Immune checkpoint receptors in cancer: redundant by design?

Available online at www.sciencedirect.com

ScienceDirect Immune checkpoint receptors in cancer: redundant by design? Jing Li, Ling Ni and Chen Dong Co-inhibitory receptors expressed on activated immune cells function to regulate T cell tolerance to self-antigens, also serving by tumor cells to escape from eradication by the host immune system. Therefore, blockade of immune checkpoint receptors (ICR) has become a promising immunotherapeutic strategy for treatment of a wide variety of cancers. However, blockade of one of the immune checkpoint receptors alone is often not sufficiently effective; co-blockade shows synergic effects in reversing immunosuppression. In this article, we summarize the expression patterns, mechanisms of action of different ICRs as well as the stages and sites they function in, and discuss how they execute non-redundant suppressive effects in anti-tumor immunity. Address Institute for Immunology and School of Medicine, Tsinghua University, Beijing 100084, China Corresponding author: Dong, Chen ([email protected])

Current Opinion in Immunology 2017, 45:37–42 This review comes from a themed issue on Tumour immunology

ICRs belong to the Ig superfamily and share some structural similarities, they function non-redundantly to suppress immune responses, since co-blockade of theses pathways can reverse immunosuppression to a greater extent than monotherapy. Here we compare the expression patterns of ICRs and their ligands in the tumor microenvironment, downstream signaling pathways, their major target cells and sites of action, revealing nonredundant roles in controlling immune responses.

CTLA-4 CTLA-4 was first identified in 1987 from mouse cytolytic T cells as a member of the immunoglobulin (Ig) superfamily [2]. Mice with genetic knockout of CTLA-4 died of excessive lymphoproliferation and severe autoimmunity in multiple organs within 3 weeks after birth, revealing its critical negative role in regulating immune response [3]. CTLA-4 is induced on Foxp3 CD4+ T and CD8+ T cells after early activation, while it is constitutively expressed on regulatory T cells (Treg). NFAT and Foxp3 are two key transcription factors regulating of CTLA-4 transcripts [4,5].

Edited by Dmitry Gabrilovich and Robert L Ferris

http://dx.doi.org/10.1016/j.coi.2017.01.001 0952-7915/ã 2017 Elsevier Ltd. All rights reserved.

Introduction Precise responses of T cells to antigens are determined by 2 signals: signal 1 comes from activation of T cell receptor (TCR) by antigens presented by MHC molecules, which confers specificity for T cell responses; signal 2 is driven by co-stimulatory or co-inhibitory receptors to augment or limit TCR-mediated activation, ensuring appropriate responses to foreign and self antigens. Co-inhibitory receptors are also regarded as immune checkpoints and are induced on activated T cells to avoid overreaction to self-antigens. Tumor antigens resemble self-antigens in some regards and tumor cells escape from eradication by effector T cells via expression of ligands for immune checkpoint receptors (ICR). Several ICRs, such as CTLA-4, PD-1, Tim-3 and LAG-3, have been identified and targeted for cancer immunotherapy to reinvigorate anti-tumor immune responses [1]. Although most of the www.sciencedirect.com

CTLA-4 functions as a negative regulator of T cell activation, by inhibiting proliferation, IL-2 production, IL-2 receptor expression and cell cycle progression of T cells [6]. Structurally, CTLA-4 resembles CD28, a costimulatory receptor, and binds to the CD28 ligands CD80 and CD86 with higher affinity. Thus, it delivers negative signals to T cells [7]. Notably, CTLA-4 is thought to function primarily to control T cell activation in the priming phase, since its ligands CD80 and CD86 are expressed on antigen-presenting cells (APC) in secondary lymphoid organs (Figure 1). CTLA-4 may exert its function in multiple cell types to control T cell activation and induce tolerance. In conventional Foxp3 CD4+ and CD8+ T cells, CTLA4 competes for the binding of CD28 to CD80 and CD86 and transduces inhibitory signals to TCR activation via its intracellular binding partners, PP2A and SHP-2. In Tregs, PKC-h is associated with the cytoplasmic tail of CTLA-4 and recruited to the immunological synapses of Treg to render suppressive activity to Treg. Moreover, CTLA-4 was reported to down-regulate CD86 and CD80 expression on the surface of APC by trans-endocytosis, depriving of co-stimulatory ligands for T cells [8]. Because of its inhibitory role in anti-tumor immune responses, CTLA-4 was the first immune checkpoint Current Opinion in Immunology 2017, 45:37–42

