PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Eur J Immunol. Author manuscript; available in PMC 2009 October 8.
Published in final edited form as:
PMCID: PMC2759376
NIHMSID: NIHMS133122

TIM-3 is Expressed on Activated Human CD4+ T Cells and Regulates Th1 and Th17 Cytokines

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.

Keywords: human, T cells, cell activation, immunosuppression

Introduction

CD4+ T helper cells, as regulators of adaptive immunity, exert their effects through the secretion of cytokines. A seminal discovery was the observation that murine T helper cells secrete cytokines that can be classified based on their biological effects on adaptive immunity [1]. In mice, T helper 1 (Th1) cytokines IFNγ and IL-2 promote macrophage activation and cytotoxic T lymphocyte (CTL) expansion and activity, whereas IL-4 and IL-5 produced by Th2 cells promote B cell expansion and IgE-mediated eosinophilic inflammation. The utility and relevance of the Th1/2 paradigm has been demonstrated in many murine models of autoimmunity, allergy, and in the context of infections with numerous pathogens [2]. More recently, a third subset of helper T cells, Th17, has been discovered and associated with several autoimmune diseases [3]. This subset is induced in mice and humans by a combination of proinflammatory cytokines (IL-6, IL-21, IL-1) plus TGFβ [4-7] and is characterized by the secretion of IL-17A, IL-17F, IL-21, and IL-22. Th17 cells appear to promote tissue inflammation by exerting effects on parenchymal/tissue cells and by chemoattraction of neutrophils to the site of inflammation [3].

TIM-3 is a molecule uniquely expressed on the surface of murine Th1 cells that negatively regulates IFNγ secretion by inducing cell death by binding to its ligand Galectin-9 [8]. TIM-3 is known to be involved in tolerance induction [9] and blockade of this molecule exacerbates EAE as well as disease in the NOD model of Type I diabetes [10, 11]. More recent studies show that TIM-3 is specifically expressed on HIV-specific T cells undergoing “exhaustion” [12]. We have shown by Taqman quantitative RT-PCR that TIM-3 mRNA expression is lower in CD4+ T cells from MS patients vs. healthy controls [13], and that blockade of TIM-3 with either monoclonal antibody or RNA interference increases the secretion of IFNγ by activated human T cells. This induction was not apparent in T cells isolated from the peripheral blood of untreated MS patients but was restored in patients undergoing treatment with IFNβ or glatiramer acetate. Thus this molecule appears to play an important role in negative CD4+ T cell regulation in both animal models and in human autoimmune disease.

In humans, Th1 and Th2 cells have been isolated from lesions of humans suffering from infectious diseases such as leprosy and leishmaniasis, with Th1 cells present in self-healing lesions and Th2 cells derived from diffuse, uncontrolled lesions [14, 15]. Th2 cells have been isolated from lesions of allergic inflammation and have been shown to infiltrate some human tumors [16-18], while Th17 cells have been isolated from the skin of subjects with contact dermatitis, the synovial membranes of subjects with rheumatoid arthritis (RA), and the gut of subjects with Crohn's disease [19-21]. Nevertheless, polarized helper T cells are not readily identified in the peripheral blood of healthy individuals, with the majority of T helper cells secreting a combination of Th1- and Th2-associated cytokines (Th0 cells). Thus, the utility of the Th1/2 paradigm with regards to human disease has been questioned [22, 23].

We reasoned that difficulties in clearly demonstrating a role of Th1, Th2, and Th17 cells in particular human inflammatory diseases may be due to the inability to precisely identify small frequencies of highly polarized T cells in human peripheral blood or tissue specimens. With this in mind, we generated a panel of human TIM-3 monoclonal antibodies and examined the expression and function of TIM-3 in human CD4+ T cells. We report that TIM-3 is expressed by a subset of activated CD4+ T cells present in human lymph nodes, but is largely absent from CD4+ cells isolated from peripheral blood. Activation with anti-CD3/28 in vitro results in induction of TIM-3, while polarization of CD4+ T cells toward Th1, Th2, or Th17 phenotypes modulates TIM-3 expression, with highest levels expressed by Th1 and Th17 cells. We demonstrate that TIM-3 appears to function as a negative regulator of Th1 and Th17 cytokines in T cells, as blocking the TIM-3 pathway with specific antibodies selectively enhances the secretion of the cytokines IFNγ, IL-17, IL-2, and IL-6, but not IL-10, IL-4, or TNF-α. In addition we find that human TIM-3 may regulate via a different molecular mechanism than murine TIM-3, since TIM-3 blockade by antibodies does not block cell death or enhance T cell proliferation but enhances cytokine expression of selected cytokines at the mRNA level.

