Initial studies suggested that polymorphisms in Tim1 and Tim3 contribute to the development of airway hyperreactivity (AHR) in an acute mouse model of asthma. This was also mirrored in human genetic studies where polymorphisms in Tim1 and Tim3 have been associated with atopic populations.
Further studies using anti-Tim1 or -Tim3 antibodies, or Tim fusion proteins, have also suggested that these molecules may function as regulators of type-1 and type-2 immunity. However, their role in the development of AHR and airway inflammation remains unclear. Given the proposed roles for Tim1 and Tim3 in type-1 and type-2 responses, we sought to determine whether these molecules were important in regulating antigen-driven lung allergy and inflammation.
We used Tim1- and Tim3-deficient mice and determined how the development of allergic lung inflammation was affected.
AHR was induced normally in the absence of both Tim1 and Tim3, although Tim1-deficient mice did show a small but significant decrease in cell infiltration in the lung and blood eosinophilia. Although Tim3 was expressed on CD4+ T cells in the allergic lung, Tim1 expression was restricted to CD86+ B cells.
Conclusions and clinical relevance
Thus, Tim1 and Tim3 are not essential for the induction of the type-2 response in lung allergy. This is contrary to what was proposed in a number of other studies using neutralizing and activating antibodies and questions the clinical relevance of Tim1 and Tim3 for novel allergy therapies.
Cite this as: J. L. Barlow, S. H. Wong, S. J. Ballantyne, H. E. Jolin and A. N. J. McKenzie, Clinical & Experimental Allergy, 2011 (41) 1012–1021.
airway hyperreactivity; asthma; eosinophils; Tim1; Tim3; type-2
T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4+ and CD8+ T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3+CD4+ and Tim-3+CD8+ T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3− counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3+CD4+ and Tim-3+CD8+ T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4+ and CD8+ T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.
Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb) infection, remains a leading cause of morbidity and mortality worldwide. While CD4+ and CD8+ T-cell effector functions producing Th1 or cytotoxic cytokines are required to mount anti-mycobacterial immunity, insufficiency or failure to mount anti-mycobacterial effector functions by CD4+ and CD8+ T cells may lead to impaired immunity against TB. Therefore, it is important to elucidate functional characteristics and regulatory pathways for Mtb-specific CD4+ and CD8+ T cells during immune responses to Mtb infection. It was postulated that T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) might represent a T-cell exhaustion marker, and expression of Tim-3 on T cells may be linked to progressive loss of secretion of cytokines. Thus, Tim-3 expression on T cells might correlate with T-cell dysfunction and disease pathogenic events. However, T-cell immune responses modulated by Tim-3 in human TB disease remain poorly understood. Here, we found that up-regulation of Tim-3 expression in active human TB disease allows CD4+ and CD8+ T cells to mount stronger, but not impaired, anti-mycobacterium effector functions. This study therefore uncovers a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and has an important implication for TB diagnostics and therapy.
Activated T helper (Th)-1 pulmonary CD4+ cells and their mediators are essential for the inflammation and granulomatous process in sarcoidosis. Recently, T-cell immunoglobulin and mucin domain (TIM) molecules were suggested to be important regulators of immune function. In this study, we wanted to investigate whether TIM molecules could play a role in sarcoidosis.
We used real-time polymerase chain reaction to investigate the differential gene expression of TIM-1 and TIM-3 as well as a few Th1 and Th2 cytokines (IL-2, IFN-γ, IL-4, IL-5 and IL-13) in CD4+ T cells isolated from bronchoalveolar lavage fluid (BALF) of patients (n = 28) and healthy controls (n = 8). Using flow cytometry, we were also able to analyse TIM-3 protein expression in 10 patients and 6 healthy controls.
A decreased TIM-3 mRNA (p < 0.05) and protein (p < 0.05) expression was observed in patients, and the level of TIM-3 mRNA correlated negatively with the CD4/CD8 T cell ratio in BALF cells of patients. Compared to a distinct subgroup of patients i.e. those with Löfgren's syndrome, BALF CD4+ T cells from non- Löfgren's patients expressed decreased mRNA levels of TIM-1 (p < 0.05). mRNA expression of IL-2 was increased in patients (p < 0.01) and non-Löfgren's patients expressed significantly higher levels of IFN-γ mRNA (p < 0.05) versus patients with Löfgren's syndrome.