38 Tumour immunology

Figure 1

m Ti

-3

y y y y y y

y

Bat3

y

y

y

Lck

+ Activation

P Fyn ITIM ITIM

PD-1

Dual blockade of PD-1 and Tim-3: Reverse T cell exhaustion

Anergy ITSM P

P

pSTAT1/T-bet

SHP2

Ras/MEK/ERK

TCR

T cells

Akt

PI3K

Dual blockade of PD-1 and CTLA-4: Reverse suppression of T cells at both priming and effector phase

PP2A

PKCη

CTLA-4 KI

EE

LE

Treg suppressive activity

Activation

Dual blockade of PD-1 and LAG-3: Reverse T cell function synergistically via blocking different inhibitory pathways

LAG-3 Current Opinion in Immunology

Non-redundant suppressive effects of checkpoint receptors. CTLA-4 suppresses PI3K/Akt downstream of TCR activation via PP2A in conventional T cells and renders suppressive activity to Treg via PKC-h. LAG-3 contains KIEELE motif and inhibits T cell activation in an unknown way. PD-1 recruits SHP-2 to its phosphorylated ITSM motif to inhibit PI3K/Akt, Ras/MEK/ERK and p-STAT1/T-bet pathways. Tim-3 lacks classical inhibitory signaling motifs but contains five conserved tyrosine residues. In the presence of ligand binding, it might release Bat3 which promotes TCR activation and recruit Fyn which induces T cell anergy. The crosstalk between phosphatases induced by PD-1 and kinases recruited by Tim-3 might cooperate to induce T cell exhaustion.

receptor to be targeted for cancer immunotherapy. Ipilumumab, the CTLA-4 blockade antibody, was approved by FDA for treatment of advanced melanoma in 2014. Besides reversing inhibitory signals interfering TCR activation, this antibody may depend on depletion of intratumoral Tregs which highly express CTLA-4 through an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism [9].

lupus-like autoimmune diseases and autoimmune dilated cardiomyopathy in adults [10,11], indicating the selectivity of its regulatory role in certain organs. Expression of PD-1 can be induced on T cells by TCR signaling and common g chain cytokines (IL-2, IL-7, IL-15, IL-21 and type I interferons) via binding of NFATc1 and STATs to promoter or transcription start site of PD-1 locus [12,13].

PD-1

Programmed death-ligand 1 (PD-L1) and Programmed death-ligand 2 (PD-L2) are the two ligands which interact with PD-1, though PD-L2 has threefold higher affinity than PD-L1. Interaction of PD-1 with its ligand upon TCR stimulation results in phosphorylation of its immunoreceptor tyrosine-based inhibition motif (ITIM)

PD-1 is another co-inhibitory receptor in the CD28 superfamily and shares 15.6% identity with the CD28 extracellular domain. Unlike severe systemic autoimmunity observed in CTLA-4 knockout mice, PD-1-deficient mice develop organ-specific inflammation, such as Current Opinion in Immunology 2017, 45:37–42

www.sciencedirect.com

Immune checkpoint receptors in cancer Li, Ni and Dong 39

and an immunoreceptor tyrosine-based switch motif (ITSM), which recruits the phosphatase SHP-2 to shut down the TCR-mediated activation of PI3K-Akt and Ras-MEK-ERK pathways and activation of T-bet/ STAT1 by dephosphorylation, thus resulting in arrest of cell cycle progression and inhibition of effector function of T cells [8,14,15]. Recently, a study revealed that PD-L2 out-competes PD-L1 for binding with PD-1, which is essential for generating prolonged Th1 immune response in malarial infections [16]. It is yet to be determined whether PD-L2 regulates PD-1: PD-L1 axis in a similar way in other disease settings such as cancer. Our previous studies suggest that PD-L1 and PD-L2 differentially regulate T cell tolerance to tissue or dietary antigens, respectively, possibly due to their different expression in DC subsets [17,18]. Although PD-1 affects both cell cycle progression and effector functions of CD4+ and CD8+ conventional T cells and development of induced Treg [19], the main site of action for the PD-1 pathway in cancer is within the tumor microenvironment itself via suppressing antigen-experienced effector CD8+ T cells [20]. Since PD-1/PD-L1 blockade can reverse T cell dysfunction and exhaustion in pre-clinical mouse tumor and infection models, inhibiting this pathway has been successfully translated to clinical cancer therapy. PD-1 inhibitors, such as nivolumab and pembrolizumab, have shown broad efficacy (response rates of 10–87%) in multiple types of cancer in clinical trials, and therefore has been FDA-approved for treatment of metastatic melanoma, non-small cell lung cancer, head and neck cancer and kidney cancer [8]. Notably, the combination of CTLA-4 and PD-1 blockade was more effective than monotherapies in treatment of melanoma and lung cancer [21,22], since they regulate T cell immune response at priming stage and effector stage, respectively, revealing non-redundant functions of PD-1 and CTLA-4.

