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1.  Tim1 and Tim3 are not essential for experimental allergic asthma 
Clinical and Experimental Allergy  2011;41(7):1012-1021.
Background
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.
Objective
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.
Method
We used Tim1- and Tim3-deficient mice and determined how the development of allergic lung inflammation was affected.
Results
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.
doi:10.1111/j.1365-2222.2011.03728.x
PMCID: PMC3132443  PMID: 21470319
airway hyperreactivity; asthma; eosinophils; Tim1; Tim3; type-2
2.  “Tim-4 expressed on antigen-presenting cells induces T cell expansion and survival” 
TIM (T-cell, immunoglobulin, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in antigen-presenting cells. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating monoclonal antibodies, we show that expression of Tim-4 protein is restricted to CD11c+ and CD11b+ cells and is upregulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on antigen-presenting cells, is a costimulatory molecule that promotes T cell expansion and survival by crosslinking Tim-1 on T cells.
PMCID: PMC2948965  PMID: 18354194
T cells; costimulation; apoptosis; cell surface molecules
3.  TIM2 Gene Deletion Results in Susceptibility to Cisplatin-Induced Kidney Toxicity 
Toxicological Sciences  2010;118(1):298-306.
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.
doi:10.1093/toxsci/kfq240
PMCID: PMC2955214  PMID: 20702592
TIM2; cisplatin; kidney; inflammation; apoptosis
4.  Tim-2 regulates T helper type 2 responses and autoimmunity 
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.
doi:10.1084/jem.20050308
PMCID: PMC2213076  PMID: 16043519
5.  The Emerging Role of T Cell Ig Mucin-1 (TIM-1) in The Mechanism of Liver Ischemia and Reperfusion Injury* 
Hepatology (Baltimore, Md.)  2010;51(4):1363-1372.
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.
doi:10.1002/hep.23442
PMCID: PMC3066468  PMID: 20091883
6.  Emerging Tim-3 functions in anti-microbial and tumor immunity 
Trends in immunology  2011;32(8):345-349.
T cell immunoglobulin-3 (Tim-3) was identified as a marker of differentiated IFN-γ-producing CD4+ T helper type 1 and CD8+ T cytotoxic type 1 cells. The interaction of Tim-3 with its ligand, galectin-9 (Gal-9), induces cell death and in vivo blockade of this interaction results in exacerbated autoimmunity and abrogation of tolerance in experimental models, establishing Tim-3 as a negative regulatory molecule. Recent studies have uncovered additional mechanisms by which Tim-3 negatively regulates T cell responses, such as by promoting the development of CD8+ T cell exhaustion and inducing expansion of myeloid-derived suppressor cells (MDSC). In contrast to this inhibitory effect on T cells, Tim-3-Gal-9 interaction promotes macrophage clearance of intracellular pathogens. Here, we will focus on the emerging role for Tim-3 in tumor and anti-microbial immunity.
doi:10.1016/j.it.2011.05.003
PMCID: PMC3164311  PMID: 21697013
7.  Tim-1 stimulation of dendritic cells regulates the balance between effector and regulatory T cells 
European journal of immunology  2011;41(6):1539-1549.
Summary
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.
doi:10.1002/eji.201040993
PMCID: PMC3129006  PMID: 21469101
Tim-1; DC; EAE; T cells; inflammation
8.  Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells 
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/ar3288
PMCID: PMC3132034  PMID: 21426565
9.  Tim-1 Signaling Substitutes for Conventional Signal 1 and Requires Costimulation to Induce T Cell Proliferation1 
Differentiation and clonal expansion of Ag-activated naive T cells play a pivotal role in the adaptive immune response. T cell Ig mucin (Tim) proteins influence the activation and differentiation of T cells. Tim-3 and Tim-2 clearly regulate Th1 and Th2 responses, respectively, but the precise influence of Tim-1 on T cell activation remains to be determined. We now show that Tim-1 stimulation in vivo and in vitro induces polyclonal activation of T cells despite absence of a conventional TCR-dependent signal 1. In this model, Tim-1-induced proliferation is dependent on strong signal 2 costimulation provided by mature dendritic cells. Ligation of Tim-1 upon CD4+ T cells with an agonist anti-Tim-1 mAb elicits a rise in free cytosolic calcium, calcineurin-dependent nuclear translocation of NF-AT, and transcription of IL-2. Because Tim-4, the Tim-1 ligand, is expressed by mature dendritic cells, we propose that interaction between Tim-1+ T cells and Tim-4+ dendritic cells might ensure optimal stimulation of T cells, when TCR-derived signals originating within an inflamed environment are weak or waning.