These findings are the first data on the expression of TIM-1 and TIM-3 molecules in sarcoidosis. The reduced TIM-3 expression in the lungs of patients may result in a defective T cell ability to control the Th1 immune response and could thus contribute to the pathogenesis of sarcoidosis. The down-regulated TIM-1 expression in non-Löfgren'spatients is in agreement with an exaggerated Th1 response in these patients.
T cell immunoglobulin and mucin protein 3 (TIM-3) is a type I cell surface protein that was originally identified as a marker for murine T helper type 1 cells. TIM-3 was found to negatively regulate murine T cell responses and galectin-9 was described as a binding partner that mediates T cell inhibitory effects of TIM-3. Moreover, it was reported that like PD-1 the classical exhaustion marker, TIM-3 is up-regulated in exhausted murine and human T cells and TIM-3 blockade was described to restore the function of these T cells. Here we show that the activation of human T cells is not affected by the presence of galectin-9 or antibodies to TIM-3. Furthermore, extensive studies on the interaction of galectin-9 with human and murine TIM-3 did not yield evidence for specific binding between these molecules. Moreover, profound differences were observed when analysing the expression of TIM-3 and PD-1 on T cells of HIV-1-infected individuals: TIM-3 was expressed on fewer cells and also at much lower levels. Furthermore, whereas PD-1 was preferentially expressed on CD45RA−CD8 T cells, the majority of TIM-3-expressing CD8 T cells were CD45RA+. Importantly, we found that TIM-3 antibodies were ineffective in increasing anti-HIV-1 T cell responses in vitro, whereas PD-L antibodies potently reverted the dysfunctional state of exhausted CD8 T cells. Taken together, our results are not in support of an interaction between TIM-3 and galectin-9 and yield no evidence for a functional role of TIM-3 in human T cell activation. Moreover, our data indicate that PD-1, but not TIM-3, is a promising target to ameliorate T cell exhaustion.
Inhibitory costimulatory receptors are a hallmark of exhausted T cells, which accumulate during chronic infection with viruses like HIV-1. Recently, TIM-3 was described as functional receptor on exhausted human T cells. Galectin-9 was reported as an inhibitory ligand for TIM-3 on murine T cells, but it was not known whether galectin-9 has a role in human T cell activation processes. We have found that the activation of human T cells is not affected by the presence of galectin-9 or antibodies to TIM-3. Furthermore, we demonstrate that galectin-9 does not serve as a ligand of human or murine TIM-3. Analysis of T cells of HIV-infected individuals regarding the expression of TIM-3 and PD-1 demonstrates that TIM-3 is expressed on fewer cells and also at much lower levels. In fact, TIM-3 expression characterizes a T cell population that is distinct from the PD-1 expressing exhausted T cells. Our results indicate that PD-1, but not TIM-3, is a promising target to ameliorate T cell exhaustion.
T cell immunoglobulin and mucin domain-2 (TIM-2) has been shown to regulate CD4 T cell activation. However, the role of TIM-2 in the autoimmune disease models has not been clarified yet. In this study, we investigated the effects of anti-TIM-2 monoclonal antibodies (mAbs) in collagen-induced arthritis (CIA) to determine whether TIM-2 contributes to the development of T helper (Th) 1 or Th17 cells and joint inflammation.
DBA/1 mice were treated with anti-TIM-2 mAbs during the early or late phase of CIA. Type II collagen (CII)-specific CD4 T-cell proliferative response and cytokine production were assessed from lymph node cell culture. The serum levels of CII-specific antibody were measured by ELISA. The expression of TIM-2 on CD4 T cells or B cells was determined by flow cytometric analysis.
Administration of anti-TIM-2 mAbs in early phase, but not late phase, significantly exacerbated the development of CIA. Although anti-TIM-2 mAbs treatment did not affect the development of Th1 or Th17 cells in the draining lymph node, the serum levels of anti-CII antibodies were significantly increased in the anti-TIM-2-treated mice. TIM-2 expression was found on splenic B cells and further up-regulated by anti-immunoglobulin (Ig)M, anti-CD40, and interleukin(IL)-4 stimulation. In contrast, CD4 T cells did not express TIM-2 even when stimulated with both anti-CD3 and anti-CD28 mAbs. Interestingly, anti-TIM-2 mAbs enhanced proliferation and antibody production of activated B cells in vitro.