Tim-3 Tim-3 was initially identified as a specific marker on CD4+ T helper 1 (Th1) and CD8+ T cytotoxic 1 (Tc1) cells [23]. Mice deficient with Tim-3 do not develop spontaneous autoimmune diseases but fail in immune tolerance induction [24]. Expression of Tim-3 was found on Th1, Tc1, Treg as well as innate immune cells including NK, DC and monocytes in response to inflammatory stimuli [25].

stimulation [27]. Recently, CEACAM1 has been reported to bind to Tim-3 in cis and in trans. CEACAM1 is coexpressed with Tim-3 on T cells, and the cis interaction stabilizes Tim-3 on the cell surface. Both cis and trans interactions drive inhibition of T cell function [28]. The complex binding of Tim-3 with different ligands might render selectivity and specificity for Tim-3 pathways to fine-tune immune responses in tissues, however, there is still controversy regarding its ultimate function. Tim-3 consists of an extracellular IgV domain, a mucin domain, a transmembrane domain and a cytoplasmic tail. Unlike CTLA-4 and PD-1, the cytoplasmic tail of Tim-3 lacks a classical inhibitory signaling motif such as ITIM and ITSM, but contains five conserved tyrosine residues that can be phosphorylated by intracellular kinases [29]. In the absence of ligand binding, Bat3 is bound to the cytoplasmic tail of Tim-3 to preserve and promote T cell signaling via recruitment of Lck [30]. Binding of Tim-3 to its ligands leads to phosphorylation of its cytoplasmic tail, release of Bat3 and possible recruitment of Fyn, which can induce T cell anergy [31]. Therefore, different signals through Tim-3 might switch the function of Tim-3 on TCR signaling via interactions with different downstream binding partners. However, the detailed signaling pathways and how the interactions with different ligands in different cell types affect the signaling need to be further investigated. Since galectin-9 and HMGB1 can be induced under inflammatory conditions, whereas expression of Tim-3 is driven by IFNb [32], control of immune responses by Tim-3 mainly takes place in inflammatory sites. Tim-3 is highly expressed in chronic viral infection (such as HIV, HCV and HBV) and in cancer [33–36] but is poorly expressed in autoimmunity, indicating a special role of Tim-3 in chronic inflammation. Notably, Tim-3 is expressed by Tregs in the tumor but not in peripheral sites, endowing them superior suppressive function [37]. Moreover, Tim-3 is co-expressed with PD-1 on virusspecific and tumor-infiltrating CD8+ T cells, which marks the most dysfunctional or exhausted CD8 T subset. PD-1 antibody-bound T cells show upregulated Tim-3 [38]. In addition, co-blockade of PD-1 and Tim-3 results in greater reinvigoration in effector T cell responses than single blockade and intracellular crosstalk of PD-1 and Tim-3 has been implicated [34,39,40], indicating that PD-1 and Tim-3 have non-redundant and cooperative functions in suppressing T cell responses.

