The TIM gene family: emerging roles in immunity and disease

© 2003 Nature Publishing Group

454 | JUNE 2003 | VOLUME 3 www.nature.com/reviews/immunol

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THE TIM GENE FAMILY: EMERGINGROLES IN IMMUNITY AND DISEASEVijay K. Kuchroo*, Dale T. Umetsu‡, Rosemarie H. DeKruyff‡ and Gordon J. Freeman§

The search for cell-surface markers that can distinguish T helper 1 (TH1) cells from TH2 cells has ledto the identification of a new gene family, encoding the T-cell immunoglobulin mucin (TIM) proteins,some of which are differentially expressed by TH1 and TH2 cells. The role of the TIM-family proteinsin immune regulation is just beginning to emerge. Here, we describe the various TIM-familymembers in mice and humans, and discuss the genetic and functional evidence for their role inregulating autoimmune and allergic diseases.

TH1-cell-specific molecule9. However, the differences in

expression of most of these molecules between TH1 and

TH2 cells are quantitative. Furthermore, many of these

molecules are present on naive or recently activated T cells and are up or downregulated by the T-cell sub-sets after differentiation. While generating clonotypicantibodies specific for mouse T-cell receptor (TCR)-transgenic T cells and defined T

H1-cell clones, V.K.K.’s

group identified four monoclonal antibodies thatstained the cell surface of T

H1 but not T

H2 cells.

Expression cloning with two of the antibodies resultedin the identification of a new gene, now known as T-cell immunoglobulin mucin 3 (TIM3), which isexpressed only by differentiated T

H1 cells10. Further

genetic and genomic analyses indicated that this genebelongs to the TIM family of genes, which are found inboth mice and humans. The gene family is known asTIM because these proteins are expressed by T cells andcontain an immunoglobulin V-like domain and amucin-like domain (FIG. 1). The first TIM gene to beidentified, TIM1, is expressed by the kidney and liver,and was initially cloned as kidney injury molecule 1(KIM1) and hepatitis A virus cellular receptor(HAVCR)11−15. We have recently shown that TIM1 isalso expressed by T cells and, as well as its role in renal-tissue injury, TIM1 might have an important role inregulating immune responses. As we discuss, recentgenetic studies in mice and humans, as well as epidemi-ological data that link infection with hepatitis A virusand atopic disease, indicate that the TIM family hasimportant immunological functions. The role of TIM

Activation of naive CD4+ T helper (TH) cells results in the

development of at least two phenotypically and function-ally distinct effector populations, T

H1 and T

H2 cells1,2. T

H1

cells produce the cytokines interferon-γ(IFN-γ), inter-leukin-2 (IL-2) and lymphotoxin, which are commonlyassociated with cell-mediated immune responses againstintracellular pathogens, delayed-type hypersensitivityreactions and induction of organ-specific autoimmunediseases, such as EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

(EAE) and type-I diabetes3−5. TH

2 cells producecytokines that are crucial for the control of extracellularhelminth infections, such as IL-4, IL-5, IL-6, IL-10 andIL-13. Production of T

H2 cytokines (classically IL-4 and

IL-13) is also observed in atopic and allergic diseasesand is usually accompanied by increased production ofIgG1 and IgE, and by activation of eosinophils and mastcells3,4. Also, each T

H-cell subset can cross-regulate the

clonal expansion and function of the other. So, preferen-tial induction of T

H2 cells can inhibit autoimmune dis-

eases, and preferential induction of TH1 cells can regu-

late the induction of asthma, atopy and allergy.Although much is known about the functional proper-ties of these subsets, few known cell-surface moleculeshave been shown to distinguish between them. Severalgroups have reported expression of the chemokinereceptors CCR5 and CXCR3 by T

H1 cells, and CCR3,

CCR4, CXCR4 and CCR8, as well as the co-stimulatoryreceptors T1 (also known as ST2) and inducible T-cellco-stimulator (ICOS), by T

H2 cells6−8. Similarly,

Chandra, a multiple-transmembrane-spanning mole-cule of unknown function, has also been identified as a

*Center for NeurologicDiseases, Brigham andWomen’s Hospital and§Department of MedicalOncology, Dana-FarberCancer Institute, HarvardMedical School, 77 AvenueLouis Pasteur, Boston,Massachusetts 02115, USA.‡Division of Immunologyand Allergy, Department of Pediatrics, StanfordUniversity School of Medicine,300 Pasteur Drive, Stanford,California 94305, USA.Correspondence to V.K.K.e-mail: [email protected]:10.1038/nri1111

EXPERIMENTAL AUTOIMMUNE

ENCEPHALOMYELITIS

(EAE). An experimental modelfor the human disease multiplesclerosis (MS). Autoimmunedisease is induced inexperimental animals byimmunization with myelinantigen or peptides derivedfrom myelin. The animalsdevelop a paralytic diseasewith inflammation anddemyelination in the brain and spinal cord.