TIM-2 signaling influences both proliferation and antibody production of B cells during the early phase of CIA, but not induction of Th1 or Th17 cells.
The newly discovered T-cell immunoglobulin mucin (TIM) gene family molecules, expressed by T cells, regulate host immunity and tolerance. Although CD4+ T cells mediate innate immunity-dominated liver ischemia-reperfusion injury (IRI), the underlying mechanisms remain obscure. We have recently documented the novel function of TIM-1 pathway in the mechanism of liver IRI and also found that TLR4 activation plays a key triggering role. Using an anti-TIM-3 Ab, we now studied the role of TIM-3 signaling in the model of partial warm liver ischemia followed by reperfusion. Anti-TIM-3 Ab therapy exacerbated the liver damage, as compared with controls. Histological examination has revealed that anti-TIM-3 Ab augmented the hepatocellular damage, increased local neutrophil infiltration, facilitated local accumulation of T cells/macrophages and promoted liver cell apoptosis. Intrahepatic neutrophil activity, induction of pro-inflammatory cytokines/chemokines and expression of cleaved caspase-3/NF-NB/TLR4 were all increased in the treatment group. In parallel, anti-TIM-3 Ab and anti-galectin-9 (Gal-9; TIM-3 ligand) Ab increased IFN-γ production in ConA-stimulated spleen T cells, and TNFα/IL-6 expression in ConA-stimulated macrophage/T cell co-culture system. Interestingly, anti-TIM-3 Ab treatment did not affect liver IRI in TLR4-deficient (KO) mice. In conclusion, TIM-3 blockade exacerbated local inflammation and liver damage, suggesting importance of TIM-3/Gal-9 signaling in the maintenance of hepatic homeostasis. TIM-3-TLR4 cross regulation determined the severity of liver IRI in TLR4-dependent manner, a novel finding of potential importance to modulate tissue innate vs. adaptive responses in liver transplant patients. Thus, harnessing physiological negative T cell co-stimulation signaling on hepatic T cells may minimize innate immunity-mediated liver tissue damage.
T-cell Immunoglobulin and Mucin domain 2 (TIM2) belongs to the receptor family of cell surface molecules expressed on kidney, liver, and T cells. Previous studies have revealed that TIM2-deficient mice (TIM2−/−) are more susceptible to the Th2-mediated immune response in an airway inflammation model. Here, we investigated the phenotypic response of TIM2−/− mice to cisplatin-induced kidney toxicity. A lethality study in male BALB/c wild-type (TIM2+/+) and TIM2−/− mice, administered with 20 mg/kg cisplatin ip, resulted in 80% mortality of TIM2−/− mice as compared with 30% mortality in the TIM2+/+ group by day 5. The TIM2−/− mice showed approximately fivefold higher injury as estimated by blood urea nitrogen and serum creatinine at 48 h that was confirmed by significantly increased proximal tubular damage assessed histologically (H & E staining). A significantly higher expression of Th2-associated cytokines, TNF-α, IL-1β, IL-6, and TGFβ, with a significant reduction of Th1-associated cytokines, RANTES and MCP-1, by 72 h was observed in the TIM2−/− mice as compared with TIM2+/+ mice. A higher baseline protein expression of caspase-3 (approximately twofold) coupled with an early onset of p53 protein activation by 48 h resulted in an increased apoptosis by 48–72 h in TIM2−/− compared with TIM2+/+. In conclusion, the increased expression of the proinflammatory and proapoptotic genes, with a higher number of apoptotic cells, and a pronounced increase in injury and mortality of the TIM2-deficient mice collectively suggest a protective role of TIM2 in cisplatin-induced nephrotoxicity.
TIM2; cisplatin; kidney; inflammation; apoptosis
T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers1–5. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition6–10. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.
