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Immunology Letters 141 (2011) 28– 35

Contents lists available at SciVerse ScienceDirect

Immunology Letters

j ourna l ho me pag e: www.elsev ier .com/ locate / immlet

eview

ovel roles for TIM-1 in immunity and infection

aul D. Rennert ∗

epartment of Molecular Discovery and Immunobiology, Biogen Idec Inc., 12 Cambridge Center, Cambridge, MA 02142, United States

r t i c l e i n f o

rticle history:eceived 7 July 2011eceived in revised form 12 August 2011ccepted 27 August 2011vailable online 2 September 2011

a b s t r a c t

T cell, immunoglobulin domain and mucin domain-1 (TIM-1) is the nominant member of a small familyof related proteins that regulate immune cell activities. TIM-1 was initially characterized in a mousecongenic analysis of Th2 T cell responses and related pathology. Data accumulated to date suggest thatTIM-1 regulates effector T cell function, and may play distinct roles in the activities of B cells, invariantNKT cells and epithelial cells. In addition, a variety of ligands for TIM-1 have been proposed. In this

eywords: cellsendritic cellshosphatidylserinesthmaepatitis A virus

review I discuss recent data that have accumulated on the function of TIM-1, propose a model to explainhow TIM-1 regulates effector T cell activity through recognition of distinct ligands, and review othersfunctions of this increasingly fascinating protein. Of considerable interest are the novel findings thatTIM-1 mediates virus entry and virulence.

© 2011 Elsevier B.V. All rights reserved.

ilovirus

. Introduction

T cell, immunoglobulin domain and mucin domain-1 (TIM-1)as initially identified as an important regulator of T cell responses

n a series of genetic studies. Congenic analysis in mouse identifiedhe TIM gene family locus as critical to conferring Th2 cytokineesponses and airway hyper-responsiveness (AHR) [1]. Impor-antly, the phenotype could be adoptively transferred with T cells1]. A human genetics study from the same group described a TIM-1olymorphism that was associated with asthma susceptibility [2].f keen interest, hepatitis A virus (HAV) seropositive status wasssociated with protection from atopic disease in individuals car-ying a 6 amino acid (AA) insertion in the TIM-1 protein [2]. Thisemarkable finding provided a molecular rationale for the “hygieneypothesis” which holds that rising rates of asthma and other atopiciseases are associated with declining rates of exposure to virusesnd other pathogens [3]. This association between a common TIM-1olymorphism, HAV-seropositivity, and asthma susceptibility wasertainly credible, since TIM-1 had originally been described as anAV cellular receptor, called HAVcr-1 [4]. Multiple studies have

eplicated the finding that the 6 AA insertion is associated with pro-ection from asthma, although without consideration of the HAVtatus of the patient population (recently reviewed in [5]). Whether

IM-1 really provides a molecular basis for the hygiene hypothesisemains unclear. With that question in mind this review will cover

∗ Tel.: +1 617 679 2986.E-mail address: paul.rennert@biogenidec.com

165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.imlet.2011.08.003

emerging data on the multiple ways in which the TIM-1 pathwaycan regulate immune responses and modulate viral infections.

1.1. TIM-1 protein structure

TIM-1 is structurally a member of the immunoglobulin super-family (IgSF) [1], containing a single IgV-like domain sitting atopa mucin domain, which in turn gives way to a short stalk domainwhich forms the membrane adjacent part of the protein (Fig. 1A).At the level of sequence homology TIM-1 most closely aligns withthe siglecs [6], as illustrated using molecular modeling (Fig. 1B),and indeed both the sequence homology (Fig. 1C) and modelingsuggest that the IgV domain should be able to bind carbohydrateusing a siglec-like sialic acid binding motif. In Fig. 1C, blue arrowsshow the three residues that siglecs use to form the sialic acidbinding site; TIM-1 shares the second and third residue and usesa unique residue (red arrow) to complete the binding site with anovel side chain, as illustrated in Fig. 1D, which shows a minimalenergy model of the TIM-1 IgV domain in complex with me-a-9-n-benzoyl-amino-9-deoxy-neu5ac (benz compound; one of a familyof synthetic sialic acid like compounds [7], and references therein).The IgV domain of TIM-1 also contains an N-linked glycosylationsite and the mucin domain contains numerous O-linked glycosy-lation sites (Fig. 1A). There is a typical transmembrane domain,and an intracellular (cytoplasmic) domain containing at least onetyrosine phosphorylation site [1]. At the time of its description

as an atopic disease susceptibility gene, TIM-1 had no known lig-ands, and thus its identification as a receptor capable of transducingtyrosine-phosphorylation dependent signals was of great interest.We and others determined that the transfection or crosslinking of