PMCID: PMC3767978  PMID: 19155484
10.  The Tim3–Galectin 9 Pathway Induces Antibacterial Activity in Human Macrophages Infected with Mycobacterium tuberculosis 
The Journal of Immunology Author Choice  2012;189(12):5896-5902.
T cell Ig and mucin domain 3 (Tim3) is an inhibitory molecule involved in immune tolerance, autoimmune responses, and antiviral immune evasion. However, we recently demonstrated that Tim3 and Galectin-9 (Gal9) interaction induces a program of macrophage activation that results in killing of Mycobacterium tuberculosis in the mouse model of infection. In this study, we sought to determine whether the Tim3–Gal9 pathway plays a similar role in human pulmonary TB. We identified that pulmonary TB patients have reduced expression of Tim3 on CD14+ monocytes in vivo. By blocking Tim3 and Gal9 interaction in vitro, we show that these molecules contribute to the control of intracellular bacterial replication in human macrophages. The antimicrobial effect was partially dependent on the production of IL-1β. Our results establish that Tim3–Gal9 interaction activates human M. tuberculosis –infected macrophages and leads to the control of bacterial growth through the production of the proinflammatory cytokine IL-1β. Data presented in this study suggest that one of the potential pathways activated by Tim3/Gal9 is the secretion of IL-1β, which plays a crucial role in antimicrobial immunity by modulating innate inflammatory networks.
doi:10.4049/jimmunol.1200990
PMCID: PMC3516679  PMID: 23180819
11.  TIM-3 Does Not Act as a Receptor for Galectin-9 
PLoS Pathogens  2013;9(3):e1003253.
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.
Author Summary
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.
doi:10.1371/journal.ppat.1003253
PMCID: PMC3605152  PMID: 23555261
12.  THE EMERGING ROLE OF THE TIM MOLECULES IN TRANSPLANTATION 
Since their discovery in 2001, the T cell immunoglobulin mucin (TIM) family members have been shown to play important roles in the regulation of immune responses. The TIM family comprises of eight genes in the mouse, three of which are conserved in humans (TIM-1, TIM-3 and TIM-4). Initially, TIM-1 and TIM-3 were thought to be expressed solely on T cells. However, emerging data suggest a much broader expression pattern where their presence on APCs confers differing functions, including the ability to mediate phagocytosis. In contrast, TIM-4 is exclusively expressed on APCs. Together, the TIM molecules provide a functional repertoire for determining the fate of T cell activation and differentiation. To date, much of the knowledge about the TIM family members has been garnered from models of asthma, allergy and autoimmunity. More recently, data from experimental models of transplantation demonstrate that TIM family members are also key in alloimmunity. This review will serve to highlight the emerging data regarding this unique family of molecules, and to identify their potential in transplantation tolerance.
doi:10.1111/j.1600-6143.2011.03727.x
PMCID: PMC3205957  PMID: 21906254
transplant immunology; T cell immunoglobulin mucin (TIM) family; T cell activation; costimulatory molecules
13.  Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+ regulatory T cells is interleukin 10 dependent 
The Journal of Experimental Medicine  2005;202(11):1539-1547.
Deficient suppression of T cell responses to allergen by CD4+CD25+ regulatory T cells has been observed in patients with allergic disease. Our current experiments used a mouse model of airway inflammation to examine the suppressive activity of allergen-specific CD4+CD25+ T cells in vivo. Transfer of ovalbumin (OVA) peptide–specific CD4+CD25+ T cells to OVA-sensitized mice reduced airway hyperreactivity (AHR), recruitment of eosinophils, and T helper type 2 (Th2) cytokine expression in the lung after allergen challenge. This suppression was dependent on interleukin (IL) 10 because increased lung expression of IL-10 was detected after transfer of CD4+CD25+ T cells, and regulation was reversed by anti–IL-10R antibody. However, suppression of AHR, airway inflammation, and increased expression of IL-10 were still observed when CD4+CD25+ T cells from IL-10 gene–deficient mice were transferred. Intracellular cytokine staining confirmed that transfer of CD4+CD25+ T cells induced IL-10 expression in recipient CD4+ T cells, but no increase in IL-10 expression was detected in airway macrophages, dendritic cells, or B cells. These data suggest that CD4+CD25+ T cells can suppress the Th2 cell–driven response to allergen in vivo by an IL-10–dependent mechanism but that IL-10 production by the regulatory T cells themselves is not required for such suppression.
doi:10.1084/jem.20051166
PMCID: PMC1350743  PMID: 16314435
14.  Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+ regulatory T cells is interleukin 10 dependent 
The Journal of experimental medicine  2005;202(11):1539-1547.