The TIM (T cell Immunoglobulin and Mucin domain-containing molecules) protein family, expressed by T cells, plays a crucial role in regulating host adaptive immunity and tolerance. However, its role in local inflammation, such as innate immunity-dominated organ ischemia and reperfusion injury (IRI) remains unknown. Liver IRI occurs frequently after major hepatic resection or liver transplantation. Using an antagonistic anti-TIM-1 Ab, we studied the role of TIM-1 signaling in the model of partial warm liver ischemia followed by reperfusion. Anti-TIM-1 Ab monotherapy ameliorated the hepatocellular damage and improved liver function due to IR, as compared to controls. Histological examination has revealed that anti-TIM-1 Ab treatment decreased local neutrophil infiltration, inhibited sequestration of T lymphocytes, macrophages, TIM-1 ligand expressing TIM-4+ cells, and reduced liver cell apoptosis. Intrahepatic neutrophil activity and induction of pro-inflammatory cytokines/chemokines were also reduced in the treatment group. In parallel in-vitro studies, anti-TIM-1 Ab suppressed IFN-γ production in Con A-stimulated spleen T cells, and diminished TNF-α/IL-6 expression in macrophage/spleen T cell co-culture system. This is the first study, which provides evidence for the novel role of TIM-1 signaling in the mechanism of liver IRI. TIM-1 regulates not only T cell activation but may also affect macrophage function in the local inflammation response. These results provide compelling data for further investigation of TIM-1 pathway in the mechanism of IRI, to improve liver function, expand the organ donor pool, and improve the overall success of liver transplantation.
Identification of the T cell immunoglobulin mucin-domain containing (Tim) gene family introduced a new family of cell surface molecules that is involved in the regulation of immune responses. We previously demonstrated that Tim-3 is expressed on terminally differentiated T helper (Th)1 cells, and serves to regulate Th1 immune responses. Here, we describe the identification and function of Tim-2, a novel member of the Tim gene family. In contrast with Tim-3, we demonstrate that Tim-2 is expressed preferentially in differentiated Th2 cells. Blockade of the Tim-2/Tim-2 ligand interaction, by administration of soluble Tim-2 fusion protein (Tim-2 immunoglobulin [Ig]), results in T cell hyperproliferation and the production of Th2 cytokines. Administration of Tim-2 Ig during the induction phase reduces the severity of experimental autoimmune encephalomyelitis, a Th1-mediated autoimmune disease model of multiple sclerosis. We propose that Tim-2, an orthologue of human Tim-1, is critical for the regulation of Th2 responses during autoimmune inflammation.
Engagement of T-cell immunoglobulin mucin (Tim)-1 on T cells with its ligand, Tim-4, on antigen presenting cells delivers positive costimulatory signals to T cells. However, the molecular mechanisms for Tim-1-mediated regulation of T-cell activation and differentiation are relatively poorly understood. Here we investigated the role of Tim-1 in T-cell responses and allograft rejection using recombinant human Tim-1 extracellular domain and IgG1-Fc fusion proteins (Tim-1-Fc). In vitro assays confirmed that Tim-1-Fc selectively binds to CD4+ effector T cells, but not dendritic cells or natural regulatory T cells (nTregs). Tim-1-Fc was able to inhibit the responses of purified CD4+ T cells that do not express Tim-4 to stimulation by anti-CD3/CD28 mAbs, and this inhibition was associated with reduced AKT and ERK1/2 phosphorylation, but it had no influence on nTregs. Moreover, Tim-1-Fc inhibited the proliferation of CD4+ T cells stimulated by allogeneic dendritic cells. Treatment of recipient mice with Tim-1-Fc significantly prolonged cardiac allograft survival in a fully MHC-mismatched strain combination, which was associated with impaired Th1 response and preserved Th2 and nTregs function. Importantly, the frequency of Foxp3+ cells in splenic CD4+ T cells was increased, thus shifting the balance toward regulators, even though Tim-1-Fc did not induce Foxp3 expression in CD4+CD25− T cells directly. These results indicate that Tim-1-Fc can inhibit T-cell responses through an unknown Tim-1 binding partner on T cells, and it is a promising immunosuppressive agent for preventing allograft rejection.
T-cell immunoglobulin and mucin domain 3 (TIM-3) is known as a negative immune regulator and emerging data have implicated TIM-3 a pivotal role in suppressing antitumor immunity. The co-stimulatory receptor CD137 is transiently upregulated on T-cells following activation and increases their proliferation and survival when engaged. Although antagonistic anti-TIM-3 or agonistic anti-CD137 antibodies can promote the rejection of several murine tumors, some poorly immunogenic tumors were refractory to this treatment. In this study, we sought to evaluate whether combined TIM-3 blockade and CD137 activation would significantly improve the immunotherapy in the murine ID8 ovarian cancer model.
