The authors of this paper report four findings: 1) that galectin-9 does not inhibit activation or induce cell death in human T cells; 2) that galectin-9 is not a ligand for either human or murine Tim-3; 3) that Tim-3 has no functional role in T cell activation; and 4) that Tim-3 expression is not significantly up-regulated on T cells from HIV-infected subjects. The authors make far-reaching and broad claims that challenge the work of over one dozen laboratories around the world based based on a limited set of data.

The data in the paper are based on only one galectin-9 reagent that was purchased from a commercial source (R&D systems) and one anti-human Tim-3 antibody (clone 2E2). Moreover, the paper is based mostly on negative data for which no appropriate controls are provided. The authors report that galectin-9 does not induce T cell death. This is contradictory to the work of many laboratories that have shown repeatedly by different means that galectin-9 triggers T cell death. The authors report next that galectin-9 does not bind either human or murine Tim-3. Galectin-9 has been found to interact with many other surface molecules including CD44, CD40, and PDI, many of which exhibit low affinity interaction with galectin-9. Unfortunately, the authors did not include any one of these characterized galectin-9 receptors as a positive control to validate whether the ELISA system they set up for studying galectin-9-Tim-3 interaction is sensitive enough to differentiate between specific and non-specific interactions with galectin-9. Indeed, the ELISA assay actually shows that everything tested binds to galectin-9. Importantly, the authors do not show appropriate control experiments that demonstrate whether the galectin-9 used in their study is functional (cell adhesion, chemotaxis, etc). The lack of such controls in the study raises a concern as to the reliability of the results presented.

The authors further perform a series of functional assays using an anti-Tim-3 antibody. The Tim-3 antibody clone 2E2 that the authors use has been shown by at least five other labs to have functional effects on human T cells (Fourcade et al., 2010; Fourcade et al., 2012; Hastings et al., 2009; Jones et al., 2008; McMahan et al., 2010; Wu et al., 2012; Yang et al., 2008; Yang et al., 2012). Indeed, similar effects on T cell function have been reported in assays with other monoclonal antibody clones (1G5, 334823) and with both Tim-3 Ig fusion protein and soluble recombinant Tim-3 (Golden-Mason et al., 2009). In the assays with samples from HIV patients, the authors examine whole PBMC and not HIV-specific (HIV antigen tetramer positive) T cells. Only a fraction of T cells within PBMC will be HIV-specific and among these only a fraction will express Tim-3 or PD-1 and exhibit exhausted phenotype. Thus, it is not surprising that the authors fail to detect the effect of Tim-3 blockade. Similarly, Tim-3 expression or lack thereof, should have been tested on tetramer reactive HIV-specific T cells, before making the claim that TIM-3 expression is not upregulated in HIV patients. Lastly, the authors do not use any other anti-Tim-3 antibodies or recombinant protein reagents in any of their assays, many of which are commercially available, to corroborate their data.

Ultimately, the findings presented counter the work of many independent laboratories around the world. These labs have shown:
-Galectin-9 induces cell death in both human and murine T cells (papers not listed due to a large body of literature in the past 15 years).
-Galectin-9 binds Tim-3
-Galectin-9 differentially kills Tim-3 positive T cells and Tim-3 negative T cells
-Galectin-9 kills Tim-3+Treg cells
-Galectin-9 induces Tim-3+ NKT cells and such effect can be neutralized by anti-Tim-3 antibody
-EBV-infected nasopharyngeal carcinoma (NPC) release exosomes containing high amounts of galectin-9 to induce apoptosis of Th1 lymphocytes. Both anti-Tim-3 and anti-galectin-9 antibodies can block such effects.
-Galectin-9-Tim-3 interaction is required for NK cell activation and IFN-γ production
-Tim-3 triggers killing of Mycobacterium tuberculosis in macrophages that is dependent on galectin-9
-Tim-3 promotion of myeloid-derived suppressor cells is dependent on galectin-9
-Galectin-9 triggers dissociation of the adaptor protein Bat-3 from the intracytoplasmic tail of Tim-3
-Galectin-9 suppresses Tim-3 positive effector T cells in HIV
- Tim-3 expression is upregulated on both CD4+ and CD8+ T cells in the context of chronic viral infection (HIV, LCMV, HCV, and HBV) and that in HIV this expression correlates positively with viral load and inversely with CD4 count
-Tim-3 blockade increases T cell responses in T cells from patients with Multiple Sclerosis, follicular B cell non-Hodgkin lymphoma, HIV, chronic HBV infection, chronic HCV infection, advanced metastatic melanoma
-Tim-3 blockade increases T cell responses in experimental disease models including chronic LCMV infection, HSV infection, Influenza A infection, graft versus host disease, and cancer (melanoma, colon, prostate, nephroblastoma, fibrosarcoma, and acute myelogenous leukemia).