LAG-3 Several molecules have been identified as ligands of Tim3 and mediate immune tolerance of different immune cell types. The C-type lectin galectin-9 binds to Tim-3+ Th1 cells to suppress type 1 immune response and ameliorate EAE [26]. High Mobility Group Protein B1 (HMGB1) interferes with responses of Tim-3+ DC to nucleic acid www.sciencedirect.com

LAG-3, another checkpoint receptor identified 25 years ago, is induced on activated CD4+, CD8+ T cells, Treg and a subset of NK cells. It resembles the CD4 coreceptor structurally. Mice deficient with LAG-3 are normal in diverse aspects of immune function in steady state [41] but show uncontrolled expansion of T cells Current Opinion in Immunology 2017, 45:37–42

40 Tumour immunology

when challenged with Staphylococcal enterotoxin B (SEB) and OVA [42]. LAG-3 binds to MHC class II with higher affinity than does CD4, and therefore mediates immunosuppression of CD4+ T cells [43]. Recently, another two ligands of LAG3, LSECtin and Galectin-3, have been identified. LSECtin was initially found to be expressed in the liver and promoted tolerance to HBV infection [44], and was later found to be expressed on human and mouse melanomas to suppress tumor-specific T cell responses [45]. Galectin-3 is highly expressed on tumor-infiltrating CD8+ T cells and might inhibit anti-tumor T cell responses via cis and trans interaction with LAG-3 [46]. LAG-3 does not contain classical signaling motifs in its cytoplasmic tail but its unique KIEELE motif is essential for its inhibitory function in effector CD4+ T cells [47]. LAG-3 suppresses proliferation and cytokine production of effector T cells and confers Treg an activated phenotype, producing high levels of IL-10 and TGF-b. Although blockade of LAG-3 can synergize with tumor vaccine to improve anti-tumor efficacy, this effect is dependent on activation of tumor-specific CD8+ T cells and does not require CD4+ T cells [48]. Whether LAG-3 regulates the functions of CD4+ effector T, Treg and NK cells in other contexts is yet to be determined. Although LAG-3 deficiency alone does not cause autoimmunity, combined LAG-3 and PD-1 deficiency induces lethal myocarditis in BALB/c mice [49]. LAG3 is co-expressed with PD-1 on exhausted CD8+ T cells during chronic LCMV infection and tumor-infiltrating CD4+ and CD8+ T cells. Importantly, co-blockade of PD-1 and LAG-3 pathways can synergize to restore

T cell function in both chronic viral infection and cancer [50,51], identifying LAG-3 as a non-redundant checkpoint receptor.

Conclusion and outlook In summary, checkpoint receptors function non-redundantly to restrain immune responses (Table 1). Although most of them are expressed on effector T cells and Tregs, they display different kinetics of expression after stimulation and have different target cells in cancer; Moreover, they interact with different downstream signaling components to mediate immunosuppression. CTLA-4 mainly controls T cell responses in secondary lymphoid organs, while other checkpoint receptors mainly function in tissues to maintain tissue tolerance and suppress immunopathology, dependent on where their ligands are expressed. PD-1, Tim-3 and LAG-3 are co-expressed on exhausted CD8+ T cells in chronic viral infection and in the tumor sites, and dual blockade of PD-1 and Tim-3, or PD-1 and LAG-3 shows greater restoration in T cell function than monotherapies, indicating they act nonredundantly, suggesting the possibility of intracellular crosstalk between these receptors. Given the clinical and biological data to date, clearly the mechanisms for intracellular crosstalk of these checkpoint receptors need to be further investigated. Recently we have seen great success of anti-PD-1 for treatment of multiple types of cancer, but there are still 60–80% of patients who do not respond to the therapy. Whether these non-responder patients can benefit from combination with blockade of other checkpoint receptors and the extent to which dual (or triple) blockade of ICRs will cause more severe toxicity in patients is emerging from recent clinical trials (ASCO 2016). These studies will ultimately unleash the power of the immune system, and improve clinical

Table 1 Comparison of non-redundant immune checkpoint receptors ICR

Ligands

Expression Ligand

Signaling motif

Downstream molecules

Main target cells

Site of action

Receptor

CTLA-4

CD80, CD86

DC, Mf

Tconv, Treg

ITIM ITSM

PP2A, SHP-2, PKC-h (Treg)

Effector T, Treg

Secondary lymphoid organs

PD-1

PD-L1, PD-L2

PD-L1: induced expression on both hematopoietic and non-hematopoietic cells PD-L2: DC, Mf

T, NK, NKT

ITIM ITSM

SHP-2

CD8 T

Tissue (pancreas islet, stroma) Tumor

Tim-3

Galectin-9, HMGB1, CEACAM1

Gal-9: eosinophil, T cells, DCs, Mf, endothelial cells HMGB1: soluble ligands in inflamed tissues CEACAM1: co-expressed with Tim-3 on T cells