P.D. Rennert / Immunology Letters 141 (2011) 28– 35 29

Fig. 1. Structure of the TIM-1 protein. (A) Schematic representation of TIM-1, showing the IgV, mucin, stalk and cytoplasmic domains, and indicating the location of O-linkedand N-linked glycosylation sites. (B) Alignment of the TIM-1 IgV domain with the IgV domain of sialoadhesin-1 using a minimal energy algorithm. The unique disulfidebridges that stabilize the large F/G–C/C′ cleft the uniquely characterizes TIM-1 are illustrated along with the conserved disulfide bridge shared with siglec-1. (C) Sequencealignment of TIM-1 (shown as Kim-1) with members of the siglec protein family, and a consensus siglec sequence (1od9.pdb). Highly conserved residues are starred, majorityconserved residues are given a “:” or “.” symbol. Color coding indicates degree of similarity among family members. Blue arrows show the three residues that siglecs use toform the sialic acid binding site; TIM-1 share the second and third residue and uses a unique residue (red arrow) to complete the binding site with a novel side chain, asshown in D. (D) Minimal energy modeling of a sialic acid like compound docking in the TIM-1 IgV domain. Note that the binding site sits atop the cation-containing cleftwhich binds PS.

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IM-1 on mouse or human T cells induced a plethora of T cell acti-ation/proliferation signals, as recently reviewed [8]. These datare consistent with the hypothesis implicit in the genetic data,.e. that TIM-1 functions by controlling T cell function, specifically,he expression of cytokines by Th2 effector T cells. However, asiscussed in Sections 1.3–1.6, the “genetic hypothesis” of TIM-1unction only partly describes the activities of this protein.

The identification of a bona fide ligand for TIM-1, capable ofransducing the described signal to T cells, has proven to be diffi-ult. The recombinant protein, whether mouse or human, appearso be inherently “sticky” unless assayed under chelating conditions,ven if only the IgV domain is expressed and purified [6,9]. Assayssing recombinant protein, e.g. TIM-1-IgV-Fc fusion proteins, areherefore difficult to analyze, and the use of recombinant TIM-1rotein in vivo is likely to produce non-specific effects. Nonethe-

ess, the successful identification of the low affinity interaction ofIM-1 and the closely related protein TIM-4 was a notable achieve-ent [10]. It appears that at least some aspects of TIM-1 function

re regulated by TIM-1/TIM-4 engagement, and perhaps also byIM-1/TIM-1 interaction. It has recently been suggested that theIM-1/TIM-4 interaction is mediated by phosphatidylserine (PS)ontaining exosomes [11] (see Section 1.2) and it has also beenuggested that TIM-4 interacts with a distinct ligand on T cells [12],urther complicating the analysis of TIM-4 function. It is worth not-ng that TIM-4 recombinant proteins are also very “sticky”, andnalyses performed using these proteins suffer the same limitationss seen with recombinant TIM-1 proteins. Several other proposedigands are not discussed here as they are assumed to interact non-pecifically with the TIM-1 protein. CD300b, a recently describedandidate ligand, is discussed in Section 1.5.

.2. TIM-1 as a phosphatidylserine binding protein

A very compelling story emerged with the solving of the crystaltructure of TIM-1 (and TIM-4) [13] and the identification of TIM-

as a PS binding protein, first described in 2007 [14], as recentlyeviewed [11]. Curiously, the PS binding site sits at the bottom ofn extensive cleft formed by the folding of the F/G and C/C′ loops.his cleft is an unusual feature of the TIM protein family, and its stabilized by unique �-strand/loop disulfide bonds (Fig. 1B). Theop of the cleft shares amino acids with the sialic acid binding motifescribed earlier, suggesting that multiple binding activities mighte mediated by this one structure. Regardless, the binding of PSy TIM-1 is well established, although the functional consequencef such interaction is unclear. PS has not been described to triggerIM-1 signaling in T cells, and it certainly does not induce phagocy-osis by T cells, as lymphocytes are not competent to carry out thisunction. Whether TIM-1 can trigger a PS-dependent autophagyignal in T cells is not known. The consequence of PS recognitiony TIM-1 expressed on other cell types is discussed in Section 1.6.

