVd reactions,which reflect the involvement of distinct effector cells.

3Lymphocyte Transformation Test

3Hypersensitivity Reactions

Type 1 or Type 2 T Cell Responses

Subset of T lymphocytes called T helper (Th) cells canrespond to different stimuli by secreting different cy-tokine patterns. Two well characterized patterns arecategorized as type 1 (Th1) and type 2 (Th2) re-sponses. Type 1 responses promote inflammationand cell mediated immunity primarily through the pro-duction of IFN-γ. Type 2 responses promote allergies

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and antibody mediated responses primarily throughproduction of IL-4 and IL-5. A type 1 response isantagonistic to a type 2 response and vice versa.

3Cytokine Assays

Type II Activation

3Macrophage Activation

Type II Error

A decision error in which a false null hypothesis is notrejected.

3Statistics in Immunotoxicology

Type II Interferon

3Interferon-γ

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