Th1, Tc1, Treg, NK, DC and Mo

Five conserved tyrosine residues

Bat-3/Fyn

Exhausted CD8 T, Treg

Tissue (brain, gut) Tumor

LAG-3

MHC II, LSECtin, Galectin-3

MHC II: APC LSECtin: liver Gal-3: CD8 TIL

CD4 T, CD8 T, NK

KIEELE

Unknown

CD8 T, Treg

Tissue (liver) Tumor

Current Opinion in Immunology 2017, 45:37–42

www.sciencedirect.com

Immune checkpoint receptors in cancer Li, Ni and Dong 41

responses in cancer patients and provide invaluable pretreatment and on-treatment specimens for translational research.

13. Terawaki S, Chikuma S, Shibayama S, Hayashi T, Yoshida T, Okazaki T, Honjo T: IFN-alpha directly promotes programmed cell death-1 transcription and limits the duration of T cellmediated immunity. J Immunol 2011, 186:2772-2779.

Conflicts of interest

14. Yokosuka T, Takamatsu M, Kobayashi-Imanishi W, HashimotoTane A, Azuma M, Saito T: Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2. J Exp Med 2012, 209:1201-1217.

The authors declare that there are no conflicts of interest to disclose.

Acknowledgements This work was supported by grant from The National Natural Science Foundation of China (NSFC) (grant number 81502462 to L.N.). C.D. is a Bayer Chair Professor at Tsinghua University.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Pardoll DM: The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012, 12:252-264.

2.

Brunet JF, Denizot F, Luciani MF, Roux-Dosseto M, Suzan M, Mattei MG, Golstein P: A new member of the immunoglobulin superfamily—CTLA-4. Nature 1987, 328:267-270.

3.

4.

Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH: Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 1995, 3:541-547. Gibson HM, Hedgcock CJ, Aufiero BM, Wilson AJ, Hafner MS, Tsokos GC, Wong HK: Induction of the CTLA-4 gene in human lymphocytes is dependent on NFAT binding the proximal promoter. J Immunol 2007, 179:3831-3840.

5.

Zheng Y, Josefowicz SZ, Kas A, Chu TT, Gavin MA, Rudensky AY: Genome-wide analysis of Foxp3 target genes in developing and mature regulatory T cells. Nature 2007, 445:936-940.

6.

Walunas TL, Bakker CY, Bluestone JA: CTLA-4 ligation blocks CD28-dependent T cell activation. J Exp Med 1996, 183:25412550.

7.

Collins AV, Brodie DW, Gilbert RJ, Iaboni A, Manso-Sancho R, Walse B, Stuart DI, van der Merwe PA, Davis SJ: The interaction properties of costimulatory molecules revisited. Immunity 2002, 17:201-210.

8. 

Schildberg FA, Klein SR, Freeman GJ, Sharpe AH: Coinhibitory pathways in the B7-CD28 ligand-receptor family. Immunity 2016, 44:955-972. This review summarizes the recent advances in the understanding of coinhibitory pathways in the B7-CD28 family, including CTLA-4 and PD-1 pathways. 9.

Jie HB, Schuler PJ, Lee SC, Srivastava RM, Argiris A, Ferrone S, Whiteside TL, Ferris RL: CTLA-4(+) regulatory T cells increased in cetuximab-treated head and neck cancer patients suppress NK cell cytotoxicity and correlate with poor prognosis. Cancer Res 2015, 75:2200-2210.

10. Nishimura H, Nose M, Hiai H, Minato N, Honjo T: Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999, 11:141-151. 11. Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, Sasayama S, Mizoguchi A, Hiai H, Minato N et al.: Autoimmune dilated cardiomyopathy in PD-1 receptordeficient mice. Science 2001, 291:319-322. 12. Austin JW, Lu P, Majumder P, Ahmed R, Boss JM: STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells. J Immunol 2014, 192:48764886. www.sciencedirect.com