.3. The activity of TIM-1 elucidated by studies using monoclonalntibodies

By virtue of their presumed specificity and well understood pro-ein biochemical properties, monoclonal antibodies (mAbs) can be

valuable tool when studying a new biological system. Somewhaturprisingly, most mAbs raised to mouse or human TIM-1 have nopparent effect on the activity of the protein in vitro or in vivo (Ren-ert, unpublished). Those that do, however, have quite dramaticffects on immune responses and in models of disease. Studies from

everal groups have focused on the activities of agonist and antag-nist anti-mouse TIM-1 mAbs. The data in part confirm the geneticypothesis put forward above, but also reveal several unexpectedspects of the function of TIM-1 on T cell populations.

tters 141 (2011) 28– 35

Using the OVA-induced lung inflammation model, we demon-strated that 4A2.2, a rat anti-mouse TIM-1 mAb, blockeddevelopment of lung disease, including mononuclear cell infiltra-tion, mucin production by goblet cells, and Th2 cytokine production[15]. Ex vivo analyses demonstrated that the effect could be tracedto T cells in the draining (bronchial) lymph nodes, which failed toproliferate in response to antigen restimulation. The 4A2.2 mAbwas shown to bind to the F/G–C/C′ cleft, potentially disrupting TIM-1 interaction with ligand(s) [9]. Although we could not demonstrateTIM-1 protein expression in this model, TIM-1 mRNA was clearlyupregulated in the setting of the inflammed lung. As it turns outthere is very little data on the expression of TIM-1 on T cells invivo, a point I will return to in Section 1.6.

Using several models, we demonstrated that an agonist ratanti-mouse TIM-1 mAb, 1H8, had a dramatic effect on immuneresponses [15]. Using a standard immunization protocol of KLH inCFA we noted a drastic increase in T cell proliferation and cytokineproduction, including both Th1 and Th2 cytokines. In the OVA-induced lung inflammation model, infiltration of the lung wasintense and both Th1 and Th2 cytokine production was dramati-cally enhanced. Therefore while Th2 cytokines were modulated byTIM-1 activity as expected, the effect was not limited to the Th2T cell subset. Somewhat similar data was generated by DeKruyffand co-workers, who showed that an agonist anti-mouse TIM-1mAb, 3B3, enhanced T cell responses to OVA immunization andwas capable of overriding intra-nasally induced tolerance to OVAin a lung challenge model. In these settings, TIM-1 was shownto be expressed on Th2 T cells, and 3B3 treatment induced T cellproliferation and increased levels of IL-4, IL-10 and IFN� [16].

Using EAE modeling, Kuchroo and co-workers also identifieddistinct antagonist and agonist anti-mouse TIM-1 mAbs [17]. Whilethe impact on disease course resembles the outcomes describedfor antagonist and agonist anti-TIM-1 activity, i.e. antagonist mAbRMT1–10 reduced the EAE disease score while agonist mAb 3B3aggravated the disease, the mechanism of action of these mAbs doesnot resemble the activities we described for 4A2.2 and 1H8. In par-ticular the antagonist mAb RMT1–10, rather than reducing overallcytokine production, instead preferentially induced Th2 cytokineexpression, which in the setting of the EAE model proved to be ther-apeutic. The agonist mAb 3B3 induced the Th1 cytokine IFN� andthe Th17 cytokine IL-17, but had no effect on IL-4 levels (which werenegligible in this model). In this setting these two different mAbswere differentially modulating T cell effector function, thereby hav-ing opposing effects on disease outcome. Whether mAb RMT1–10demonstrated true antagonist activity in this model, in the con-text of the aforementioned genetic hypothesis, is unclear, since Th2cytokine production was induced rather than blocked. Attempts toreproduce these findings using the same EAE model and treatmentwith the RMT1–10 mAb or the 4A2.2 mAb were unsuccessful, andthus we could not directly compare the activities of these two mAbsin the EAE setting.