NATURE REVIEWS | IMMUNOLOGY VOLUME 3 | JUNE 2003 | 455

R E V I E W S

structural similarity to the TIM molecules, in that it con-tains two immunoglobulin domains and one mucindomain17. The MADCAM1 immunoglobulin domainbinds to α

4β

7integrin and, although there is only a low

level of identity between TIM proteins and MADCAM1,both mouse and human TIM4 contain an RGD MOTIF,indicating that they might also have an integrin ligand.

The cytoplasmic domain of TIM gene-family mem-bers consists of 42−77 amino acids and is the mosthighly conserved domain between mouse and humanorthologues10. The TIM1, TIM2 and TIM3 proteinscontain a predicted intracellular tyrosine-kinase phos-phorylation motif. The cytoplasmic domain of TIM1has two splice variants18 — the TIM1a variant is mainlyexpressed by human liver and lacks a tyrosine-kinasephosphorylation motif, indicating that it might func-tion as a ‘decoy’ receptor, whereas the TIM1b variant ismainly expressed by human kidney and contains twotyrosine residues, including a highly conserved tyrosine-kinase phosphorylation motif, RAEDNIY. TIM2 con-tains an intracellular tyrosine-kinase phosphorylationmotif, RTRCEDGVY, which is tyrosine phosphorylatedafter T-cell activation19. This indicates that TIM2 is afunctional receptor that transduces signals through thephosphorylated tyrosine residue. Similarly, TIM3 con-tains a distinct, conserved tyrosine-kinase phosphoryla-tion motif and an SRC-homology 2 (SH2)-domainbinding motif in the cytoplasmic tail. By contrast, thecytoplasmic domain of TIM4 is different from the otherfamily members and has no conserved tyrosines, indicat-ing that it might also be a decoy receptor. The structural

proteins in T-cell differentiation, T-cell effector func-tion and diseases such as autoimmunity, allergy andasthma are just beginning to be understood10,16.

Structure of the TIM genes and proteinsSo far, the TIM family consists of eight genes on mousechromosome 11B1.1 and three genes on the SYNTENIC

human chromosome 5q33.2, with no other interveninggenes (FIG. 2). The three human TIM genes are most sim-ilar to mouse Tim1, Tim3 and Tim4 (FIG. 3). In addition,there are TIM pseudogenes on other chromosomes, asindicated by a lack of introns or a clearly discernabletranslation-initiation site.

The immunoglobulin V domain of all TIM-familymembers contains four conserved cysteines, in additionto the two conserved cysteines in the immunoglobulindomain, indicating that the structure is highly con-served and crosslinked. The size of the mucin domainvaries considerably between members, but, they are allrich in threonine, serine and proline. The mucin-likestructure is predicted to have an extended conformationand be heavily O-glycosylated. TIM3 has the shortestmucin domain and only a moderate level of predictedglycosylation. The mucin domain is encoded by 2−4exons, and some heterogeneity in the usage of theseexons has been observed.

As discussed later, a ligand for mouse Tim2, but notTim3, has been shown to be semaphorin 4A (Sema4A).However, the ligands for the other TIM proteins have notyet been identified. The cell adhesion molecule mucosaladdressin cell-adhesion molecule 1 (MADCAM1) has

Tim1

32

Tim2

30

Tim3

5

Tim4

43

TIM1(HAVCR1)

56

TIM3

1

TIM4

38

Immunoglobulin V domain

Mucin domain

N-linked glyosylation site

O-linked glycosylation site

Transmembrane domain

Cytoplasmic domain

Tyrosine-kinasephosphorylation motif

Plasma membrane

RGD motif

a bMouse Human

Figure 1 | Schematic representation of TIM-protein structures. Glycosylation sites were predicted using NetOglyc and NetNglyc (see further information web site)on the basis of sequence context and surface accessibility. HAVCR, hepatitis A virus cellular receptor; TIM, T-cell immunoglobulin mucin.

SYNTENY

Colinearity in the order of genes(or of other DNA sequences) ina chromosome region of twospecies.

RGD MOTIF

A peptide motif that consists ofthe amino acids arginine, glycineand aspartic acid, and is found inmany ligands that bindintegrins.



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ALLERGEN-INDUCED AIRWAY

HYPERREACTIVITY

(AHR). An experimental modelof human allergic asthma inwhich mice are first sensitized to,then challenged with, allergen.This leads to airwayhyperreactivity, a cardinalfeature of asthma in humans.