15. Li J, Jie HB, Lei Y, Gildener-Leapman N, Trivedi S, Green T, Kane LP, Ferris RL: PD-1/SHP-2 inhibits Tc1/Th1 phenotypic responses and the activation of T cells in the tumor microenvironment. Cancer Res 2015, 75:508-518. 16. Karunarathne DS, Horne-Debets JM, Huang JX, Faleiro R, Leow CY, Amante F, Watkins TS, Miles JJ, Dwyer PJ, Stacey KJ et al.: Programmed death-1 ligand 2-mediated regulation of the PD-L1 to PD-1 axis is essential for establishing CD4(+) T cell immunity. Immunity 2016, 45:333-345. 17. Martin-Orozco N, Wang YH, Yagita H, Dong C: Cutting edge: programmed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens. J Immunol 2006, 177:8291-8295. 18. Zhang Y, Chung Y, Bishop C, Daugherty B, Chute H, Holst P, Kurahara C, Lott F, Sun N, Welcher AA et al.: Regulation of T cell activation and tolerance by PDL2. Proc Natl Acad Sci U S A 2006, 103:11695-11700. 19. Francisco LM, Salinas VH, Brown KE, Vanguri VK, Freeman GJ, Kuchroo VK, Sharpe AH: PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. J Exp Med 2009, 206:3015-3029. 20. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V et al.: PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014, 515:568-571. 21. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL,  Lao CD, Schadendorf D, Dummer R, Smylie M, Rutkowski P et al.: Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med 2015, 373:23-34. This article reports the combincational therapy of checkpoint blockades, anti-CTLA-4 plus anti-PD-1 in the clinical trial, revealing the non-redundant suppressive effects of CTLA-4 and PD-1 in anti-tumor immunity. 22. Antonia SJ, Gettinger SN, Goldman J, Brahmer J, Borghaei H, Chow LQ, Ready NE, Gerber DE, Juergens R, Shepherd F et al.: ORAL01.03: CheckMate 012: safety and efficacy of first-line nivolumab and ipilimumab in advanced NSCLC: topic: medical oncology. J Thorac Oncol 2016, 11:S250-S251. 23. Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, Manning S, Greenfield EA, Coyle AJ, Sobel RA et al.: Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 2002, 415:536-541. 24. Sabatos CA, Chakravarti S, Cha E, Schubart A, Sanchez-Fueyo A, Zheng XX, Coyle AJ, Strom TB, Freeman GJ, Kuchroo VK: Interaction of Tim-3 and Tim-3 ligand regulates T helper type 1 responses and induction of peripheral tolerance. Nat Immunol 2003, 4:1102-1110. 25. Anderson AC, Joller N, Kuchroo VK: Lag-3, Tim-3, and TIGIT:  co-inhibitory receptors with specialized functions in immune regulation. Immunity 2016, 44:989-1004. This review summaizes the functions, signaling and roles in disease of LAG-3, Tim-3 and TIGIT. 26. Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury SJ, Zheng XX, Strom TB, Kuchroo VK: The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol 2005, 6:1245-1252. 27. Chiba S, Baghdadi M, Akiba H, Yoshiyama H, Kinoshita I, Dosaka-Akita H, Fujioka Y, Ohba Y, Gorman JV, Colgan JD et al.: Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1. Nat Immunol 2012, 13:832-842. Current Opinion in Immunology 2017, 45:37–42

42 Tumour immunology

28. Huang YH, Zhu C, Kondo Y, Anderson AC, Gandhi A, Russell A, Dougan SK, Petersen BS, Melum E, Pertel T et al.: CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature 2015, 517:386-390. 29. Lee J, Su EW, Zhu C, Hainline S, Phuah J, Moroco JA, Smithgall TE, Kuchroo VK, Kane LP: Phosphotyrosinedependent coupling of Tim-3 to T-cell receptor signaling pathways. Mol Cell Biol 2011, 31:3963-3974. 30. Rangachari M, Zhu C, Sakuishi K, Xiao S, Karman J, Chen A, Angin M, Wakeham A, Greenfield EA, Sobel RA et al.: Bat3 promotes T cell responses and autoimmunity by repressing Tim-3-mediated cell death and exhaustion. Nat Med 2012, 18:1394-1400. 31. Davidson D, Schraven B, Veillette A: PAG-associated FynT regulates calcium signaling and promotes anergy in T lymphocytes. Mol Cell Biol 2007, 27:1960-1973. 32. Boivin N, Baillargeon J, Doss PM, Roy AP, Rangachari M: Interferon-beta suppresses murine Th1 cell function in the absence of antigen-presenting cells. PLoS One 2015, 10: e0124802. 33. Jones RB, Ndhlovu LC, Barbour JD, Sheth PM, Jha AR, Long BR, Wong JC, Satkunarajah M, Schweneker M, Chapman JM et al.: Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection. J Exp Med 2008, 205:2763-2779. 34. McMahan RH, Golden-Mason L, Nishimura MI, McMahon BJ, Kemper M, Allen TM, Gretch DR, Rosen HR: 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 2010, 120:4546-4557. 35. Wu W, Shi Y, Li J, Chen F, Chen Z, Zheng M: Tim-3 expression on peripheral T cell subsets correlates with disease progression in hepatitis B infection. Virol J 2011, 8:113.