Mice with established ID8 tumor were intraperitoneally injected with single or combined anti-TIM-3/CD137 monoclonal antibody (mAb); mice survival was recorded, the composition and gene expression of tumor-infiltrating immune cells in these mice was analyzed by flow cytometry and quantitative RT-PCR respectively, and the function of CD8+ cells was evaluated by ELISA and cytotoxicity assay.
Either anti-TIM-3 or CD137 mAb alone, although effective in 3 days established tumor, was unable to prevent tumor progression in mice bearing 10 days established tumor, however, combined anti-TIM-3/CD137 mAb significantly inhibited the growth of these tumors with 60% of mice tumor free 90 days after tumor inoculation. Therapeutic efficacy was associated with a systemic immune response with memory and antigen specificity, required CD4+ cells and CD8+ cells. The 2 mAb combination increased CD4+ and CD8+ cells and decreased immunosuppressive CD4+FoxP3+ regulatory T (Treg) cells and CD11b+Gr-1+ myeloid suppressor cells (MDSC) at tumor sites, giving rise to significantly elevated ratios of CD4+ and CD8+ cells to Treg and MDSC; This is consistent with biasing local immune response towards an immunostimulatory Th1 type and is further supported by quantitative RT-PCR data showing the increased Th1-associated genes by anti-TIM-3/CD137 treatment. The increased CD8+ T cells produced high level of IFN-γ upon tumor antigen stimulation and displayed antigen-specific cytotoxic activity.
To our knowledge, this is the first report investigating the effects of anti-TIM-3/CD137 combined mAb in a murine ovarian cancer model, and our results may aid the design of future trials for ovarian cancer immunotherapy.
Microglia/macrophages are known to play important roles in initiating brain inflammation after spontaneous intracerebral hemorrhage (ICH). T cell immunoglobulin and mucin domain-3 (Tim-3) have been proven to play a critical part in several inflammatory diseases through regulation of both adaptive and innate immune responses. Tim-3 can be expressed by microglia/macrophages and regulates their function in the innate immune response. However, the effect of Tim-3 on inflammatory responses following ICH is unclear.
In this study, we investigated Tim-3 expression, the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and brain water content in peri-hematomal brain tissue at 12 hours and at 1, 3, 5, and 7 days post-ICH in wild type (WT) ICH and Tim-3−/− ICH mice. The numbers of Tim-3 positive cells,astrocytes, neutrophils and microglia/macrophages were detected using immunofluorescence staining. Cytokines were measured by ELISA. Double immunoflurorescence labeling was performed to identify the cellular source of Tim-3 expression. Mouse neurological deficit scores were assessed through animal behavior.
Expression of Tim-3 increased early in mouse peri-hematomal brain tissue after autologous blood injection, peaked at day 1, and was positively correlated with the concentrations of TNF-α, IL-1β, and brain water content. Tim-3 was predominantly expressed in microglia/macrophages. Compared with WT mice, Tim-3−/− mice had reduced ICH-induced brain inflammation with decreased TNF-α and IL-1β, cerebral edema and neurological deficit scores. Moreover, Tim-/- inhibited activation of microglia/macrophages. The number of activated microglia/macrophages in Tim-3−/− ICH mice was much lower than that in WT ICH mice.
Our findings demonstrate that Tim-3 plays an important role in brain inflammation after ICH, and may be a potential treatment target.
T cell immunoglobulin; Mucin domain 3; Brain inflammation; Microglia; Macrophages; TNF-α; IL-1β
It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed on T cells serves to positively costimulate T cell responses. However, crosslinking of Tim-1 by its ligand Tim-4 resulted in either activation or inhibition of T cell responses, thus raising the issue of whether Tim-1 can have a dual function as a costimulator. To resolve this issue, we tested a series of monoclonal antibodies specific for Tim-1 and identified two antibodies that showed opposite functional effects. One anti–Tim-1 antibody increased the frequency of antigen-specific T cells, the production of the proinflammatory cytokines IFN-γ and IL-17, and the severity of experimental autoimmune encephalomyelitis. In contrast, another anti–Tim-1 antibody inhibited the generation of antigen-specific T cells, production of IFN-γ and IL-17, and development of autoimmunity, and it caused a strong Th2 response. Both antibodies bound to closely related epitopes in the IgV domain of the Tim-1 molecule, but the activating antibody had an avidity for Tim-1 that was 17 times higher than the inhibitory antibody. Although both anti–Tim-1 antibodies induced CD3 capping, only the activating antibody caused strong cytoskeletal reorganization and motility. These data indicate that Tim-1 regulates T cell responses and that Tim-1 engagement can alter T cell function depending on the affinity/avidity with which it is engaged.