Genetic linkage of the TIM locus to autoimmunityAutoimmune diseases are complex genetic disordersthat have been linked to several chromosome regions inboth mice and humans20. In mouse models of type-1diabetes and EAE, up to 18 susceptibility loci have beenidentified, and the locus on chromosome 11, whichincludes the TIM gene family, has shown a marked asso-ciation with susceptibility to autoimmunity in manydisease models. Linkage analyses have shown that thereis an association between the chromosome 11 locus andsusceptibility to autoimmune diseases in several straincombinations, including the EAE-susceptible (SJL) andresistant (B10.S) strain combination, and diabetes-susceptible (non-obese diabetic, NOD) and resistant(C57BL/6) mice20. For EAE susceptibility, the linkedlocus, which contains the Tim gene family, is Eae6 andfor diabetes, the locus has been identified as Idd4. Bothof these loci overlap on chromosome 11, and the shar-ing of susceptibility loci between many strains of micehas been interpreted as an indication of the existenceof common autoimmune genes that affect many auto-immune diseases21,22. As the Il4 cytokine cluster, whichcontains the genes encoding IL-4, IL-5 and IL-13, islocated in the Eae6−Idd4 interval, it had been assumedthat the linkage to autoimmune susceptibility might bedue to the Il4 gene family. However, the genetic intervalhas not been reduced to a small enough size to identifythe gene. As we discuss, genetic and functional data nowindicate that the Tim gene family, which is located in thelinked genetic interval on mouse chromosome 11,might have an important role in susceptibility toautoimmune diseases.

Genetic linkage of the TIM locus to asthmaSimilar to autoimmune diseases, asthma is a complexgenetic trait and its severity is influenced by many envi-ronmental and genetic factors that interact with eachother in non-additive ways, complicating the identifica-tion of asthma-susceptibility genes. Asthma suscepti-bility has been linked to several chromosome regions,but with a resolution no better than 5−10 cM, in whichinterval there are usually hundreds of candidate genes.Moreover, because genetic variation in any single geneis likely to have only modest effects on the overallpathogenesis of asthma, and because gene−gene andgene−environment interactions confuse the analysis,positional cloning of susceptibility genes is difficult.Nevertheless, asthma susceptibility has been linked togenetic intervals on human chromosomes 5, 6, 11, 12,14 and 20 (REFS 23−26). Of these, chromosome 5q23−35has received the greatest attention, because it contains alarge number of candidate genes, including thoseencoding IL-9, IL-12 p40 and the α-adrenergic receptor,and the IL4 cytokine cluster27−30. However, the large sizeof the linked region of chromosome 5q complicates itsanalysis and a gene that affects asthma has not yet beenconclusively identified from this site.

To analyse the human chromosome region 5q23−35for asthma-susceptibility genes, McIntire et al.16 used amouse model of asthma — ALLERGEN-INDUCED AIRWAY

HYPERREACTIVITY (AHR) — and focused on the syntenic

and motif variation in the TIM family indicates thatthe intracellular signalling pathways that are engagedby individual TIM proteins after ligand binding mightbe distinct.

Finally, kidney epithelial cells produce a soluble TIM1protein. Soluble TIM1 is probably produced by cleavageof cell surface TIM1 by a metalloproteinase, as metallo-proteinase inhibitors can inhibit the production of solu-ble TIM1 (REF. 18) (BOX 1). The production of solubleTIM1 has not been examined in immune cells.

Homo sapiensChromosome 5

156.6 156.5 156.4 156.3 Megabases

Mega-bases

TIM3

Tim3 Tim5 Tim6 Tim8 Tim7 Tim2 Tim1 Tim4

TIM4TIM1/HAVCR1

46.8 46.9 47.0 47.1 47.2

11B1.1

Mus musculusChromosome 11

5q33.2

Figure 2 | Genomic organization of human and mouse TIM genes. Mouse Tim5, Tim6, Tim7and Tim8 are predicted genes. The direction and extent of messenger RNA transcription areindicated by arrows. TIM, T-cell immunoglobulin mucin.

mouse Tim7

mouse Tim6

mouse Tim8

mouse Tim5

mouse Tim2mouse Tim1

rat Kim1

mouse Tim4human TIM4

mouse Tim3

human TIM3

human TIM1

monkey Havcr1

Figure 3 | Dendrogram showing sequence similarity in the TIM gene family. TIM proteinsequences were aligned using ClustalW. Sequence similarity is proportional to the length of the linethat connects two sequences and was visualized as an unrooted tree using Drawtree. Drawtree isa component of The Biology Workbench, University of California, San Diego. HAVCR, hepatitis Avirus cellular receptor; Kim1, kidney injury molecule 1; TIM, T-cell immunoglobulin mucin.



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progeny. In the N2 mice, both of these traits segregatedinto high and low responders with a bimodal distribu-tion, confirming that genetic variation in a single locuson chromosome 11 regulates both the production ofT

H2-cell cytokines and AHR16. This single region was

defined and is now known as the T-cell and airway-phenotype regulator (Tapr). This locus was geneticallyseparable from the Il4 cytokine gene cluster and othernearby cytokine genes, such as that encoding IL-12 p40,and the Tim gene family was showed to be located inthis region. Marked polymorphisms in Tim1 and Tim3were identified that are associated with the differentionof cells to a T

