Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1^–/– donors. PD-L1–deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1^–/– donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell–mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.     

Ibrutinib treatment ameliorates murine chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4⁺ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell–driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell–mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials.     

CSF-1–dependant donor-derived macrophages mediate chronic graft-versus-host disease

Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17–dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80⁺CSF-1R⁺CD206⁺iNOS^–). Cutaneous cGVHD developed in a CSF-1/CSF-1R–dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r^–/– mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80⁺ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti–CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD.     

Epstein-Barr Virus–induced Molecule 1 Ligand Chemokine Is Expressed by Dendritic Cells in Lymphoid Tissues and Strongly Attracts Naive T Cells and Activated B Cells

Movement of T and B lymphocytes through secondary lymphoid tissues is likely to involve multiple cues that help the cells navigate to appropriate compartments. Epstein-Barr virus– induced molecule 1 (EBI-1) ligand chemokine (ELC/MIP3β) is expressed constitutively within lymphoid tissues and may act as such a guidance cue. Here, we have isolated mouse ELC and characterized its expression pattern and chemotactic properties. ELC is expressed constitutively in dendritic cells within the T cell zone of secondary lymphoid tissues. Recombinant ELC was strongly chemotactic for naive (L-selectin^hi) CD4 T cells and for CD8 T cells and weakly attractive for resting B cells and memory (L-selectin^lo) CD4 T cells. After activation through the B cell receptor, the chemotactic response of B cells was enhanced. Like its human counterpart, murine ELC stimulated cells transfected with EBI-1/CC chemokine receptor 7 (CCR7). Our findings suggest a central role for ELC in promoting encounters between recirculating T cells and dendritic cells and in the migration of activated B cells into the T zone of secondary lymphoid tissues.     

Rapamycin-treated human endothelial cells preferentially activate allogeneic regulatory T cells

Human graft endothelial cells (ECs) can act as antigen-presenting cells to initiate allograft rejection by host memory T cells. Rapamycin, an mTOR inhibitor used clinically to suppress T cell responses, also acts on DCs, rendering them tolerogenic. Here, we report the effects of rapamycin on EC alloimmunogenicity. Compared with mock-treated cells, rapamycin-pretreated human ECs (rapa-ECs) stimulated less proliferation and cytokine secretion from allogeneic CD4⁺ memory cells, an effect mimicked by shRNA knockdown of mTOR or raptor in ECs. The effects of rapamycin persisted for several days and were linked to upregulation of the inhibitory molecules PD-L1 and PD-L2 on rapa-ECs. Additionally, rapa-ECs produced lower levels of the inflammatory cytokine IL-6. CD4⁺ memory cells activated by allogeneic rapa-ECs became hyporesponsive to restimulation in an alloantigen-specific manner and contained higher percentages of suppressive CD4⁺CD25^hiCD127^loFoxP3⁺ cells that did not produce effector cytokines. In a human-mouse chimeric model of allograft rejection, rapamycin pretreatment of human arterial allografts increased graft EC expression of PD-L1 and PD-L2 and reduced subsequent infiltration of allogeneic effector T cells into the artery intima and intimal expansion. Preoperative conditioning of allograft ECs with rapamycin could potentially reduce immune-mediated rejection.     

Immunoregulatory mechanisms triggered by viral infections protect from type 1 diabetes in mice

Type 1 diabetes (T1D) is an autoimmune disease that is caused by the destruction of insulin-producing β cells. Viral infections induce immune responses that can damage β cells and promote T1D or on the other hand prevent the development of the disease. However, the opposing roles of viral infections in T1D are not understood mechanistically. We report here that viruses that do not inflict damage on β cells provided protection from T1D by triggering immunoregulatory mechanisms. Infection of prediabetic NOD mice with Coxsackie virus B3 or lymphocytic choriomeningitis virus (LCMV) delayed diabetes onset and reduced disease incidence. Delayed T1D onset was due to transient upregulation of programmed cell death–1 ligand 1 (PD-L1) on lymphoid cells, which prevented the expansion of diabetogenic CD8⁺ T cells expressing programmed cell death–1 (PD-1). Reduced T1D incidence was caused by increased numbers of invigorated CD4⁺CD25⁺ Tregs, which produced TGF-β and maintained long-term tolerance. Full protection from T1D resulted from synergy between PD-L1 and CD4⁺CD25⁺ Tregs. Our results provide what we believe to be novel mechanistic insight into the role of viruses in T1D and should be valuable for prospective studies in humans.     