T cell immunoglobulin- and mucin domain–containing molecule (TIM)3 is a T helper cell (Th)1–associated cell surface molecule that regulates Th1 responses and promotes tolerance in mice, but its expression and function in human T cells is unknown. We generated 104 T cell clones from the cerebrospinal fluid (CSF) of six patients with multiple sclerosis (MS) (n = 72) and four control subjects (n = 32) and assessed their cytokine profiles and expression levels of TIM3 and related molecules. MS CSF clones secreted higher amounts of interferon (IFN)-γ than did those from control subjects, but paradoxically expressed lower levels of TIM3 and T-bet. Interleukin 12–mediated polarization of CSF clones induced substantially higher amounts of IFN-γ secretion but lower levels of TIM3 in MS clones relative to control clones, demonstrating that TIM3 expression is dysregulated in MS CSF clones. Reduced levels of TIM3 on MS CSF clones correlated with resistance to tolerance induced by costimulatory blockade. Finally, reduction of TIM3 on ex vivo CD4+ T cells using small interfering (si)RNA enhanced proliferation and IFN-γ secretion, directly demonstrating that TIM3 expression on human T cells regulates proliferation and IFN-γ secretion. Failure to up-regulate T cell expression of TIM3 in inflammatory sites may represent a novel, intrinsic defect that contributes to the pathogenesis of MS and other human autoimmune diseases.
T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a newly identified negative immunomodulator that is up-regulated on dysfunctional T cells during viral infections. The expression and function of Tim-3 on human innate immune responses during HCV infection, however, remains poorly characterized. In this study, we report that Tim-3 is constitutively expressed on human resting CD14+ monocyte/macrophages (M/MØ) and functions as a cap to block IL-12, a key pro-inflammatory cytokine linking innate and adaptive immune responses. Tim-3 expression is significantly reduced and IL-12 expression increased upon stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848. Notably, Tim-3 is over-expressed on un-stimulated as well as TLR-stimulated M/MØ, which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects. Up-regulation of Tim-3 and inhibition of IL-12 are also observed in M/MØ incubated with HCV-expressing hepatocytes, as well as in primary M/MØ or monocytic THP-1 cells incubated with HCV core protein, an effect that mimics the function of complement C1q and is reversible by blocking the HCV core/gC1qR interaction. Importantly, blockade of Tim-3 signaling significantly rescues HCV-mediated inhibition of IL-12, which is primarily expressed by Tim-3 negative M/MØ. Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation. Conversely, blocking PD-1 or silencing SOCS-1 gene expression also decreases Tim-3 expression and enhances IL-12 secretion and STAT-1 phosphorylation. These findings suggest that Tim-3 plays a crucial role in negative regulation of innate immune responses, through crosstalk with PD-1 and SOCS-1 and limiting STAT-1 phosphorylation, and may be a novel target for immunotherapy to HCV infection.
T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10–expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1dhiCD5+. A low-affinity TIM-1–specific antibody that normally promotes tolerance in mice, actually accelerated (T cell–mediated) immune responsiveness in the absence of B cells. TIM-1+ B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-1+ regulatory B cells (Bregs) were induced by TIM-1–specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-10+ Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs.
Dendritic cells (DCs) are the central architects of the immune response, inducing inflammatory or tolerogenic immunity, dependent upon their activation status. As such, DCs are highly attractive therapeutic targets and may hold the potential to control detrimental immune responses. TIM-4, expressed on antigen presenting cells, has complex functions in vivo, acting both as a costimulatory molecule and a phosphatidylserine (PS) receptor. The effect of TIM-4 costimulation on T cell activation remains unclear.