H1 or T

H2 phenotype and AHR (BOX 2). The

polymorphisms in Tim1 are located in the signal andmucin-like domains, whereas the polymorphisms iden-tified in Tim3 are clustered in the immunoglobulindomain16. Importantly, these polymorphisms com-pletely co-segregate with Tapr, which indicates thatpolymorphisms in human TIM1 and/or TIM3 mightunderlie the marked association between asthma sus-ceptibility and human chromosome 5q. This idea is sup-ported by the fact that variations in the coding region ofhuman TIM1 are seen in human genome sequences andexpressed sequence tag (EST) databases. This highdegree of variation distinguishes TIM1 and other TIM-family members from many other candidate asthma-susceptibility genes, such as the cytokines and cytokinereceptors. The association between TIM1 and asthmasusceptibility is further supported by reports of linkageof mite-sensitive childhood asthma to a marker that isapproximately 0.5 Mb from TIM1 and TIM3 (REF. 29),although a transmission-disequilibrium test that wascarried out recently to analyse TIM1 polymorphisms ina Japanese population did not show an association ofTIM1 with atopic asthma35. Whether TIM1 will showany linkage with asthma and atopy in other populationsis presently being investigated. Nevertheless, the identifi-cation of TIM1 is the first example of the use of congenicmice to define genes that might contribute to the devel-opment of allergic asthma.Validation of the role of TIM1in human asthma awaits further sequencing of TIM1 inhumans and association analysis of genetic variations ofTIM1 (and TIM3 and TIM4), including variations inthe promoter regions and introns, in additional subjectswith asthma and allergy.

TIM1 in IL-4 regulation, asthma and allergyAs the TIM proteins were positionally cloned using amouse model of allergic asthma, it is probable that theTIM proteins also regulate the development of T

H2-

biased immune responses. To explore the specific func-tions of the TIM proteins and the effect of the polymor-phic variants of the TIM proteins, TCR-transgenic miceon the C.D2 Es−HBA background were generated, andcompared with TCR-transgenic mice on the BALB/cbackground16. When stimulated with specific antigenand dendritic cells (DCs), naive BALB/c TCR-transgenicT cells were found to produce markedly higher amountsof IL-4 and IL-13 than the C.D2 Es−HBA TCR-trans-genic T cells, in a manner that was independent of themouse strain of the antigen-presenting cells (APCs).

region on mouse chromosome 11. This approach offeredseveral potential advantages: environmental variationcould be controlled, many phenotypes could be testedsimultaneously and different inbred mouse strains couldbe used31. The mice used in this model included BALB/cmice, which produce high levels of IL-4 and developsevere AHR after sensitization and challenge with aller-gen, as well as DBA/2 mice, which produce low levels ofIL-4 and have normal airway responses after similar sen-sitization and challenge with allergen. Moreover, a con-genic mouse, known as C.D2 Es−HBA — in whichhaemoglobin A (HBA) is a chromosome marker used todefine the genetic interval — was generated, which has adiscrete genomic interval that is inherited from theDBA/2 chromosome 11 on the BALB/c genomic back-ground32−34. Use of these congenic mice simplified thegenetic analysis, as it was possible to characterize the con-genic locus without the effects of EPISTASIS by many othergenes that influence the asthmatic phenotype. Mostimportantly, inheritance of this DBA/2 chromosome 11segment converted the BALB/c mouse into one that pro-duced markedly less IL-4 and IL-13 in response to sensiti-zation with antigen. In addition, the C.D2 Es−HBA mice,when sensitized and challenged with allergen, showednormal airway reactivity in response to the allergen,similar to DBA/2 mice. These results indicated that theDBA/2-derived region of C.D2 Es−HBA chromosome11, which has large regions of conserved synteny withhuman chromosome 5q23−35, contains a gene(s) thatreduced the production of IL-4 and IL-13 in respone toantigen, and converted the BALB/c AHR-susceptible phe-notype into a DBA/2-like AHR-resistant phenotype. So,the use of the congenic C.D2 Es−HBA mouse allowed agenetic interval that is linked to disease susceptibility to bestudied in the absence of genetic variation outside theregion, by converting a complex genetic trait into a simpleMendelian trait.

Linkage analysis of IL-4 production and AHR withspecific genetic markers was carried out by breedingBALB/c and C.D2 Es−HBA mice and generating N2

EPISTASIS

An interaction between non-allelic genes, such that one genemasks, interferes with orenhances the expression of theother gene.

Box 1 | TIM1 and kidney injury

T-cell immunoglobulin mucin 1 (TIM1), also known as kidney injury molecule 1(KIM1), is minimally expressed by healthy kidney but highly upregulated in theproximal tubule epithelial cells in post-ischaemic kidneys11,12. Surviving epithelial cellsde-differentiate, proliferate and then reconstitute the morphology of the polarizedtubule. In kidney epithelial-cell lines, the extracellular domain of TIM1 is cleaved nearthe membrane by a metalloproteinase, releasing a soluble form18. This might explain thepresence of soluble TIM1 in the urine of patients with acute tubular necrosis12. Bailly et al.18 speculate that soluble TIM1 might bind to integrins and ‘quench’ their adhesivefunction, allowing kidney epithelial cells to move freely during regeneration. Theproduction of soluble TIM1 has not yet been examined in immune cells. But curiously,a gene encoding a disintegrin and metalloprotease protein 33 (ADAM33) on humanchromosome 20 has been shown to be an asthma-susceptibility gene26. Allelic variants of TIM1 and ADAM33 might synergize for asthma susceptibility. In addition to TIM1,ischaemic kidneys highly express several other proteins that are involved in theregulation of T

H2 cells, including inducible co-stimulatory molecule (ICOS) ligand41

and osteopontin42. It is tempting to speculate that the injured kidney expresses proteinsthat direct incoming inflammatory cells towards a more protective T

H2-cell response

rather than a destructive TH

1-cell response.