Several labs have published models of allograft toleranceand rejection in which TIM-1 was shown to play diverseroles. Zayegh and co-workers, using the anti-mouse TIM-1 mAbRMT1–10, studied the role of TIM-1 in alloreactivity [18]. Usinga major MHC mismatch cardiac allograft rejection model, theydemonstrated that RMT1–10 reduced the number of allospe-cific IFN� producing T cells without affecting the number ofallospecific IL-5 producing T cells. When administered withsubtherapeutic doses of rapamycin, RMT1–10 resulted in reduc-tion of allospecific IFN� producing T cells, enhancement ofallospecific IL-5 producing T cells, and long term allograft

acceptance. Remarkably, long term allograft acceptance requiredthe presence of Treg cells, as shown in adoptive transfer experi-ments. This suggests that the protection conferred by treatmentwith mAb RMT1–10 is associated with Th2-cytokine mediated

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nhancement of Treg activity [18]. In keeping with this hypoth-sis, Strom and co-workers showed that the agonist anti-mouseIM-1 mAb 3B3 enhanced Th1 and Th17 T cell responses andrevented the productive generation of Tregs in an islet transplan-ation model. Indeed, 3B3 treatment was sufficient to override isletraft tolerance induced by costimulatory pathway blockade [19].inally, modulation of TIM-1 function with mAb RMT1–10, alongith CTLA4-Ig and anti-CD40L treatment, restored allograft tol-

rance in a Tbet knockout mouse model in which costimulatorylockade alone was insufficient to induce tolerance [20]. In thisodel RMT1–10 treatment was associated with blockade of IL-17,

FN� and the Th2 cytokines IL-5 and IL-13 and was associated withiminished CD8 T cell activity. Therefore this study demonstratediverse roles for the TIM-1 pathway in modulating T cell activityreducing Th1, Th2, Th17 and CD8 T cell responses).

These studies suggest that in the mouse, TIM-1 activity is differ-ntially modulated by specific monoclonal antibodies in the contextf diverse immune responses. The activities of TIM-1 thereforeppear to be broader than would be expected in the context of theenetic hypothesis outlined above. One complication is that not allntibodies mentioned above have been thoroughly characterized inhe literature, and it is formally possible that some of the commer-ially available mAbs can bind non-specifically to other antigens21].

In a humanized mouse model of dust mite allergic asthma,e evaluated the therapeutic activity of a panel of mouse anti-uman TIM-1 monoclonal antibodies. As a candidate therapeutice selected mAb A6G2, an anti-human TIM-1 IgV-domain specificAb having an epitope that overlapped with that identified for

he anti-mouse TIM-1 mAb 4A2.2, described earlier. Thus, A6G2ound to an epitope which included the cleft formed by the fold-

ng of the F/G and C/C′ loops. As noted above this cleft mediatesinding to PS. We also showed that this cleft mediated bindingo dendritic cells in a cation-independent manner (i.e. in a man-er distinctly different from PS binding) in both the mouse anduman systems [9]. PBMC from moderate to severe dust mite asth-atic patients were used to chimerize SCID mice. The mice were

hen subjected to repetitive challenge with the dust mite allergenerP1. Mice thus challenged developed pronounced human cell

nflammation in the lung, expressed human Th2 cytokines in theung and draining lymphoid tissue, and were found to have an AHReaction to methacholine challenge. Treatment with mAb A6G2,ut not with irrelevant control mAbs, ablated these pathogenicesponses. The mechanism of action was found to be similar to thateen in the murine model described earlier, with reduced inflam-ation, reduced Th2 responses (at both the transcription factor and

ytokine levels) and normalization of the airways response to chal-enge. In addition we used PBMC isolated directly from the patientso show that mAb A6G2 prevented productive interaction of T cellsith allergen loaded myeloid dendritic cells, thus linking the mech-

nism of action to the dendritic cell binding assay described above.ur biochemical characterization studies of the mAbs along with

he in vivo activity data therefore supported a model in which TIM--mediated T cell/dendritic cell interaction played a critical role, aoint I will return to in Section 2.

.4. The activity of TIM-1 elucidated by genetic studies

In contrast with the dramatic results obtained with various anti-urine TIM-1 mAbs, and in contrast with the dramatic results

f the original congenic analysis [1], gene-deletion (knock-out)r overexpression (transgenic) of TIM-1 in mice has had little

henotypic effect. Indeed, at least different 5 labs have made TIM-1nock-out mice and a similar number have made transgenic mice,ut only very a few reports have been published. A phenotypiccreen of a large collection of knockout mouse lines revealed no

tters 141 (2011) 28– 35 31

phenotype associated with TIM-1 gene deletion, although theseanalyses were performed on a mixed 129/B6 background [22] (seeonline supplemental Table 2 for reference 22). Similarly, TIM-1gene-deficient mice had no discernable phenotype related to Th2immune responses, including production of Th2 cytokines by Tcells, or in vivo response to Schistosoma challenge [21]. McKenzieand co-workers have also shown that TIM-1 deficient mice have anattenuated response to OVA-induced lung inflammation but canstill develop pathology (as measured by mucus production andAHR) [23]. TIM-1-deficient mice did have reduced inflammationof the lung tissue and bronchial fluid, which was due to reducedlymphocyte and eosinophil cell numbers, and required a higherdose of methacholine to induce airway resistance. However, thiseffect could not be traced to a defect in Th2 cytokine production.Indeed, TIM-1 protein was not detected on T cells in this model, butcould only be found on activated B cells, which do not contributeto disease in the OVA-induced lung inflammation model.