Here, we demonstrate that antibody blockade of DC-expressed TIM-4 leads to increased induction of iTregs from naïve CD4+ T cells, both in vitro and in vivo. iTreg induction occurs through suppression of IL-4/STAT6/Gata3 induced Th2 differentiation. In addition, blockade of TIM-4 on previously activated DCs still leads to increased iTreg induction. iTregs induced under TIM-4 blockade have equivalent potency to control and upon adoptive transfer, significantly prolong skin allograft survival in vivo. In RAG−/− recipients of skin allografts adoptively transferred with CD4+ T cells, we show that TIM-4 blockade in vivo is associated with a three-fold prolongation in allograft survival. Furthermore, in this mouse model of skin transplantation, increased induction of allospecific iTregs and a reduction in T effector responses were observed, with decreased Th1 and Th2 responses. This enhanced allograft survival and pro-tolerogenic skewing of the alloresponse is critically dependent upon conversion of naïve CD4+ to Tregs in vivo. Collectively, these studies identify blockade of DC-expressed TIM-4 as a novel strategy which holds the capacity to induce regulatory immunity in vivo.
transplantation; costimulatory molecules; T cell differentiation; Treg; dendritic cells
The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.
Background and objective
T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) represents a novel mechanism of T-cell dysfunction in chronic viral diseases. However, the role of Tim-3 in the pathogenesis of chronic hepatitis B (CHB) is not well understood. We investigated Tim-3 expression on peripheral T cell subsets and analyzed the relationship between Tim-3 expression and disease progression in HBV infection.
peripheral blood samples were obtained from CHB patients (n = 40), including 23 patients with moderate CHB [MCHB] and 17 with severe CHB [SCHB]. Control samples were obtained from nine acute hepatitis B patients (AHB) and 26 age-matched healthy subjects. The expression of Tim-3 on T cells was determined by flow cytometry.
Tim-3 expression was elevated on peripheral CD4+ and CD8+ T cells from AHB and CHB patients compared to those from healthy controls. The percentage of Tim-3+ T cells was further increased in SCHB patients relative to MCHB patients and showed a positive correlation with conventional markers for liver injury (alanine aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (TB) and international normalized ratio (INR) level). The frequency of Tim-3-expressing T cells was negatively correlated with T-bet mRNA expression and plasma interferon-gamma (INF-gamma) levels. Further, Tim-3 expression on CD4+ or CD8+ T cells was reduced in CHB patients with disease remission after antiviral treatment and in AHB patients during the convalescence phase.
Our results suggest that over-expression of Tim-3 is involved in disease progression of CHB and that Tim-3 may participate in skewing of Th1/Tc1 response, which contributes to persistency of HBV infection.
Studies in mice and humans have revealed that the T cell, immunoglobulin, mucin (TIM) genes are associated with several atopic diseases. TIM-1 is a type I membrane protein that is expressed on T cells upon stimulation and has been shown to modulate their activation. In addition to a recently described interaction with dendritic cells, TIM-1 has also been identified as a phosphatidylserine recognition molecule, and several protein ligands have been proposed. Our understanding of its activity is complicated by the possibility that TIM-1 possesses multiple and diverse binding partners. In order to delineate the function of TIM-1, we generated monoclonal antibodies directed to a cleft formed within the IgV domain of TIM-1. We have shown here that antibodies that bind to this defined cleft antagonize TIM-1 binding to specific ligands and cells. Notably, these antibodies exhibited therapeutic activity in a humanized SCID model of experimental asthma, ameliorating inflammation, and airway hyperresponsiveness. Further experiments demonstrated that the effects of the TIM-1–specific antibodies were mediated via suppression of Th2 cell proliferation and cytokine production. These results demonstrate that modulation of the TIM-1 pathway can critically influence activated T cells in a humanized disease model, suggesting that TIM-1 antagonists may provide potent therapeutic benefit in asthma and other immune-mediated disorders.
We show that Tim-1, initially reported to be expressed on CD4+ T cells, is constitutively expressed on dendritic cells (DC) and that its expression further increases after DC maturation. Tim-1 signaling into DC upregulates costimulatory molecule expression and proinflammatory cytokine production, thereby promoting effector T cell responses, while inhibiting Foxp3+ Treg responses. By contrast, Tim-1 signaling in T cells only regulates Th2 responses. Using a high-avidity/agonistic anti-Tim-1 antibody as a co-adjuvant enhances the immunogenic function of DC, decreases the suppressive function of Treg cells, and substantially increases proinflammatory Th17 responses in vivo. The treatment with high-but not low-, avidity anti-Tim-1 not only worsens experimental autoimmune encephalomyelitis (EAE) in susceptible mice but also breaks tolerance and induces EAE in a genetically resistant strain of mice. These findings indicate that Tim-1 has an important role in regulating DC function, and thus shifts the balance between effector and regulatory T cells towards an enhanced immune response. By understanding the mechanisms by which Tim-1 regulates DC and T cell responses, we may clarify the potential utility of Tim-1 as a target of therapy against autoimmunity, cancer and infectious diseases.