R E V I E W S

The expression of Tim1 was shown to be inducedduring the earliest stages of these in vitro responses(within 7 hours), which indicates that the BALB/c alleleof Tim1 might promote early expression of T

H2

cytokines, whereas the C.D2 Es−HBA allele of Tim1might lead to the downregulation of T

H2 cytokines.

Furthermore, under TH2-polarizing conditions, Tim1

messenger RNA is expressed by TH2-cell lines, but not

by TH1 cells, indicating that Tim1 might be expressed by

TH2 but not by T

H1 cells, in contrast to Tim3, which is

preferentially expressed by TH

1 cells10. Similarly, inhumans, TIM1 mRNA was expressed by T

H2- but not

TH1-cell lines, whereas TIM3 expression was detected in

TH1 but not T

H2 cells (G.J.F.,V.K.K., D.T.U. and R.H.D.,

unpublished observations). These data indicate thatTIM1 and TIM3 are reciprocally expressed by T

H1 and

TH

2 cells during T-cell differentiation. However, howpolymorphic alleles of TIM1 and TIM3 in humans affectthe development of both asthma and autoimmunity isnot clear as yet.

Hepatitis A virus and TIM1Human TIM1 was initially identified as the cellularreceptor for hepatitis A virus. This is intriguing, asseveral studies have noted that infection with hepatitisA virus is associated with protection against the devel-opment of asthma in humans, in both Europe36 andthe United States37, indicating that stimulation ofTIM1 might directly regulate the development ofT

H2-biased immune responses. In one study of more

than 30,000 individuals from the United States, theprevalence of hay fever and asthma in individuals thatare seropositive for hepatitis A virus was less than onethird of that in seronegative individuals after adjust-ing for socioeconomic factors37. However, becausehepatitis A virus is transmitted through the faecal ororal route, infection with hepatitis A virus has beenthought to be simply a marker of poor hygiene, whichhas also been hypothesized to protect against thedevelopment of asthma38. The binding of hepatitis Avirus to TIM1 on the surface of differentiating T

H2

cells might directly alter T-cell differentiation and theT

H1- versus T

H2-cell balance, and promote protection

against TH2-cell differentiation and atopy. This mech-

anism, which is consistent with the idea that TIM pro-teins regulate the development of T

H1- versus T

H2-cell

differentiation in mice, provides a potential molecularexplanation for the hygiene hypothesis. So, infectionwith hepatitis A virus — the incidence of which wasnearly 100% in Western countries before 1970 on thebasis of serological data — might have protected mostindividuals in the past against the development of atopy.However, due to improved hygiene, the incidence ofinfection with hepatitis A virus has been greatlyreduced, and hepatitis A virus now infects only about10−30% of individuals in Western countries. Thisreduction in the incidence of hepatitis A virus infectionmight have resulted in a marked loss of the hepatitis Avirus protective effect and a marked increase in theprevalence of atopic diseases, as predicted by thehygiene hypothesis.

Furthermore, BALB/c and C.D2 Es−HBA TCR-trans-genic T cells produced equivalent amounts of IL-2 andcomparable levels of proliferation in response to specificantigen during the secondary cultures. These resultsshowed that although C.D2 Es−HBA and BALB/c T cellswere similarly activated, the levels of T

H2 cytokines they

produced were distinct — BALB/c CD4+ T cells devel-oped a stronger T

H2-cell response than C.D2 Es−HBA

CD4+ T cells. These findings indicate that the Taprlocus might regulate T

H-cell differentiation during pri-

mary antigen-specific responses — a time when earlyexpression of cytokines is crucial in determining thesubsequent T

H-cell phenotype. However, further analy-

sis is required to determine whether this is due to allelicvariation in the TIM genes.

Box 2 | Marked polymorphisms in Tim1 and Tim3

Comparison of the sequences of the coding regions of the three T-cell immunoglobulinmucin (Tim) genes in BALB/c mice and C.D2 Es–HBA−DBA congenic mice indicated marked polymorphisms in Tim1 and Tim3, but not Tim2. In Tim1, thesepolymorphisms encode three amino-acid differences and a 15 amino-acid deletion in the C.D2 Es–HBA−DBA Tim1 gene. Seven predicted amino-acid differences wereidentified in Tim3 (REF. 16). The polymorphisms in Tim1 are located in the signal andmucin-like domains, whereas the polymorphisms that were identified in Tim3 areclustered in the immunoglobulin domain16. Genomic sequencing confirmed that thesepolymorphisms, including the deletion, are true polymorphisms and not splice variants.In glycoproteins with immunoglobulin and mucin domains, variations in either domainmight affect receptor−ligand interactions, as shown for mucosal addressin cell adhesionmolecule 1 (MADCAM1). Furthermore, although the predicted signal-cleavage site ofTim1 is unaltered by the polymorphism in the signal sequence, it is possible that thepolymorphism might affect glycosylation, as has been shown for a signal-peptidevariant of cytotoxic T lymphocyte antigen 4 (CTLA4)43, or the efficiency of cleavageand/or trafficking of the receptor to the cell surface. As human TIM1 is also the hepatitisA virus cellular receptor (HAVCR), polymorphisms in TIM1 might also alter thepathogenesis of hepatitis A virus in humans14, as discussed in the main text.