The analyses of the TIM-1 knockout mice may have been com-plicated by the proximity of the mouse TIM-2 gene, which isclose enough to segregate with TIM-1 when backcrossing to dif-ferent mouse strains (Rennert, unpublished). Thus it is importantto demonstrate that normal expression of TIM-2 protein, which is anegative regulator of T cell immune responses [24], is not impactedby the TIM-1 knockout. Such experiments are notably lacking. Alsothe TIM-1-deficient mouse was generated on a C57Bl/6 backgroundbefore backcrossing onto Balb/c for six generations and it is atleast formally possible that residual C57Bl/6 genetic backgroundinfluenced the behavior of the model. Regardless, the demonstra-tion that transgenic overexpression of TIM-1 on T cells (or both Tand B cells) had no notable phenotype is puzzling and difficult tointerpret. One possibility is that the monoclonal antibody studiesdescribed earlier were impacted by mAb-mediated cell depletion(in the case of antagonist mAbs) or aberrant induction of TIM-1signaling (in the case of agonist mAbs). Further study is certainlyrequired to understand these disparate results. One aspect thatthis work makes quite clear is that TIM-4 appears to have TIM-1independent functions, related to its expression on myeloid cellsand phagocytic activity, as genetic depletion of the TIM-4 genecauses pronounced phenotypes including defective cell clearance[25,26].

1.5. CD300b as a TIM-1 binding protein

We had predicted from monoclonal antibody epitope mappingand binding studies that TIM-1 should have a ligand expressedon dendritic cells that was neither PS nor a carbohydrate moiety[9]. Recently such a ligand was identified as LMIR5, also knownas CD300b [27]. Using LMIR5-Fc, this group performed expressioncloning from A20 cells, a B cell line known to express high levelsof TIM-1 protein [15]. From this expression cloning they identifiedTIM-1 as a ligand for LMIR5. Importantly, LMIR5 did not bind to PS,indicating that this interaction was not mediated indirectly throughthe phospholipid. Furthermore, interaction of TIM-1 with LMIR5could be blocked by anti-TIM-1 antibody or by specific mutationof residues contained within the cleft formed by the folding of theF/G and C/C′ loops. Thus LMIR5, which is expressed by myeloid lin-eage cells, has the attributes expected of a TIM-1 ligand expressedby myeloid dendritic cells. Some questions remain, however, sinceLMIR5 was also identified as a binding partner for TIM-4. Further,LMIR5 heterodimerizes with other CD300 family members, and itis unclear whether there is a specific form required for TIM-1 bind-

ing or activation. Thus, TIM-1 as expressed on T cells still doesnot have a clearly identified ligand that can trigger the diverse Tcell activation signals induced by overexpression or crosslinking ofTIM-1.

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.6. TIM-1 function on iNKT cells, B cells and epithelial cells

As mentioned in Section 1.3, identification of TIM-1 expres-ion on T cells is exceedingly difficult. We have found that 3 orore rounds of differentiation to a Th2 phenotype will induce TIM-

expression on mouse T cells, but even then the expression isariable, and most of the protein is found intracellularly (Rennert,npublished). Murine thymocytes can also be induced to expressIM-1 using CD3/CD28 stimulation, and a small proportion of acti-ated B cells express cell surface TIM-1 [15]. In the human systemt has been very challenging to identify TIM-1 expression on Tells using flow cytometry, Western blot, immunoprecipitation ormmunohistochemistry, while TIM-1 mRNA is readily detected; weave been not been able to detect cell surface expressed TIM-1 onouse or human dendritic cells or macrophages, nor on human B

ells (Rennert, unpublished).Recent observations illustrate a distinct role for TIM-1. Invari-

nt NKT (iNKT) cells are a T cell subset expressing an invariantCR, which respond to antigen in a CD1d-restricted manner. iNKTells respond to antigenic stimulation by secreting large amounts ofytokines, notably IL-4 and IFN�. These cells are suggested to play