IL-33 acts as a costimulatory signal to generate alloreactive Th1 cells in graft-versus-host disease

Antigen-presenting cells (APCs) integrate signals emanating from local pathology and program appropriate T cell responses. In allogeneic hematopoietic stem cell transplantation (alloHCT), recipient conditioning releases damage-associated molecular patterns (DAMPs) that generate proinflammatory APCs that secrete IL-12, which is a driver of donor Th1 responses, causing graft-versus-host disease (GVHD). Nevertheless, other mechanisms exist to initiate alloreactive T cell responses, as recipients with disrupted DAMP signaling or lacking IL-12 develop GVHD. We established that tissue damage signals are perceived directly by donor CD4⁺ T cells and promoted T cell expansion and differentiation. Specifically, the fibroblastic reticular cell–derived DAMP IL-33 is increased by recipient conditioning and is critical for the initial activation, proliferation, and differentiation of alloreactive Th1 cells. IL-33 stimulation of CD4⁺ T cells was not required for lymphopenia-induced expansion, however. IL-33 promoted IL-12–independent expression of Tbet and generation of Th1 cells that infiltrated GVHD target tissues. Mechanistically, IL-33 augmented CD4⁺ T cell TCR-associated signaling pathways in response to alloantigen. This enhanced T cell expansion and Th1 polarization, but inhibited the expression of regulatory molecules such as IL-10 and Foxp3. These data establish an unappreciated role for IL-33 as a costimulatory signal for donor Th1 generation after alloHCT.     

Potentiating Functional Antigen-specific CD8+ T Cell Immunity by a Novel PD1 Isoform-based Fusion DNA Vaccine

Understanding and identifying new ways of mounting an effective CD8⁺ T cell immune response is important for eliminating infectious pathogens. Although upregulated programmed death-1 (PD1) in chronic infections (such as HIV-1 and tuberculosis) impedes T cell responses, blocking this PD1/PD-L pathway could functionally rescue the “exhausted” T cells. However, there exists a number of PD1 spliced variants with unknown biological function. Here, we identified a new isoform of human PD1 (Δ42PD1) that contains a 42-nucleotide in-frame deletion located at exon 2 domain found expressed in peripheral blood mononuclear cells (PBMCs). Δ42PD1 appears to function distinctly from PD1, as it does not engage PD-L1/PD-L2 but its recombinant form could induce proinflammatory cytokines. We utilized Δ42PD1 as an intramolecular adjuvant to develop a fusion DNA vaccine with HIV-1 Gag p24 antigen to immunize mice, which elicited a significantly enhanced level of anti-p24 IgG1/IgG2a antibody titers, and important p24-specific and tetramer⁺CD8⁺ T cells responses that lasted for ≥7.5 months. Furthermore, p24-specific CD8⁺ T cells remain functionally improved in proliferative and cytolytic capacities. Importantly, the enhanced antigen-specific immunity protected mice against pathogenic viral challenge and tumor growth. Thus, this newly identified PD1 variant (Δ42PD1) amplifies the generation of antigen-specific CD8⁺ T cell immunity when used in a DNA vaccine.     

Hypoxia-inducible factor activity promotes antitumor effector function and tissue residency by CD8+ T cells

Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8⁺ T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8⁺ T cells induced HIF-1α/HIF-2α–dependent differentiation of tissue-resident memory–like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8⁺ TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.     

Absence of mouse 2B4 promotes NK cell–mediated killing of activated CD8+ T cells,leading to prolonged viral persistence and altered pathogenesis