TH1

TH2

NaiveT-cell

ApoptosisInactivation

a

b

c

Hepatitis A virus

Figure 4 | Possible mechanisms by which hepatitis A virusmight reduce the development of atopic diseases. a |Hepatitis A virus might productively infect T helper 2 (TH2) cellspreferentially and eliminate them. b | Hepatitis A virus mightbind to T-cell immunoglobulin mucin 1 (TIM1) and block thedevelopment of TH2 cells. c | Hepatitis A virus might bind topolymorphic variants of TIM1 on naive T cells and enhance thedevelopment of TH1 cells.



R E V I E W S

Tim2 and its ligand semaphorin 4ATim2 is also expressed by mouse CD4+ T cells during T-cell activation16, but, it is not known whether Tim2 isdifferentially expressed after differentiation into T

H1- or

TH2-cell subsets. The structure of mouse Tim2 is closely

related to Tim1 and the encoding gene is located withthe other family members on mouse chromosome 11.There is no structural orthologue of Tim2 in humans,indicating that Tim2 might be functionally redundantwith one of the other Tim molecules. Sema4A wasrecently identified as the ligand for Tim2 (REF. 19).Sema4A is expressed by activated macrophages, B cellsand DCs, and at a low level by activated T cells, but notby naive T cells. A Sema4A−immunoglobulin fusionprotein used to crosslink Tim2 on T cells in vitro orwhen administered in vivo enhanced T-cell activationand clonal expansion, and resulted in the secretion ofIL-2, IFN-γand IL-4 by the responding T cells (FIG. 5).So, crosslinking of Tim2 by the Sema4A−immunoglob-ulin fusion protein did not promote T

H1 or T

H2-cell dif-

ferentiation, indicating that Tim2 might, in fact, nothave a role in skewing T cells to the T

H1- or T

H2-cell

pathway, but whether Tim2 is differentially expressedafter T cells have terminally differentiated was notaddressed in the study. Binding of mouse Tim2 to itsligand, Sema4A, has been shown to lead to tyrosinephosphorylation of Tim2 (REF. 19), indicating an activesignalling role for Tim proteins. The functional role ofTim2 in vivo was also examined by administration ofantibodies specific for Sema4A and this treatment wasfound to inhibit the development of EAE when admin-istered early, but not late. In vivo administration of anti-bodies specific for Sema4A inhibits EAE not by immunedeviation or skewing of the cytokine balance, but byinhibiting the generation of antigen-specific T cells andproduction of IFN-γand IL-4. These studies indicatethat the interaction of Tim2 with its ligand Sema4A iscrucial for early clonal expansion and cytokine produc-tion by responding T cells, but not for their effectorfunction (FIG. 5). Inhibition of this interaction limitsclonal expansion of T cells and the production ofcytokines.

TIM3 in macrophage activation and autoimmunityTIM3 protein is not expressed by naive T cells but isupregulated as they differentiate into T

H1 cells10. Long-

term TH2-cell clones and T

H2-cell lines that were gener-

ated in the presence of IL-4 and antibody specific forIL-12 did not express TIM3, even after several rounds ofpolarization. Furthermore, TIM3 is not expressed onthe surface of T

H1 cells immediately as they acquire the

IFN-γ-producing phenotype; it takes two to threerounds of polarization in vitro for protein expression tobe detectable on the cell surface. This indicates that notall IFN-γ-producing cells are TIM3 positive. During anin vivo immune response, few T cells (<2%) expressTIM3 on the cell surface, and most TIM3-positive cellsare seen in the spleen but not in the draining lymphnodes. So, the cells that express TIM3 might exit thelymph nodes and traffic to the spleen and other periph-eral tissue sites. Whether TIM3 is a marker for terminal

The specific immunological mechanisms by whichinteraction between hepatitis A virus and TIM1might alter T

H-cell differentiation and limit the devel-

opment of asthma are not clear. It is possible thathepatitis A virus productively infects T

H2 cells, which

seem to preferentially express TIM1. Alternatively,hepatitis A virus might bind to differentiating T

Hcells

and provide a signal that promotes TH

1-cell differen-tiation. Or, hepatitis A virus, by binding to TIM1,might interfere with the binding of TIM1 to its lig-and, which would normally provide a T

H2-inducing

signal (FIG. 4). Polymorphisms in TIM1 might alter thebinding affinity of hepatitis A virus for TIM1, andregulate these effects, possibly enhancing T

H1-cell

development. These possibilities require furtherstudy, but are supported by other examples of virusinteractions with virus receptors that might regulateT

H-cell differentiation39,40.