role in innate immune responses. In a recent study it was shownhat mouse iNKT cells constitutively express TIM-1, as determinedy staining with mAb 3B3 [28]. This observation confirms thatf another group who demonstrated TIM-1 expression on mouseNKT cells using mAbs RMT1–4 and RMT1–10 [29]. Neither studyrovided RNA data to support the observation that TIM-1 pro-ein was present in these cells although the flow cytometric datappear convincing. Functionally, TIM-1 expressed on iNKT cellsound apoptotic cells through PS, inducing cell activation, prolifer-tion and cytokine secretion [28]. Furthermore, apoptotic epithelialells induced by intranasal treatment with anti-FAS mAb were ableo trigger lung inflammation and AHR. This effect required iNKTells as shown in CD1d-deficient mice and also required TIM-1s suggested by blockade of TIM-1 with a novel anti-mouse mAb,D10. The agonist mAb 3B3 in contrast was shown to activate iNKTells when given in concert with �-GalCer (which stimulates iNKTells) thus secreting IL-4 and IFN�. Somewhat similar results wereeported when iNKT cells were stimulated with mAb RMT1–4 (butot mAb RMT1–10) in the presence of �-GalCer, but in this case IL-

was secreted while IFN� was downregulated [29]. As seen in theontext of putatively T cell-mediated biologies, the activity of TIM-

on iNKT cells seems to be differentially modulated by differentAbs.Several groups have identified B cell activity mediated by TIM-1.

erminal center B cells were identified as a prominent B cell typexpressing TIM-1, and B cells in general were shown to require BCRngagement to upregulate TIM-1 [21]. However, TIM-1-deficientice had no obvious defects in B cell activity, germinal center size

r antibody production [21,23]. In contrast a critical role for TIM-1n regulating antibody production was proposed based on studiessing yet another novel anti-mouse TIM-1 mAb, RMT1–17, a puta-ive agonist [30]. Also, studies using B cells showed that TIM-1 canssociate with the signaling molecule Fyn, and that Fyn can phos-horylate TIM-1 at its conserved tyrosine residue [31]. This activityarallels the similar activity of the T cell restricted Src-family kinaseck. The roles that TIM-1 may play in B cell function remain to beetermined.

TIM-1 is known in the kidney and oncology literature as kidney-njury-molecule-1, based on its expression in injured and cancerousidney samples [32]. In the kidney, TIM-1 is expressed on lumi-al epithelial cells of the proximal tubules, and in this regard the

unction of the protein has remained mysterious. In the contextf PS-binding activity, it has recently been proposed that TIM-1s upregulated in the kidney in order to clear the lumen of apop-otic material through recognition of PS [33]. In this paper it was

tters 141 (2011) 28– 35

demonstrated that expression of TIM-1 conferred a phagocytic phe-notype on kidney epithelial cells. The recent description of TIM-1expression in other mucosal sites suggests that TIM-1 may servethis role more widely, or that it has yet other, to date undescribed,activities [34].

1.7. TIM-1 and viral infection

As mentioned in Section 1, TIM-1 has been described as a hep-atitis A virus cellular receptor (HAVcr-1) [4]. HAV is a picornovirus,which is a single positive-sense strand RNA virus contained withina capsid protein. Early work on this virus/protein interaction indi-cated that the IgV domain was most critical for viral uncoatingand subsequent cell infectivity [35,36]. Rough epitope mappingof a protective mAb 190/4 suggested that an N-glycosylation sitein the TIM-1 IgV domain contributed to the motif and might beinvolved in virus interaction. Subsequent work [35] (and refer-ences therein) provided data that both the IgV and mucin domainsof the protein contributed to viral uncoating – these studies werepotentially confounded by the sticky nature of recombinant TIM-1 proteins, as noted in Section 1.1. While TIM-1 mRNA can bedetected in liver tissue lysates [4], expression of TIM-1 on liverresident cell types has not been demonstrated, nor is there evi-dence that HAV utilizes TIM-1 to infect hepatocytes. More recentwork has demonstrated, remarkably, that HAV severity in a pedi-atric patient population was associated with TIM-1 polymorphisms[37]. In this study it was shown that human TIM-1 protein con-taining a mucin domain insertion polymorphism bound HAV moreefficiently. This is the same polymorphism initially identified asprotective in the study associating asthma susceptibility with TIM-1 in HAV seropositive patients (insertion at position 157: MTTTVP).It was further shown that this long form of TIM-1, when expressedby NKT cells, induced greater cytolytic damage associated withhepatitis, thereby functionally linking HAV recognition by TIM-1 to disease outcome. The requirement for TIM-1 to mediate theHAV-induced NKT cell response was shown using an anti-humanmAb 1D12. Thus in the setting of HAV infection now we havemultiple proposed roles for TIM-1: first as a cellular receptor onepithelial cells, second as a viral recognition receptor on NKT cellsand theoretically as an immune cell response modulator on T andB cells. How the mucin domain insertion polymorphism influ-ences the response to HAV infection among these cell types isunclear.