Deficient suppression of T cell responses to allergen by CD4+CD25+ regulatory T cells has been observed in patients with allergic disease. Our current experiments used a mouse model of airway inflammation to examine the suppressive activity of allergen-specific CD4+CD25+ T cells in vivo. Transfer of ovalbumin (OVA) peptide-specific CD4+CD25+ T cells to OVA-sensitized mice reduced airway hyperreactivity (AHR), recruitment of eosinophils, and T helper type 2 (Th2) cytokine expression in the lung after allergen challenge. This suppression was dependent on interleukin (IL) 10 because increased lung expression of IL-10 was detected after transfer of CD4+CD25+ T cells, and regulation was reversed by anti-IL-10R antibody. However, suppression of AHR, airway inflammation, and increased expression of IL-10 were still observed when CD4+CD25+ T cells from IL-10 gene-deficient mice were transferred. Intracellular cytokine staining confirmed that transfer of CD4+CD25+ T cells induced IL-10 expression in recipient CD4+ T cells, but no increase in IL-10 expression was detected in airway macrophages, dendritic cells, or B cells. These data suggest that CD4+CD25+ T cells can suppress the Th2 cell-driven response to allergen in vivo by an IL-10-dependent mechanism but that IL-10 production by the regulatory T cells themselves is not required for such suppression.
doi:10.1084/jem.20051166
PMCID: PMC1350743  PMID: 16314435
15.  The Tim3-Galectin 9 pathway induces antibacterial activity in human macrophages infected with M. tuberculosis 
T cell immunoglobulin and mucin-(Tim)-3 domain is an inhibitory molecule involved in immune tolerance, autoimmune responses, and antiviral immune evasion. However, we recently demonstrated that Tim3 and Galectin-9 (Gal9) interaction induces a program of macrophage activation that results in killing of Mycobacterium tuberculosis (M.tb) in the mouse model of infection. In this study we sought to determine whether Tim3-Gal9 pathway plays a similar role in human pulmonary tuberculosis. We identified that pulmonary tuberculosis (PTB) patients have reduced expression of Tim3 on CD14+ monocytes in vivo. By blocking Tim3 and Gal9 interaction in vitro, we show that these molecules contribute to the control of intracellular bacterial replication in human macrophages. The antimicrobial effect was partially dependent on the production of IL-1β. Our results establish that Tim3/Gal9 interaction activates human M.tb-infected macrophages and leads to the control of bacterial growth through the production of the pro-inflammatory cytokine IL-1β. Data presented here suggest that one of the potential pathways activated by Tim3/Gal9 is the secretion of IL-1β, which plays a crucial role in antimicrobial immunity by modulating innate inflammatory networks.
doi:10.4049/jimmunol.1200990
PMCID: PMC3516679  PMID: 23180819
Tim3; Galectin 9; pulmonary tuberculosis; mycobacterium tuberculosis; IL-1β
16.  Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice 
The Journal of Clinical Investigation  2011;121(9):3645-3656.
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.
doi:10.1172/JCI46274
PMCID: PMC3163958  PMID: 21821911
17.  Apoptotic Cells Activate NKT Cells through T Cell Ig-Like Mucin-Like–1 Resulting in Airway Hyperreactivity 
T cell Ig-like mucin-like–1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1–dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma.
doi:10.4049/jimmunol.1001116
PMCID: PMC3114419  PMID: 20889552
18.  Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1479-5876-11-215
PMCID: PMC3853027  PMID: 24044888
19.  Differential engagement of Tim-1 during activation can positively or negatively costimulate T cell expansion and effector function 
The Journal of Experimental Medicine  2007;204(7):1691-1702.
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.
doi:10.1084/jem.20062498
PMCID: PMC2118637  PMID: 17606630
20.  TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity 
Immunological reviews  2010;235(1):172-189.