Sema4Ass

APC

CD3

TCR

MHC

T-cell activation ↑T-cell clonal expansion ↑IL-2, IL-4, IFN-γ ↑

PP

?

Tim2

Y Y

T cell

Figure 5 | Tim2−Sema4A interactions. Semaphorin 4A(Sema4A) is a member of the transmembrane-type semaphorinfamily, which contain a 500 amino-acid sema domain, as wellas immunoglobulin, transmembrane and cytoplasmic domains.Sema4A is expressed as a disulphide-linked homodimer bydendritic cells and activated B cells. T-cell immunoglobulinmucin 2 (TIM2) contains immunoglobulin V, mucin andtransmembrane domains followed by a cytoplasmic domain,which contains consensus tyrosine-phosphorylation sites.Ligation of the T-cell receptor (TCR) induces expression of TIM2by T cells. Interaction of Sema4A with TIM2 results in tyrosinephosphorylation of TIM2 and the recruitment of as-yet-unidentified signalling molecules, resulting in enhanced T-cellactivation and production of cytokines. APC, antigen-presentingcell; IFN-γ, interferon-γ; IL-2, interleukin-2.



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Transwell experiments showed that the macrophageactivation and clonal expansion that is induced byTim3-specific antibodies requires a cognate interac-tion between T

H1 cells and macrophages, indicating

that TIM3 protein expressed on the surface of TH

1cells might regulate macrophage function directly. Asone of the main functions of T

H1 cells is to activate

macrophages and eradicate intracellular pathogens,the ability of TIM3 to regulate the activation ofmacrophages is likely to be important in a normalimmune response. So, in addition to IFN-γ,which isknown to activate macrophages, an interaction withTIM3 expressed by T

H1 cells must either directly or

indirectly have an important role in regulatingmacrophage activation.

To study the role of TIM3 in the development of aT

H1-cell-mediated autoimmune disease, we adminis-

tered Tim3-specific antibody to SJL mice that wereimmunized to induce the development of EAE10.Administration of Tim3-specific antibody accelerateddisease onset, increased severity and mortality, andincreased weight loss. These mice often showed atypicalsigns of disease, including extensive demyelinatinglesions that were filled with activated macrophages thatwere actively phagocytosing myelin. Preliminary datashowed that Tim3 was expressed by only 2−3% ofT cells in the peripheral lymphoid tissue, but by almostall the T cells that were invading the brain parenchymaat the onset of disease. Furthermore, administration ofTim3-specific antibody in vivo resulted in the rapid exitof Tim3-positive encephalitogenic T cells from thelymph nodes into the brain, indicating that Tim3might also have a role in the trafficking of T cells to thetarget tissues (H. Waldner and V.K.K., unpublishedobservations). A role for TIM3 in T-cell homing is sup-ported by the observation that TIM3 is structurallyrelated to the adhesion molecule MADCAM1.Therefore, in addition to regulating the activation ofother immune cells in the lymphoid compartment,TIM3 might also be involved in the exit of differenti-ated T

H1 cells from lymphoid organs to the target tis-

sues. We speculate that the accelerated progression ofEAE that is observed in the mice treated with Tim3-specific antibody might be due to a combination ofincreased influx of T

H1 cells into the brain together

with activated macrophages that mediate target-tissueinjury and demyelination in the CNS. The mechanismby which Tim3-specific antibody in vivo mediatesmacrophage activation or promotes cell trafficking isnot yet clear.

Model of TIM3 interactionAlthough there is only a limited amount of functionaldata on this new gene family, emerging data indicatethat the TIM gene family might have important func-tional and biological effects (BOX 3). For example,although TIM3 is differentially expressed by T

H1 cells,

administration of antibodies specific for TIM3 duringan ongoing immune response results in marked acti-vation and clonal expansion of macrophages. Howdoes the administration of an antibody specific for a

TH1-cell differentiation or for T cells that are trafficking

out of the lymph nodes into the periphery and targetorgans remains to be determined. TIM3 is alsoexpressed by differentiated type 1 CD8+ cytotoxic T cells(T

C1 CELLS), but it does not seem to be expressed by any

cell type other that T cells and possibly a small numberof natural killer (NK) cells. Low-level expression ofTIM3 mRNA has been seen in macrophages, but pro-tein expression by these cells has not been detectedusing flow cytometry10.

The functional role of TIM3 in vivo is just beginningto be understood. Administration of antibody specificfor Tim3 during an ongoing immune response enhancedthe activation and clonal expansion of macrophages10.

Box 3 | TIM gene family is involved in asthma and allergic disease

• The T-cell immunoglobulin mucin (TIM) genes are located on human chromosome5q33 — a region that has been repeatedly linked to asthma susceptibility and isassociated with marked allelic variation.

• TIM1 is expressed by CD4+ T cells, which have a crucial role in the development ofallergen-induced airway hyperreactivity (AHR) and in the pathogenesis of asthma44.