Results

Generation of human TIM-3 monoclonal antibodies

To generate monoclonal antibodies against human TIM-3, we immunized TIM-3 deficient mice with hTIM-3 fusion protein and screened supernatants for reactivity to hTIM-3 using both ELISA and flow cytometry. Three monoclonal antibodies (1G5, 2E2, and 4A4) were ultimately selected for further analysis based on their selective ability to bind to immobilized hTIM-3 fusion protein in ELISA assays (Fig. 1, A) and to stain Chinese hamster ovary (CHO) cells transfected with human TIM-3 (Fig. 1, B). All three antibodies exhibited a variable degree of non-specific binding to immobilized murine TIM-3 or human CTLA-4 fusion proteins especially at high doses (10µg of more); this was most apparent for the 4A4 antibody. Nevertheless, none of the antibodies bound to cells transfected with murine TIM-3 or human CTLA-4 in flow cytometric based assays (data not shown). To further confirm the binding specificity of the antibodies and determine their relative affinities for TIM-3, the binding on/off rate kinetics of the antibodies to immobilized human TIM-3/IgG1 Fc fusion protein were determined. The kD values of the 1G5, 2E2, and 4A4 antibodies were 234.68 nM, 0.46 nM, and 1.46 nM, respectively (Fig. 1, C).

Figure 1
Characterization of human anti-TIM-3 monoclonal antibodies. A, Three purified anti-TIM-3 mAbs (1G5, 2E2, and 4A4) were tested for binding to recombinant human TIM-3-Ig or human CTLA-4 Ig fusion proteins by ELISA. Comparative binding to TIM-3-Ig and CTLA-4-Ig ...

To map the reactivity of the antibodies to specific regions of the molecule, we transfected 293HEK cells with DNA constructs containing different domains of human TIM-3. We found that 1G5 and 2E2 antibodies bound to 293 cells expressing the IgV domain of TIM-3 (Fig. 1, D), indicating their specificity for epitopes in this region of the molecule.

We attempted to use the TIM-3 antibodies for Western blot analysis. However, we were unable to detect TIM-3 in lysates from TIM-3 transfectants or activated human T cells that expressed high levels of TIM-3 as revealed by FACS and quantitative PCR. Thus the TIM-3 monoclonal antibodies are highly effective for FACS staining but not Western blot.

TIM-3 expression on ex vivo human T cells

We next examined the expression of TIM-3 on the surface of ex vivo CD4+ and CD8+ T cells. Consistent with our previous observations with murine T cells, we were unable to identify peripheral blood CD4+ T cells that expressed surface TIM-3 when analyzed ex vivo (Fig. 2, A). However, we did consistently detect a low frequency (typically 1-2%) of CD8+ T cells that expressed surface TIM-3 ex vivo (Fig. 2, A and data not shown). Quantitative RT-PCR analysis of mRNA isolated from ex vivo CD4+ and CD8+ T cells indicated that while TIM-3 could not readily be detected on the surface of CD4+ T cells, they did express TIM-3 mRNA at levels slightly lower than that in CD8+ T cells (Fig. 2, B). These data are consistent with murine studies in which very few, if any, T cells express TIM-3 on their surface ex vivo. We stained other cell types from peripheral blood with anti-TIM-3-PE and observed expression of TIM-3 on monocytes, in agreement with our previous work [24] (not shown).

Figure 2
TIM-3 expression on human CD4+ T cells. A, PBMCs obtained from a healthy individual were stained with antibodies to CD3 (to identify T cells), CD4, and TIM-3 (2E2). Isotype control staining for TIM-3 expression is represented in the left panel. T cells ...