Summary
The TIM (T cell/transmembrane, immunoglobulin, and mucin) gene family plays a critical role in regulating immune responses, including allergy, asthma, transplant tolerance, autoimmunity, and the response to viral infections. The unique structure of TIM immunoglobulin variable region domains allows highly specific recognition of phosphatidylserine (PtdSer), exposed on the surface of apoptotic cells. TIM-1, TIM-3, and TIM-4 all recognize PtdSer but differ in expression, suggesting that they have distinct functions in regulating immune responses. TIM-1, an important susceptibility gene for asthma and allergy, is preferentially expressed on T-helper 2 (Th2) cells and functions as a potent costimulatory molecule for T-cell activation. TIM-3 is preferentially expressed on Th1 and Tc1 cells, and generates an inhibitory signal resulting in apoptosis of Th1 and Tc1 cells. TIM-3 is also expressed on some dendritic cells and can mediate phagocytosis of apoptotic cells and cross-presentation of antigen. In contrast, TIM-4 is exclusively expressed on antigen-presenting cells, where it mediates phagocytosis of apoptotic cells and plays an important role in maintaining tolerance. TIM molecules thus provide a functional repertoire for recognition of apoptotic cells, which determines whether apoptotic cell recognition leads to immune activation or tolerance, depending on the TIM molecule engaged and the cell type on which it is expressed.
doi:10.1111/j.0105-2896.2010.00903.x
PMCID: PMC2914464  PMID: 20536563
TIM; phosphatidylserine; asthma; galectin-9; phagocytosis; costimulation; tolerance
21.  The Role of T Cell Immunoglobulin Mucin Domains 1 and 4 in a Herpes Simplex Virus-Induced Behçet's Disease Mouse Model 
Mediators of Inflammation  2013;2013:903948.
The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. TIM-1 plays an important role in the regulation of immune responses and the development of autoimmune diseases. TIM-4 is a natural ligand of TIM-1, and the interaction of TIM-1 and TIM-4 is involved in the regulation of T helper (Th) cell responses and modulation of the Th1/Th2 cytokine balance. Behçet's disease (BD) is a chronic, multisystemic inflammatory disorder with arthritic, intestinal, mucocutaneous, ocular, vascular, and central nervous system involvement. Tim-1 expression was lower in a herpes simplex virus-induced BD mouse model compared to that in asymptomatic BD normal (BDN) mice. Tim-4 expression was higher in BD mice than that in BDN mice. In this study, we investigated the Tim expression in a BD mouse model with BD-like symptoms. Tim-1 and Tim-4 expression was regulated by an expression vector or siRNA injected into the BD mouse model. The Tim-1 vector injected into BD mice resulted in changes in BD-like symptoms and decreased the severity score. Treatment with Tim-4 siRNA also improved BD-like symptoms and decreased the severity score accompanied by upregulation of regulatory T cells. We showed that regulating Tim-1 or Tim-4 affected BD-like symptoms in mice.
doi:10.1155/2013/903948
PMCID: PMC3888750  PMID: 24453431
22.  TIM-3 is Expressed on Activated Human CD4+ T Cells and Regulates Th1 and Th17 Cytokines 
European journal of immunology  2009;39(9):2492-2501.
Summary
TIM-3 is a molecule selectively expressed on a subset of murine IFNγ-secreting Th1 cells but not Th2 cells, and regulates Th1 immunity and tolerance in vivo. At this time little is known about the role of TIM-3 on human T cells. To determine if TIM-3 similarly identifies and regulates Th1 cells in humans, we generated a panel of monoclonal antibodies specific for human TIM-3. We report that TIM-3 is expressed by a subset of activated CD4+ cells, and that anti-CD3/28 stimulation increases both the level of expression as well as the number of TIM-3+ T cells. We also find that TIM-3 is expressed at high levels on in vitro polarized Th1 cells, and is expressed at lower levels on Th17 cells. In addition, human CD4+ T cells secreted elevated levels of IFNγ, IL-17, IL-2, and IL-6, but not IL-10, IL-4, or TNFα, when stimulated with anti-CD3/28 in the presence of TIM-3-specific, putative antagonistic antibodies. This was not mediated by differences in proliferation or cell death, but rather by induction of cytokines at the transcriptional level. These results suggest that TIM-3 is a negative regulator of human T cells and regulates Th1 and Th17 cytokine secretion.
doi:10.1002/eji.200939274
PMCID: PMC2759376  PMID: 19676072
human; T cells; cell activation; immunosuppression
23.  TIM3+FOXP3+ regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer 
Oncoimmunology  2013;2(4):e23849.