Tim-1; DC; EAE; T cells; inflammation
CD8+ cytotoxic T lymphocytes (CTL) are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g. IFN-γ) to aid this response. However, during cancer and chronic viral infections, such as with Human Immunodeficiency Virus, this CTL response is progressively impaired due to a process called T-cell exhaustion. Previous work has shown that the glycoprotein, T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) plays a functional role in establishing T-cell exhaustion. Tim-3 is highly upregulated on virus and tumor antigen-specific CD8+ T-cells and antagonizing Tim-3 helps restore function of CD8+ T cells. However, very little is known of how Tim-3 signals in CTLs. In this study, we assessed the role of Tim-3 at the immunological synapse as well as its interaction with proximal TCR signaling molecules in primary human CD8+ T cells. Tim-3 was found withinCD8+T cell lipid rafts at the immunological synapse. Blocking Tim-3 resulted in a significantly greater number of stable synapses being formed between Tim-3hi CD8+T cells and target cells, suggesting that Tim-3 plays a functional role in synapse formation. Further, we confirmed that Tim-3 interacts with Lck, but not the phospho-active form of Lck. Finally, Tim-3 colocalizes with receptor phosphatases CD45 and CD148, an interaction that is enhanced in the presence of the Tim-3 ligand, galectin-9. Thus, Tim-3 interacts with multiple signaling molecules at the immunological synapse and characterizing these interactions could aid in the development of therapeutics to restore Tim-3-mediated immune dysfunction.
Studies of asthma have been limited by a poor understanding of how non-allergic environmental exposures such as air pollution and infection are translated in the lung into inflammation and wheezing.
Our goal was to understand the mechanism of non-allergic asthma that lead to airway hyperreactivity (AHR), a cardinal feature of asthma independent of adaptive immunity.
We examined mouse models of experimental asthma, in which AHR was induced by Respiratory Syncytial Virus (RSV) infection or ozone exposure, using mice deficient in TIM1/HAVCR1, an important asthma susceptibility gene.
TIM1−/− mice failed to develop airways disease when infected with RSV or when repeatedly exposed to ozone, a major component of air pollution. On the other hand, the TIM1−/− mice developed allergen-induced experimental asthma, as previously shown. The RSV- and ozone-induced pathways were blocked by treatment with caspase inhibitors, indicating an absolute requirement for programmed cell death and apoptosis. TIM-1-expressing, but not TIM-1-deficient, natural killer T (NKT) cells responded to apoptotic airway epithelial cells by secreting cytokines, which mediated the development of airway hyperreactivity.
We defined a novel pathway in which TIM-1, a receptor for phosphatidylserine expressed by apoptotic cells, drives the development of asthma by sensing and responding to injured and apoptotic airway epithelial cells.
TIM-1; NKT; apoptosis; asthma
T cell immunoglobulin mucin-1 (Tim-1) is a transmembrane protein postulated to be a key regulator of Th2-type immune responses. This hypothesis is based in part upon genetic studies associating Tim-1 polymorphisms in mice with a bias toward airway hyperresponsiveness and the development of Th2-type CD4+ T cells. Tim-1 is expressed by Th2 CD4+ T cells on which it has been proposed to function as a co-stimulatory molecule. Tim-1 is also expressed by B cells, macrophages, and dendritic cells, but its role in responses by these cell types has not been firmly established. We generated Tim-1 deficient mice to determine the role of Tim-1 in a murine model of allergic airway disease that depends on the development and function of Th2 effector cells and results in the generation of AHR. We found antigen-driven recruitment of inflammatory cells into airways is increased in Tim-1 deficient mice relative to wild-type mice. In addition, we observed increased antigen-specific cytokine production by splenocytes from antigen-sensitized Tim-1 deficient mice relative to those from controls. These data support the conclusion that Tim-1 functions in pathways that suppress recruitment of inflammatory cells into the airways and the generation or activity of CD4+ T cells.
Tim-1; asthma; Th2