In order to analyze TIM-3 expression in the primary immune compartment, we stained cell suspensions isolated from pancreatic lymph nodes of normal subjects (post autopsy) with antibodies specific for CD4, CD25, and TIM-3 (Figure 2, C). In six of six subjects, we found a significant percentage of CD4+ cells that expressed TIM-3 (9.4% of total CD4+ cells, +/- 2.2, average +/- S.D). These cells expressed higher levels of CD25 than CD4+/TIM-3- cells (Figure 2, D), suggesting they are activated.

To determine if activation upregulates TIM-3 expression, we stimulated pancreatic lymph node suspensions from 3 of the six subjects for 2 days with plate bound anti-CD3/28 and stained with antibodies to CD4 and TIM-3 (Figure 2, C, right panels). We found that the number of TIM-3-expressing cells, as well as the expression level of TIM-3, increased with stimulation (26.8 +/- 13.5% of total CD4+ cells, mean +/- S.D).

TIM-3 is not associated with cytokine production by ex vivo CD4+ T cells

To determine if there is a correlation between TIM-3 expression and cytokine production on memory T cells derived ex vivo, we performed intracellular cytokine staining of T cells stimulated for 12 hours with PMA/Ionomycin and co-stained with anti-TIM-3 (Fig. 3, A). We found a small percentage of cells became TIM-3+ (about 4.5%). About one third of these cells were positive for IFNγ; however, the majority of IFNγ-producers were TIM-3 negative. This is consistent with the observation in mice that TIM-3 is downregulated on Th1 cells after initial activation/differentiation. In contrast, very few of the TIM-3+ cells stained for IL-17A or IL-4. Thus TIM-3 was more closely associated with IFNγ than other cytokines in short term stimulated ex vivo T cells, although it was not expressed by the majority of cytokine producing cells.

Figure 3
Expression of TIM-3 by short-term stimulated ex vivo CD4+ T cells does not correlate with cytokine production. A, total CD4+ T cells were isolated from PBMC of a normal healthy donor by negative selection, and stimulated with PMA plus ionomycin for ...

To further determine if TIM-3+ T cells are associated with production of IFNγ or other cytokines, we FACS sorted CD4+/TIM-3+ and CD4+/TIM-3- cells (Figure 3, B) from human spleen cell suspensions and stimulated them in vitro with anti-CD3/28. We assessed proliferation and cytokine production 48 hours post-stimulation (Figure 3, C). We found that TIM-3+/CD4+ T cells were completely unresponsive to stimulation, as evidenced by the inability to proliferate or produce cytokines. This is not surprising, as TIM-3+ cells appear to be in an activated state (Figure 2, D) and thus further activation in vitro could lead to activation induced cell death or anergy.

As the TIM-3 molecule was originally identified based on its unique expression by polarized murine Th1 but not Th2 cells [11] we assessed the ability of the TIM-3 antibodies to stain IFNγ-secreting CD4+ T cells using intracellular staining of CD4+ T cells stimulated under Th1, Th2, or Th17-polarizing conditions for 7 days (Fig. 4, A). We found that TIM-3 expression was induced by anti-CD3/28 stimulation (Th0), with a majority of cells expressing the protein after several days of stimulation. We found that TIM-3 expression was slightly enhanced by Th1 polarization (Th0 plus IL-12 and anti-IL-4) as compared to Th0 after seven days of stimulation (Fig. 4, A). Approximately half of the Th1-polarized cells were actively secreting IFNγ as shown by intracellular staining; most of these cells were TIM-3+. Th2 polarized cells (IL-4 plus anti-IL-12) showed reduced TIM-3 expression as compared to Th1 and Th0 cells, although they do express some TIM-3.

Figure 4
TIM-3 expression is modulated under Th1, Th2, and Th17 polarizing conditions. A, Naïve CD4+ T cells (CD4+/CD62L+/CD25-/CD45RA+) were sorted from peripheral blood of a normal donor and activated with plate-bound anti-CD3/28 for 7 days under Th1 ...