T-cell immunoglobulin mucin 3 (TIM3) is an inhibitory molecule that has emerged as a key regulator of dysfunctional or exhausted CD8+ T cells arising in chronic diseases such as cancer. In addition to exhausted CD8+ T cells, highly suppressive regulatory T cells (Tregs) represent a significant barrier against the induction of antitumor immunity. We have found that the majority of intratumoral FOXP3+ Tregs express TIM3. TIM3+ Tregs co-express PD-1, are highly suppressive and comprise a specialized subset of tissue Tregs that are rarely observed in the peripheral tissues or blood of tumor-bearing mice. The co-blockade of the TIM3 and PD-1 signaling pathways in vivo results in the downregulation of molecules associated with TIM3+ Treg suppressor functions. This suggests that the potent clinical efficacy of co-blocking TIM3 and PD-1 signal transduction cascades likely stems from the reversal of T-cell exhaustion combined with the inhibition of regulatory T-cell function in tumor tissues. Interestingly, we find that TIM3+ Tregs accumulate in the tumor tissue prior to the appearance of exhausted CD8+ T cells, and that the depletion of Tregs at this stage interferes with the development of the exhausted phenotype by CD8+ T cells. Collectively, our data indicate that TIM3 marks highly suppressive tissue-resident Tregs that play an important role in shaping the antitumor immune response in situ, increasing the value of TIM3-targeting therapeutic strategies against cancer.
doi:10.4161/onci.23849
PMCID: PMC3654601  PMID: 23734331
antibody; immunotherapy; inhibitory receptor; regulatory T cells; T-cell exhaustion
24.  The costimulatory role of TIM molecules 
Immunological reviews  2009;229(1):259-270.
Summary
The T-cell immunoglobulin domain and mucin domain (TIM) family, including TIM-1, TIM-2, TIM-3 and TIM-4, is a relatively newly described group of molecules with a conserved structure and important immunological functions, including T cell activation, induction of T-cell apoptosis and T-cell tolerance, and the clearance of apoptotic cells. TIM-1 costimulates T-cell activation and enhances cytokine production. In humans, TIM-1 also serves as a susceptibility gene for allergy and asthma. TIM-3, expressed on T cells and dendritic cells, regulates T-cell apoptosis and immune tolerance. In contrast, TIM-4, which is expressed primarily on antigen-presenting cells and which is a receptor for phosphatidylserine, regulates T-cell activation and tolerance, in part by mediating the uptake and engulfment of apoptotic cells. The TIM molecules thus have surprisingly broad activities affecting multiple aspects of immunology.
doi:10.1111/j.1600-065X.2009.00772.x
PMCID: PMC3217781  PMID: 19426227
TIM; costimulation; T cell; allergy; tolerance; autoimmunity
25.  T Cell Immunoglobulin Mucin Domain (TIM)-3 Promoter Activity in a Human Mast Cell Line 
Immune Network  2012;12(5):207-212.
T cell immunoglobulin mucin domain (TIM)-3 is an immunomodulatory molecule and upregulated in T cells by several cytokines. TIM-3 also influences mast cell function but its transcriptional regulation in mast cells has not been clarified. Therefore, we examined the transcript level and the promoter activity of TIM-3 in mast cells. The TIM-3 transcript level was assessed by real-time RT-PCR and promoter activity by luciferase reporter assay. TIM-3 mRNA levels were increased in HMC-1, a human mast cell line by TGF-β1 stimulation but not by stimulation with interferon (IFN)-α, IFN-λ, TNF-α, or IL-10. TIM-3 promoter -349~+144 bp region relative to the transcription start site was crucial for the basal and TGF-β1-induced TIM-3 promoter activities in HMC-1 cells. TIM-3 promoter activity was increased by overexpression of Smad2 and Smad4, downstream molecules of TGF-β1 signaling. Our results localize TIM-3 promoter activity to the region spanning -349 to +144 bp in resting and TGF-β1 stimulated mast cells.
doi:10.4110/in.2012.12.5.207
PMCID: PMC3509165  PMID: 23213314
T cell immunoglobulin mucin domain-3; TGF-β1; Transcription; Mast cells; Smad

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