• TIM1 is transcribed during primary antigen stimulation — a period of time that iscrucial in influencing T-cell differentiation and commitment to the production ofT helper 2 (T

H2) cytokines and the development of AHR16.

• TIM1 is expressed by TH

2 cells as they develop a TH

2-cell phenotype, but is notexpressed by T

H1 cells. (G. J. F.,V. K. K., D. T. U. and R. H. D., unpublished observations)

• Human TIM1 functions as a receptor for hepatitis A virus, and epidemiological datashow that infection with this virus is associated with protection from asthma andallergy13,36,37.

TH1 TH1

TIM3L

Macrophage or DC

CD4

TCRTIM3

T or Bcell

a b

?

?

Peptide–MHC complex

? ?

Figure 6 | Hypothetical mechanisms by which the interaction between TIM3 and TIM3ligand induces macrophage activation. a | If the T-cell immunoglobulin mucin 3 ligand (TIM3L)is expressed by macrophages or dendritic cells (DCs), a direct interaction between TIM3 that isexpressed by T helper 1 (TH1) cells and TIM3L might result in the activation of macrophages orDCs. b | If TIM3L is expressed by another T cell (or B cell), the interaction between TIM3 andTIM3L might result in the activation of macrophages or DCs indirectly by either relieving inhibitionor by providing an activating signal(s) by cognate interactions and induction of soluble mediators.TCR, T-cell receptor.

TC1 CELLS

CD8+ T cells that produce Thelper 1 cytokines, particularlyinterferon-γ.



R E V I E W S

Concluding remarksAs it seems that TIM1 and TIM3 are differentiallyexpressed by effector T

H2 and T

H1 cells, respectively, tar-

geting of these molecules by infectious agents or drugsmight affect the balance of T

H1- and T

H2-cell responses

and alter susceptibility to various immune-mediated dis-eases, such as autoimmunity, asthma and allergic reac-tions. An inverse relationship between infection withhepatitis A virus and asthma is a good example of howTIM molecules might be exploited by nature to alter thecourse of a disease. These data also provide a potentialmechanism to account for the ‘hygiene hypothesis’, whichproposes that previous exposure to infectious pathogensmight affect the development of asthma, allergies andautoimmune diseases38. If the TIM molecules that areexpressed by differentiated T

H1 or T

H2 cells are also used

as receptors by other infectious agents, this might result inthe clonal expansion or deletion of effector T

H1 and T

H2

cells, resulting in a change in susceptibility or resistanceto immune-mediated diseases. The data on the TIMfamily also provide an opportunity to design drugs thatcan selectively target effector T

H1 or T

H2 cells, as the

TIM molecules are expressed by differentiated T cells,and affect the disease course by inhibiting T

H1- or T

H2-

effector cells and their functions. Such a drug would alsohave the advantage of shifting the balance between T

H1

and TH2 cells, thereby potentially providing long-lasting

regulation of immune-mediated diseases.

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molecule that is expressed by TH

1 cells result in theactivation and clonal expansion of macrophages? Onepossible explanation is that a cognate interactionbetween TIM3 on the surface of T

H1 cells and its poten-

tial ligand on the cell surface of macrophages results inmacrophage activation and clonal expansion (FIG. 6).The TIM3-specific antibody could crosslink TIM3 onthe surface of T

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signal in the macrophages, resulting in their clonalexpansion and activation. Alternatively, if TIM3 nor-mally induces a negative signal to inhibit macrophagesfrom over-activation during a T

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TIM3-specific antibody might block this negative sig-nal and result in macrophage activation. Both of thesepotential mechanisms require a cognate interactionbetween T

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scenarios, macrophages are predicted to express a lig-and for TIM3. It is also possible that TIM3 might have aligand on another cell type besides macrophages (suchas T cells or B cells), which normally keep macrophagesin check, so blocking this T-cell−T-cell or T-cell−B-cellinteraction would subsequently result in macrophageclonal expansion and activation (FIG. 6). On the basis ofthe available functional data, it is predicted that one ofthe primary immune functions of TIM molecules thatare expressed on the surface of differentiated T cellsmight be to regulate the activation of other immunecells by a cognate interaction.



R E V I E W S

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Acknowledgements:We would like to thank D. Schlesinger and J. McIntire for their valu-able help in computer searches and input to the manuscript.

Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/CCR3 | CCR4 | CCR5 | CCR8 | CD4 | CXCR3 | CXCR4 | ICOS |IFN-γ | IL-2 | IL-4 | IL-5 | IL-6 | IL-9 | IL-10 | IL-12 p40 | IL-13 |MADCAM1 | Sema4A | TIM1 | Tim1 | Tim2 | TIM3 | Tim3

FURTHER INFORMATIONPrediction of mucin type O-glycosylation sites:http://www.cbs.dtu.dk/services/NetNGlyc/The Biology Workbench, University of California:http://workbench.sdsc.edu/Access to this interactive links box is free online.

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The TIM gene family: emerging roles in immunity and disease