Collectively, these studies employ multiple different experimental methodologies including antibody blockade, recombinant protein/Ig fusion protein blockade, siRNA knock-down, transgenesis, and genetic deletion to probe either the receptor (Tim-3) or ligand (galectin-9) end of the pathway. They also use multiple different reagents from both commercial sources and academic laboratories including anti-human Tim-3 antibody clones (2E2,1G5,4A4,334823 and polyclonal goat antibody), anti-murine Tim-3 antibody clones (2C12, RMT3-23), recombinant Tim-3 (academic source), recombinant galectin-9 (academic source), Tim-3 Ig fusion protein (academic and commercial source), and si RNA.

It is true that there are still many things about Tim-3 that are not known and that additional research is required to increase our understanding of the mechanisms by which this important molecule regulates T cell function. However, this paper misleads the field by making large claims based on a limited set of data that are not substantiated by the experiments presented. As such the paper does not warrant the challenge it poses to the work of multiple independent laboratories around the world, all of which undertook well-controlled studies with qualified reagents and have been reproduced many times over in multiple experimental settings. A list of publications that support the findings that Tim-3 is a receptor for galectin-9 and that Tim-3 has functional effects on T cell responses follows.


Vijay K. Kuchroo, Chen Zhu, Ana C. Anderson, Sam Behar, Arlene Sharpe, Brad Jones, and Terry Strom, Harvard Medical School
Rafi Ahmed, Emory University
Hassane Zarour, University of Pittsburgh
Mitsuomi Hirashima, Kagawa University
Bruce Blazar, University of Minnesota
Lishomwa Ndhlovu, University of Hawaii
David Hafler, Yale Medical School
Mark Smyth, Queensland Institute of Medical Research
Mario Ostrowski, University of Toronto
Helen Horton, Seattle Biomedical Research Institute
Barry Rouse, University of Tennessee

Selected papers supporting Tim-3:Gal-9 interaction and functional role of Tim-3 on T cells:

Dardalhon V, Anderson AC, Karman J, Apetoh L, Chandwaskar R, Lee DH, Cornejo M, Nishi N, Yamauchi A, Quintana FJ, Sobel RA, Hirashima M, Kuchroo VK. Tim-3/galectin-9 pathway: regulation of Th1 immunity through promotion of CD11b+Ly-6G+ myeloid cells. J Immunol. 2010 Aug 1;185(3):1383-92.
Elahi, S., Dinges, W.L., Lejarcegui, N., Laing, K.J., Collier, A.C., Koelle, D.M., McElrath, M.J., and Horton, H. (2011). Protective HIV-specific CD8+ T cells evade Treg cell suppression. Nat Med 17, 989-995.
Elahi, S., Niki, T., Hirashima, M., and Horton, H. (2012). Galectin-9 binding to Tim-3 renders activated human CD4+ T cells less susceptible to HIV-1 infection. Blood 119, 4192-4204.
Fourcade J, Sun Z, Benallaoua M, Guillaume P, Luescheer IF, Sander C, Kirkwood JM, Kuchroo V, Zarour HM. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. J Exp Med. 2010 Sep 27;207(10):2175-86.
Fourcade, J., Sun, Z., Pagliano, O., Guillaume, P., Luescher, I.F., Sander, C., Kirkwood, J.M., Olive, D., Kuchroo, V., and Zarour, H.M. (2012). CD8(+) T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1. Cancer Res 72, 887-896.
Golden-Mason L, Palmer BE, Kassam N, Townshend-Bulson L, Livingston S, McMahon BJ, Castelblanco N, Kuchroo V, Gretch DR, Rosen HR. Negative immune regulator Tim-3 is over-expressed on T cells in hepatitis C virus infection and its blockade rescues dysfunctional CD4+ and CD8+ T cells. J Virol 2009;83:9122-30.
Gupta S. Thornley TB, Gao W, Larocca R, Turka LA, Kuchroo VK, Strom TB. Allograft rejection is restrained by short-lived TIM-3+PD-1+Foxp3+ Tregs. J Clin Invest. 2012 Jul 2;122(7):2395-404.
Hastings W, Anderson DE, Kassam N, Koguchi K, Greenfield EA, Kent SC, Zheng XX, Strom TB, Hafler DA, Kuchroo VK. TIM-3 is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. Eur J Immunol. 2009 Sep;39(9): 2492-501.
Jayaraman P, Sada-Ovalle I, Beladi S, Anderson AC, Dardalhon V, Hotta C, Kuchroo VK, Behar SM. Tim3 binding to galectin-9 stimulates antimicrobial immunity. J Exp Med. 2010 Oct 25;207(11):2343-54.
Jin H-T, Anderson AC, Tan WG, West EE, Ha SJ, Araki K, Freeman GJ, Kuchroo VK, Ahmed R. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc Natl Acad Sci USA. 2010 Aug 17;107(33):14733-8. Epub 2010 Aug 2.
Jones RB, Ndhlovu LC, Barbour JD, Sheth PM, Jha AR, Long BR, Wong JC, Satkunarajah M, Schweneker M, Chapman JM, Gyenes G, Vali B, Hyrcza MD, Yue FY, Kovacs C, Sassi A, Loutfy M, Halpenny R, Persad D, Spotts G, Hecht FM, Chun TW, McCune JM, Kaul R, Rini JM, Nixon DF, Ostrowski MA. 2008. Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection. J. Exp. Med. 205: 2763-79.
Klibi J, Niki T, Riedel A, Pioche-Durieu C, Souquere S, Rubinstein E, Le Moulec S, Guigay J, Hirashima M, Guemira F, Adhikary D, Mautner J, Busson P. Blood diffusion and Th1-suppressive effects of galectin-9-containing exosomes released by Epstein-Barr virus-infected nasopharyngeal carcinoma cells. Blood. 2009 Feb 26;113(9):1957-66.
Lee J, Sue EW, Zhu C, Hainline S, Phuah J, Moroco JA, Smithgall TE, Kuchroo VK, Kane LP. Phosphotyrosine-dependent coupling of tim-3 to T-cell receptor signaling pathways. Mol Cell Biol. 2011 Oct;31(19):3963-74. Epub 2011 Aug 1.
McMahan RH, Golden-Mason L, Nishimura MI, McMahon BJ, Kemper M, Allen TM, Gretch DR, Rosen HR. 2010. Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity. J Clin Invest 120: 4546-57
Mujib S, Jones RB, Lo C, Aidarus N, Clayton K, Sakhdari A, Benko E, Kovacs C, Ostrowski MA. Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway. J Immunol. 2012 Apr 15;188(8):3745-56.