We have recently shown that treatment with IL-21 plus TGFβ drives Th17 differentiation in naïve human CD4+ T cells [5]. In order to determine if TIM-3 is expressed on human Th17 cells, we stimulated sorted naïve T cells with anti-CD3/28 plus this combination of cytokines for seven days, and stained cells with anti-TIM-3-PE (Fig. 4, A and C). We observed high expression of TIM-3 on activated T cells that were not producing IL-17, as well as the few that were IL-17+. Analysis of supernatants by ELISA showed IL-17A production in the Th17 condition only (Fig. 4, B). Activation of central memory T cells with anti-CD3/28 plus IL-6/IL-1β resulted in the induction of IL-17 in a slightly larger number of T cells (about 3%) half of which were TIM-3+ (Fig. 4, C). These data are in agreement with studies that show the proportion of IL-17 producing cells in human peripheral blood is low [25], despite the presence of significant amounts of IL-17 as detected by ELISA. In addition, we found that induced TIM-3 expression was lower in stimulated central memory T cells as compared to activated naïve T cells isolated from the same donor (Fig 4, C and D).

TIM-3 Antibodies Enhance Cytokine Secretion by Activated CD4+ T Cells

To evaluate the function of TIM-3 on human CD4+ T cells, we stimulated freshly isolated cells from a total of nine healthy donors with anti-CD3/CD28 monoclonal antibodies in the presence or absence of purified, dialyzed 2E2 antibody, and measured proliferation, cell death, and cytokine secretion. We confirmed that treatment of unstimulated T cells with 2E2 had no effect on proliferation or activation (data not shown). In agreement with our previous work [13], we consistently observed enhancement of IFNγ secretion in the presence of 2E2 antibody with little effect on T cell proliferation or cell survival (Fig. 5, A). Interestingly, enhancement of IFNγ was inversely correlated with strength of TCR signal, as 2E2 treatment at lower doses of anti-CD3 consistently showed greater enhancement as compared to control Ig (3.4-fold average with 0.1 μg/ml anti-CD3, compared to 2.8- and 2.3-fold for 0.5 μg/ml and 1.0 μg/ml anti-CD3, respectively).

Figure 5
Stimulation of CD4+ T cells in the presence of TIM-3 mAb enhances production of Th1 and Th17 cytokines but not proliferation or cell death. A, Ex vivo CD4+ T cells were stimulated with graded doses of anti-CD3/28 for 48 hours in the presence of 2E2 or ...

We also observed enhancement of the cytokines IL-17A, IL-6, and IL-2 with 2E2 treatment (Fig. 5, A and B). IL-6 was enhanced to almost the same extent as IFNγ (2.7-, 2.6-, and 3-fold at 0.1 μg/ml, 0.5 μg/ml, and 1.0 μg/ml anti-CD3, respectively), although the pattern was reversed in that higher doses induced greater amounts of cytokine. IL-17A and IL-2 were also enhanced by 2E2 treatment, about 1.5-fold as compared to control antibody. In contrast the Th2 cytokines IL-4 and IL-10, and TNFα were not significantly induced by TIM-3 blockade (Fig. 5, B). We confirmed the effects of enhanced cytokine induction using 1G5 antibody, and obtained similar results.

To determine if TIM-3 ligands are present on in vitro activated CD4+ T cells, we stained cells with anti-Galectin-9-PE (gift of Hirashima Mitsuomi) and TIM-3-Ig-PE (Figure 6). We found that unstimulated cells did not express Galectin-9, but that a small subset of cells expressed this protein after 2 days of anti-CD3/28 treatment (approximately 3%). A similarly small percentage of activated cells stained with TIM-3-Fc, while a much larger proportion of cells expressed TIM-3. Thus both TIM-3 and its ligand Galectin-9 are expressed by activated human CD4+ T cells.

Figure 6
TIM-3 and Galectin-9 are expressed by in vitro-activated CD4+ T cells. CD4+ T cells were isolated from peripheral blood of a normal donor and stimulated with plate-bound anti-CD3/28 for 2 days. Unstimulated (upper panels) and stimulated (lower panels) ...