Persistent viral infections are often associated with inefficient T cell responses and sustained high-level expression of inhibitory receptors, such as the NK cell receptor 2B4 (also known as CD244), on virus-specific T cells. However, the role of 2B4 in T cell dysfunction is undefined, and it is unknown whether NK cells contribute to regulation of these processes. We show here that persistent lymphocytic choriomeningitis virus (LCMV) infection of mice lacking 2B4 resulted in diminished LCMV-specific CD8⁺ T cell responses, prolonged viral persistence, and spleen and thymic pathologies that differed from those observed in infected wild-type mice. Surprisingly, these altered phenotypes were not caused by 2B4 deficiency in T cells. Rather, the entire and long-lasting pathology and viral persistence were regulated by 2B4-deficient NK cells acting early in infection. In the absence of 2B4, NK cells lysed activated (defined as CD44^hi) but not naive (defined as CD44^lo) CD8⁺ T cells in a perforin-dependent manner in vitro and in vivo. These results illustrate the importance of NK cell self-tolerance to activated CD8⁺ T cells and demonstrate how an apparent T cell–associated persistent infection can actually be regulated by NK cells.     

Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease

Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of dopamine-containing neurons. Mounting evidence suggests that dopaminergic cell death is influenced by the innate immune system. However, the pathogenic role of the adaptive immune system in PD remains enigmatic. Here we showed that CD8⁺ and CD4⁺ T cells but not B cells had invaded the brain in both postmortem human PD specimens and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD during the course of neuronal degeneration. We further demonstrated that MPTP-induced dopaminergic cell death was markedly attenuated in the absence of mature T lymphocytes in 2 different immunodeficient mouse strains (Rag1^–/– and Tcrb^–/– mice). Importantly, similar attenuation of MPTP-induced dopaminergic cell death was seen in mice lacking CD4 as well as in Rag1^–/– mice reconstituted with FasL-deficient splenocytes. However, mice lacking CD8 and Rag1^–/– mice reconstituted with IFN-γ–deficient splenocytes were not protected. These data indicate that T cell–mediated dopaminergic toxicity is almost exclusively arbitrated by CD4⁺ T cells and requires the expression of FasL but not IFNγ. Further, our data may provide a rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD.     

PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in regulating T cell responses during chronic infection in mice

The inhibitory receptor programmed death 1 (PD-1) is upregulated on antigen-specific CD8⁺ T cells during persistent viral infections. Interaction with PD-1 ligand 1 (PD-L1) contributes to functional exhaustion of responding T cells and may limit immunopathology during infection. PD-L1 is expressed on both hematopoietic and nonhematopoietic cells in tissues. However, the exact roles of PD-L1 on hematopoietic versus nonhematopoietic cells in modulating immune responses are unclear. Here we used bone marrow chimeric mice to examine the effects of PD-L1 deficiency in hematopoietic or nonhematopoietic cells during lymphocytic choriomeningitis virus clone 13 (LCMV CL-13) infection. We found that PD-L1 expression on hematopoietic cells inhibited CD8⁺ T cell numbers and function after LCMV CL-13 infection. In contrast, PD-L1 expression on nonhematopoietic cells limited viral clearance and immunopathology in infected tissues. Together, these data demonstrate that there are distinct roles for PD-L1 on hematopoietic and nonhematopoietic cells in regulating CD8⁺ T cell responses and viral clearance during chronic viral infection.     

An Asia-specific variant of human IgG1 represses colorectal tumorigenesis by shaping the tumor microenvironment

Emerging studies have focused on ways to treat cancers by modulating T cell activation. However, whether B cell receptor signaling in the tumor microenvironment (TME) can be harnessed for immunotherapy is unclear. Here, we report that an Asia-specific variant of human IgG1 containing a Gly396 to Arg396 substitution (hIgG1-G396R) conferred improved survival of patients with colorectal cancer (CRC). Mice with knockin of the murine functional homolog mIgG2c-G400R recapitulated the alleviated tumorigenesis and progression in murine colon carcinoma models. Immune profiling of the TME revealed broad mobilizations of IgG1⁺ plasma cells, CD8⁺ T cells, CD103⁺ DCs, and active tertiary lymphoid structure formation, suggesting an effective antitumor microenvironment in hIgG1-G396R CRC patients. Mechanistically, this variant potentiated tumor-associated antigen–specific (TAA-specific) plasma cell differentiation and thus antibody production. These elevated TAA-specific IgG2c antibodies in turn efficiently boosted the antibody-dependent tumor cell phagocytosis and TAA presentation to effector CD8⁺ T cells. Notably, adoptive transfer of TAA-specific class-switched memory B cells harboring this variant exhibited therapeutic efficacy in murine tumor models, indicating their clinical potential. All these results prompted a prospective investigation of hIgG1-G396R in patients with CRC as a biomarker for clinical prognosis and demonstrated that manipulating the functionality of IgG1⁺ memory B cells in tumors could improve immunotherapy outcomes.     