40. Fourcade J, Sun Z, Benallaoua M, Guillaume P, Luescher 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, 207:2175-2186. 41. Miyazaki T, Dierich A, Benoist C, Mathis D: Independent modes of natural killing distinguished in mice lacking Lag3. Science 1996, 272:405-408. 42. Workman CJ, Vignali DA: The CD4-related molecule, LAG-3 (CD223), regulates the expansion of activated T cells. Eur J Immunol 2003, 33:970-979. 43. Huard B, Prigent P, Tournier M, Bruniquel D, Triebel F: CD4/major histocompatibility complex class II interaction analyzed with CD4- and lymphocyte activation gene-3 (LAG-3)-Ig fusion proteins. Eur J Immunol 1995, 25:2718-2721. 44. Liu B, Wang M, Wang X, Zhao D, Liu D, Liu J, Chen PJ, Yang D, He F, Tang L: Liver sinusoidal endothelial cell lectin inhibits CTL-dependent virus clearance in mouse models of viral hepatitis. J Immunol 2013, 190:4185-4195. 45. Xu F, Liu J, Liu D, Liu B, Wang M, Hu Z, Du X, Tang L, He F: LSECtin expressed on melanoma cells promotes tumor progression by inhibiting antitumor T-cell responses. Cancer Res 2014, 74:3418-3428. 46. Kouo T, Huang L, Pucsek AB, Cao M, Solt S, Armstrong T, Jaffee E: Galectin-3 shapes antitumor immune responses by suppressing CD8+ T cells via LAG-3 and inhibiting expansion of plasmacytoid dendritic cells. Cancer Immunol Res 2015, 3:412-423. 47. Workman CJ, Dugger KJ, Vignali DA: Cutting edge: molecular analysis of the negative regulatory function of lymphocyte activation gene-3. J Immunol 2002, 169:5392-5395.

36. Sakuishi K, Apetoh L, Sullivan JM, Blazar 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, 207:2187-2194.

48. Grosso JF, Kelleher CC, Harris TJ, Maris CH, Hipkiss EL, De Marzo A, Anders R, Netto G, Getnet D, Bruno TC et al.: LAG-3 regulates CD8+ T cell accumulation and effector function in murine self- and tumor-tolerance systems. J Clin Invest 2007, 117:3383-3392.

37. Sakuishi K, Ngiow SF, Sullivan JM, Teng MW, Kuchroo VK, Smyth MJ, Anderson AC: TIM3+FOXP3+ regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer. Oncoimmunology 2013, 2:e23849.

49. Okazaki T, Okazaki IM, Wang J, Sugiura D, Nakaki F, Yoshida T, Kato Y, Fagarasan S, Muramatsu M, Eto T et al.: PD-1 and LAG-3 inhibitory co-receptors act synergistically to prevent autoimmunity in mice. J Exp Med 2011, 208:395-407.

38. Koyama S, Akbay EA, Li YY, Herter-Sprie GS, Buczkowski KA, Richards WG, Gandhi L, Redig AJ, Rodig SJ, Asahina H et al.: Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun 2016, 7:10501.

50. Blackburn SD, Shin H, Haining WN, Zou T, Workman CJ, Polley A, Betts MR, Freeman GJ, Vignali DA, Wherry EJ: Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol 2009, 10:29-37.

39. Jin HT, 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 U S A 2010, 107:14733-14738.

Current Opinion in Immunology 2017, 45:37–42

51. Woo SR, Turnis ME, Goldberg MV, Bankoti J, Selby M, Nirschl CJ, Bettini ML, Gravano DM, Vogel P, Liu CL et al.: Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape. Cancer Res 2012, 72:917-927.

www.sciencedirect.com