Also in the context of viral control, a very interesting studyexamined the distribution of TIM-1 haplotypes (as defined in[38]) in a population of Thai women [39]. The cohorts exam-ined included HIV-positive patients, patients known to have beenexposed to HIV but remaining seronegative, and normal patients(screened blood donors). TIM-1 haplotypes did not vary amongcohorts, suggesting that TIM-1 does not play a role in HIV infec-tion. However, a specific haplotype defined as D3-A, was associatedwith higher CD4+ T cell counts, less AIDS-related symptoms, andsignificantly better survival. The D3-A haplotype lacks the 6 AAinsertion discussed in Section 1 and more thoroughly in Sec-tion 2. Therefore, the polymorphism in the TIM-1 mucin domainhas diverse effects on the immune responses to viral infec-tions.

TIM-1 was recently shown to be a cellular receptor medi-ating infectivity of a second family of viruses, the filoviridae,which are the enveloped negative-sense strand RNA virusesEbola and Marburg [34]. A bioinformatics approach was used toidentify genes associated with viral infectivity, indicating a pos-

itive correlation between TIM-1 gene expression and infectivity.Gain of function experiments (TIM-1 transfection) and loss offunction experiments (TIM-1 siRNA knock-down) confirmed therole of TIM-1 in mediating filovirus infection of epithelial cells.

P.D. Rennert / Immunology Letters 141 (2011) 28– 35 33

Fig. 2. Activities of the TIM-1 pathway. (A) T cells are proposed to unfold TIM-1 on the cell surface upon activation through the TCR. TIM-1 then senses the status of theinteracting APC. If the APC is undergoing apoptosis the T cell stops its activation protocol. If instead TIM-1 interacts with LMIR5 or an as yet unknown ligand, it signals insupport of the T cell activation cascade. (B) iNKT cell presumably regulate TIM-1 unfolding in a similar fashion to T cells, yet the consequence of interaction of TIM-1 withPS may be very different, triggering iNKT activation and cytokine secretion. iNKT cells also use TIM-1 to bind HAV, resulting in increased cytolytic activity. The degree ofbinding of TIM-1 to HAV by iNKT cells appears to be regulated by TIM-1 polymorphisms. (C) Epithelial cells express relatively high levels of TIM-1 protein, suggesting that atleast a portion of the cell surface TIM-1 is unfolded at any given time on these cells. Lumenal epithelial cells of the proximal tubules in the kidney express TIM-1, especiallyafter injury, and this expression seems designed to induce phagocytosis by the epithelium of cells undergoing apoptosis, thereby clearing the lumen of debris during injury,infection or chronic disease. Other TIM-1-positive epithelial cells, likely on mucosal surfaces, are infected with HAV via the TIM-1 protein. A variety of cell types, perhapsa filovi

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IM-1 was demonstrated to interact with Ebola virus glycopro-ein in a specific manner. Anti-human TIM-1 mAbs directed tohe IgV domain blocked virus infection. The anti-human TIM-1

Ab ARD5 completely blocked infection in several model sys-ems. This blockade was effective even in experiments usingildtype Ebola virus to infect cells under BL-4 conditions. Impor-

antly, TIM-1 expression on human mucosal epithelial cells fromhe trachea, cornea and conjunctiva was shown by immunohisto-hemistry, demonstrating expression at potential routes of viralntry.

ruses such as Ebola, that use TIM-1 as a cellular receptor.