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates antitumor immune responses

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti–PD-1 immunotherapy. We showed that phospho-eIF4E–deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E–mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8⁺ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1⁺PD-1⁺CD8⁺ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti–PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti–PD-1 immunotherapy.     

Coinhibitory receptor PD-1H preferentially suppresses CD4+ T cell–mediated immunity

T cell activation is regulated by the interactions of surface receptors with stimulatory and inhibitory ligands. Programmed death-1 homolog (PD-1H, also called VISTA) is a member of the CD28 family of proteins and has been shown to act as a coinhibitory ligand on APCs that suppress T cell responses. Here, we determined that PD-1H functions as a coinhibitory receptor for CD4⁺ T cells. CD4⁺ T cells in mice lacking PD-1H exhibited a dramatically increased response to antigen stimulation. Furthermore, delivery of a PD-1H–specific agonist mAb directly inhibited CD4⁺ T cell activation both in vitro and in vivo, validating a coinhibitory function of PD-1H. In a murine model of acute hepatitis, administration of a PD-1H agonist mAb suppressed CD4⁺ T cell–mediated acute inflammation. PD-1H–deficient animals were highly resistant to tumor induction in a murine brain glioma model, and depletion of CD4⁺ T cells, but not CD8⁺ T cells, promoted tumor formation. Together, our findings suggest that PD-1H has potential as a target of immune modulation in the treatment of human inflammation and malignancies.     

Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation

Animal models of hematopoietic stem cell transplantation have been used to analyze the turnover of bone marrow–derived cells and to demonstrate the critical role of recipient antigen-presenting cells (APC) in graft versus host disease (GVHD). In humans, the phenotype and lineage relationships of myeloid-derived tissue APC remain incompletely understood. It has also been proposed that the risk of acute GVHD, which extends over many months, is related to the protracted survival of certain recipient APC. Human dermis contains three principal subsets of CD45⁺HLA-DR⁺ cells: CD1a⁺CD14⁻ DC, CD1a⁻CD14⁺ DC, and CD1a⁻CD14⁺FXIIIa⁺ macrophages. In vitro, each subset has characteristic properties. After transplantation, both CD1a⁺ and CD14⁺ DC are rapidly depleted and replaced by donor cells, but recipient macrophages can be found in GVHD lesions and may persist for many months. Macrophages isolated from normal dermis secrete proinflammatory cytokines. Although they stimulate little proliferation of naive or memory CD4⁺ T cells, macrophages induce cytokine expression in memory CD4⁺ T cells and activation and proliferation of CD8⁺ T cells. These observations suggest that dermal macrophages and DC are from distinct lineages and that persistent recipient macrophages, although unlikely to initiate alloreactivity, may contribute to GVHD by sustaining the responses of previously activated T cells.     

Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen–specific CD8+ T cell dysfunction in melanoma patients