Muthukumarana PA, Zheng XX, Rosengard BR, Strom TB, Metcalfe SM, In primed Allo-tolerance, TIM-3-Ig rapidly suppresses TGFbeta, but has no immediate effect on Foxp3. Transpl Int. 2008 Jun;21(6):593-7.
Ndhlovu, L.C., Lopez-Verges, S., Barbour, J.D., Jones, R.B., Jha, A.R., Long, B.R., Schoeffler, E.C., Fujita, T., Nixon, D.F., and Lanier, L.L. (2012). Tim-3 marks human natural killer cell maturation and suppresses cell-mediated cytotoxicity. Blood 119, 3734-3743.
Rangachari M, Zhu C, Sakuishi K, Xiao S, Karman J, Chen A, Angin M, Wakeham A, Greenfield EA, Sobel RA, Okada H, McKinnon PJ, Mak TW, Addo MM, Anderson AC, Kuchroo VK. Bat3 protects T cell responses by repressing Tim-3-mediated exhasustion and death. Nat Med. 2012 Sep;18(9):1394-400.
Sada-Ovalle I, Chavez-Galan L, Torre-Bouscoulet L, Nava-Gamino L, Barrera L, Jayaraman P, Torres-Rojas M, Salazar-Lezama MA, Behar SM. The Tim3-galectin-9 pathway induces antibacterial activity in human macrophages infected with Mycobacterium tuberculosis. J Immunol. 2012. Dec 15;189(12):5896-902.
Sakuishi K, Apetoh L, Sullivan JM, Blazer BR, Kuchroo VK, Anderson AC. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med. 2010 Sep 27;207(10):2187-94.
Sehrawat S, Reddy PB, Rajasagi N, Suryawanshi A, Hirashima M, Rouse BT. Galectin-9/TIM-3 interaction regulates virus-specific primary and memory CD8 T cell response. PLoS Pathog. 2010 May 6;6(5):e1000882.
Sharma S, Sundararajan A, Suryawanshi A, Kumar N, Veiga-Parga T, Kuchroo VK, Thomas PG, Sangster MY, Rouse BT. T cell immunoglobulin and mucin protein-3 (Tim-3)/Galectin-9 interaction regulates influenza A virus-specific humoral and CD8 T-cell responses. Proc Natl cad Sci U S A. 2011 Nov 22;108(47):19001-6.
Su EW, Lin JY, Kane LP. TIM-1 and TIM-3 proteins in immune regulation. Cytokine. 2008 Oct;44(1):9-13.
Tang ZH, Liang S, Potter J, Jiang X, Mao HQ, Li Z. Tim-3/Galectin-9 Regulate the Homeostasis of Hepatic NKT Cells in a Murine Model of Nonalcoholic Fatty Liver Disease. J Immunol. 2013 Feb 15;190(4):1788-96
Veenstra RG, Taylor PA, Zhou Q, Panoskaltsis-Mortari A, Hirashima M, Flynn R, Liu D, Anderson AC, Strom TB, Kuchroo VK, Blazar BR. Contrasting acute graft-versus-host-disease effects of Tim-3/galectin-9 pathway blockade dependent upon the presence of donor regulatory T cells. Blood. 2012 Jul 19;120(3):682-90.
Wu, W., Shi, Y., Li, S., Zhang, Y., Liu, Y., Wu, Y., and Chen, Z. (2012). Blockade of Tim-3 signaling restores the virus-specific CD8(+) T-cell response in patients with chronic hepatitis B. Eur J Immunol 42, 1180-1191.
Yang L, Anderson DE, Kuchroo J, Hafler DA. Lack of TIM-3 immunoregulation in multiple sclerosis. J Immunol. 2008 Apr 1;180(7):4409-14.
Yang, Z.Z., Grote, D.M., Ziesmer, S.C., Niki, T., Hirashima, M., Novak, A.J., Witzig, T.E., and Ansell, S.M. (2012). IL-12 upregulates TIM-3 expression and induces T cell exhaustion in patients with follicular B cell non-Hodgkin lymphoma. J Clin Invest 122, 1271-1282.
Zhou Q, Munger ME, Veenstra RG, Weigel BJ, Hirashima M, Munn DH, Murphy WJ, Azuma M, Anderson AC, Kuchroo VK, Blazar BR. Coexpression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion pehnotype in mice with disseminated acute myelogenous leukemia. Blood. 2011 Apr 28;117(17):4501-10.
Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury S, Zheng XX, Strom TB, and Kuchroo VK. The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol 2005; 6:1245-52.