2. Conclusions

The diverse activities described for the TIM-1 protein can beviewed as a continuum of responses to diverse ligands, as shownin Fig. 2, in which TIM-1 interaction and response to ligand(s) isproposed to be context and celltype specific. Based on the siglec-

like motif present in TIM-1, it seems likely that TIM-1 presentationon the cell surface is regulated by carbohydrate interaction, as istypical of siglecs and some other IgSF members (LY49 and theKIR receptors, LILR/ILTs, and others) [40]. Upon cell activation,

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4 P.D. Rennert / Immunolo

is-binding carbohydrates release TIM-1, exposing the mucin andgV domains (Fig. 2A). T cells may then use TIM-1 to establish thetatus of antigen-presenting cells (APC), such as dendritic cells,ith which they interact (Fig. 2A), and thereby avoid inappropriate

esponses to cells undergoing apoptosis. This model puts forwardhe hypothesis that TIM-1 serves a check-point function in effec-or T cell proliferation, and that this check-point is regulated byhe recognition of PS [41]. In contrast, iNKT cells may use PS as anctivation signal in tissues such as the lung, and in such a settingpoptotic cells and debris would exacerbate lung disease (Fig. 2B).idney epithelial cells may use TIM-1 to recognize and respond topoptotic, PS-positive, cells and cellular debris, activating phago-ytic function and thus keeping the kidney lumen clear. Both iNKTells and epithelial cells use TIM-1 to bind to HAV, with dramaticallyifferent effects: iNKT cells react by initiating a cytolytic response,hile epithelial cells, presumably of the oral mucosa, may become

nfected (Fig. 2C). Mucosal and other epithelial cells can be infectedy Ebola and Marburg viruses via the TIM-1 protein (Fig. 2C). Impor-antly, each of these activities may be differentially affected by theIM-1 insertion polymorphism, now shown to be associated withoth atopic disease (perhaps via T cell biology) and the severityf HAV infection (via iNKT cell biology). Such frameworks in ournderstanding of TIM-1 function will evolve as new data on therotein emerge.

As noted in Section 1, human TIM-1 exhibits a common poly-orphism which is characterized as the presence or absence

f 6 AA. By comparison with great ape nucleotide sequences,t appears that the 6 AA insertion polymorphism is the ances-ral form, while the absence of 6 AA (in actuality, the deletionolymorphism) evolved subsequently [38]. Recent studies sug-est that selective pressure may operate on human TIM-1 becausef its multi-faceted role in response to viral pathogens. On thether hand, polymorphisms of TIM-1 may influence the devel-pment of atopic conditions, including asthma. These disparateunctions of TIM-1 in immunity and infection may very wellnderlie its proposed role as a contributor, at the molecular

evel, to the hygiene hypothesis, which puts forward the con-ept the atopic disease is inversely associated with pathogenxposure. That the TIM-1 gene appears to be subject to balanc-ng selection in higher primates, including human, supports this

odel [38].Such models must be viewed with caution, as mouse stud-

es have yielded complicated and somewhat conflicting results.n particular the activity of different mAbs is difficult to recon-ile with a single unifying model of TIM-1 function. A concertedffort to compare the activities of different panels of anti-mouseIM-1 mAbs in a single model system is sorely needed, as are addi-ional specificity studies on the available mAbs. TIM-1-deficientnd TIM-1 transgenic mice have not yielded the expected pro-ound effects on T cell biology that would have been expectedrom the early congenic analysis and subsequent mAb-based stud-es. As noted in Section 1.4, TIM-1 deficient mouse models maye impacted by unexpected effects on TIM-2 or other genes inhe tightly packed TAPR (T cell and airway phenotype regula-or) locus; regardless, the transgenic mouse model results cannote easily reconciled with our understanding of TIM-1 and T celliology.

A great deal has been learned since the association of TIM-1ith the immune system was first described in 2001 [1]. A decade

ater we find the field still in its early days, with many unanswereduestions. As often happens, the picture may become clearer withnalyses of clinical studies using anti-human TIM-1 mAbs in asthma

nd atopic dermatitis. Similar clarity might also be gained form these of anti-human TIM-1 mAbs for the prevention of viral infection,r the control of viral hepatitis. With luck we will see the results ofuch studies during the next decade of TIM-1 exploration.

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tters 141 (2011) 28– 35

Acknowledgements

The author would like to thank the following Biogen Idec col-laborators, and their associates, for their relentless efforts on theTIM-1 program: Veronique Bailly, Yen-Ming Hsu, Graham Farring-ton, Ellen Garber, Henry Hess, Patricia McCoon, Steven Miklasz,Dania Rabah, Sambasiva Rao, and Martin Scott. The modeling workshown in Fig. 1 was performed and analyzed by Alexey Lugovskoy.

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