The paradoxical coexistence of spontaneous tumor antigen–specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1–specific CD8⁺ T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1–specific CD8⁺ T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1⁺ NY-ESO-1–specific CD8⁺ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3⁺PD-1⁺ NY-ESO-1–specific CD8⁺ T cells are more dysfunctional than Tim-3⁻PD-1⁺ and Tim-3⁻PD-1⁻ NY-ESO-1–specific CD8⁺ T cells, producing less IFN-γ, TNF, and IL-2. Tim-3–Tim-3L blockade enhanced cytokine production by NY-ESO-1–specific CD8⁺ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3–Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1–specific CD8⁺ T cells upon prolonged antigen stimulation and acted in synergy with PD-1–PD-L1 blockade. Collectively, our findings support the use of Tim-3–Tim-3L blockade together with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.There is ample evidence that patients with melanoma can develop immune responses directed against antigens expressed by their own tumor (Boon et al., 2006). Among these antigens, cancer-germline antigens (CGAs) are expressed by tumors of many different histological types, including melanoma, but not by normal tissues, except testis. Because germ cells in testis do not express HLA molecules on their surface (Haas et al., 1988), CGAs represent strictly tumor-specific T cell targets (Boon et al., 2006). Among CGAs, NY-ESO-1 has been shown to stimulate spontaneous cellular and humoral responses that are detectable only in patients with advanced NY-ESO-1–expressing cancer (Stockert et al., 1998; Jäger et al., 2000; Mandic et al., 2005; Fourcade et al., 2008). Understanding the failure of spontaneous NY-ESO-1–specific T cell responses to promote regression of NY-ESO-1⁺ tumors is therefore critical for the design of novel therapeutic interventions aimed at overcoming tumor-induced immune escape.We have previously shown that the large majority of spontaneous NY-ESO-1–specific CD8⁺ T cells up-regulates programmed death 1 (PD-1) expression (Fourcade et al., 2009), which appears to be associated with T cell exhaustion/dysfunction in chronic viral infections in animals and humans (Barber et al., 2006; Day et al., 2006; Petrovas et al., 2006; Trautmann et al., 2006). We observed that PD-1 up-regulation on spontaneous NY-ESO-1–specific CD8⁺ T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines ex vivo upon stimulation with cognate antigen. Blockade of the PD-1–programmed death ligand 1 (PD-L1) pathway in combination with prolonged antigen stimulation with PD-L1⁺ APCs or melanoma cells augmented the frequencies of cytokine-producing, proliferating, and total NY-ESO-1–specific CD8⁺ T cells. Our findings are in line with previous studies of PD-1 expression by HIV- and SIV-specific CD8⁺ T cells, demonstrating that PD-1 is a regulator of antigen-specific CD8⁺ T cell expansion in the context of chronic antigen exposure, although it does not exhibit a major impact upon their functionality on a cell-per-cell basis (Petrovas et al., 2006, 2007). To further determine whether other molecular pathways are involved in tumor antigen–specific T cell dysfunction, we studied T cell immunoglobulin and mucin-domain–containing molecule 3 (Tim-3) expression on spontaneous NY-ESO-1–specific CD8⁺ T cells from patients with advanced melanoma and investigated whether Tim-3 up-regulation defines a subgroup of dysfunctional tumor antigen–specific CD8⁺ T cells. Tim-3 is a transmembrane protein constitutively expressed on Th1/Tc1 cells in mice and humans (Monney et al., 2002). Several lines of evidence support the role of Tim-3 as an inhibitory molecule that down-regulates effector Th1/Tc1 cell responses. In mice, blocking the Tim-3–Tim-3L pathway resulted in hyperproliferation of Th1-type cells and abrogated the induction of peripheral and transplantation tolerance (Sabatos et al., 2003; Sánchez-Fueyo et al., 2003). Tim-3 interacts with its ligand galectin-9 to induce cell death in Th1 cells (Zhu et al., 2005). In humans, Tim-3 expression is defective in CD4⁺ T cells producing high levels of IFN-γ, as well as those isolated from cerebrospinal fluid of patients with multiple sclerosis (Koguchi et al., 2006). Recently, Tim-3 up-regulation has been reported in HIV-specific and HCV-specific CD8⁺ T cells in patients with progressive HIV infection and chronic hepatitis C, respectively (Jones et al., 2008; Golden-Mason et al., 2009). Tim-3⁺ HIV- and HCV-specific CD8⁺ T cells were distinct from the PD-1⁺ CD8⁺ T cells and exhibited T cell dysfunction. However, it is unknown whether tumor antigen–specific CD8⁺ T cells in patients with advanced cancers express Tim-3.In this study, we show that a fraction of PD-1⁺ NY-ESO-1–specific CD8⁺ T cells, which represents the large majority of circulating NY-ESO-1–specific CD8⁺ T cells in patients with advanced melanoma, up-regulates Tim-3 expression. Tim-3⁺PD-1⁺ NY-ESO-1–specific CD8⁺ T cells are highly dysfunctional compared with Tim-3⁻PD-1⁺ and Tim-3⁻PD-1⁻ NY-ESO-1–specific CD8⁺ T cells. Tim-3–Tim-3L pathway blockade alone or in combination with PD-1–PD-L1 pathway blockade enhanced NY-ESO-1–specific CD8⁺ T cell numbers and functions. Collectively, our findings support the use of Tim-3–Tim-3L blockade in association with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.