To determine if enhancement of cytokine production by TIM-3 antibodies is mediated at the level of transcription, we performed reverse transcription Taqman PCR on T cells stimulated in the presence of control antibody, 2E2, or 1G5, using primers and probes specific for Tbet, GATA3, and RORγC (Fig. 7, A). These transcription factors are known to regulate Th1, Th2, and Th17 development, respectively. We did not find consistent differences in the expression of these factors between control Ig- and TIM-3 antibody treated T cells, except for a slight decrease in the expression of GATA3. However, we did find enhanced expression of IFNγ, IL-6, and IL-17A mRNA by TIM-3 antibody treated cells that mirrored the results seen by ELISA and cytometric bead array (Fig. 7, B). The effects on IFNγ transcription were more consistent across the subjects than IL-6 or IL-17A. Thus TIM-3 appears to regulate cytokine expression at the level of transcription without affecting the mRNA expression of master transcription factors Tbet, RORγC, and GATA3.

Figure 7
TIM-3 antibodies regulate cytokine production at the level of transcription. A, CD4+ T cell stimulation in the presence of TIM-3 mAbs does not change expression of T-Bet, RORγC, or GATA3. RNA was isolated from ex vivo CD4+ T cells (unstim) or ...

Discussion

The generation of TIM-3 specific monoclonal antibodies has allowed us to probe TIM-3 expression and function on human CD4+ T cells in a more thorough fashion than was previously possible. While TIM-3 has been well studied and characterized in mice, its role in human immune responses is less clear. In the course of our studies we have found similarities and differences between human and murine TIM-3. In agreement with our observations of murine TIM-3, virtually no CD4+ T cells present in the peripheral blood of healthy human subjects express surface TIM-3. In contrast to mouse T cells, which express TIM-3 only after several rounds of Th1 polarization, the majority of in vitro activated human CD4+ cells become TIM-3+ after a few days of stimulation with anti-CD3/28. Thus it appears TIM-3 is expressed by cells that have been stimulated strongly via TCR plus costimulation through signal 2 (CD28). In support of this we observed TIM-3 expression was correlated to dose of anti-CD3 (data not shown). It is possible that TIM-3 is expressed mainly on activated T cells present in sites other than peripheral blood, such as lymph notes, spleen, skin, or mucosa. In support of this, we have observed TIM-3 expression on activated (CD25+) populations of CD4+ T cells isolated from pancreatic lymph nodes (Fig. 2, C and D). This is in agreement with our observations of TIM-3 expressed by mouse T cells in the CNS of mice in the early stages of EAE [11].

We observed an apparent paradox in that TIM-3 does not appear to be correlated with cytokine production in CD4+ T cells examined directly ex vivo (Figure 3), whereas TIM-3 is coexpressed with IFNγ and IL-17 by naïve T cells stimulated in vitro with anti-CD3/28 +/- polarizing cytokines for 7 days (Figure 4). We believe this is consistent with the hypothesis that TIM-3 is a negative regulator of T cells that have previously undergone activation. Thus ex vivo TIM-3+ cells will be resistant to reactivation, perhaps through activation induced cell death or anergy, and will not proliferate or produce cytokines. Stimulation/polarization of naïve, TIM-3-negative cells in vitro, on the other hand, results in activation, secretion of cytokines, and subsequent expression of TIM-3, which will then render them resistant to further stimulation.

Administration of TIM-3-Ig has been shown to enhance Th1 responses in vivo and prevent peripheral T cell tolerance [9, 10]. Extending these observations, we have recently identified Galectin-9 as a ligand of TIM-3, and demonstrated that Galectin-9 induces apoptosis in Tim-3+ Th1 cells but not Th2 cells [8]. Thus, engagement of Tim-3 on murine Th1 cells negatively regulates Th1-derived IFN-γ secretion by inducing apoptosis in Th1 cells. Our data now show that activated human CD4+ T cells express TIM-3 and Galectin-9, and that addition of TIM-3 antibodies during T cell activation augments secretion of IFNγ, IL-17, IL-6, and IL-2, but not IL-4, IL-10 or TNFα. Thus it appears that human TIM-3 regulates the secretion of specific cytokines (Th1 and Th17 to some extent) but does not does not have a global effect on cytokine production.

Since treatment of murine T cells with Galectin-9 results in reduced production of IFNγ via TIM-3-regulated cell death [8], we hypothesize that the TIM-3 monoclonal antibodies we describe herein are antagonistic, and are blocking a negative signal leading to enhanced production of cytokines. To clarify this, we attempted blocking experiments with activated CD4+ T cells using TIM-3-Ig, but found no enhancement of cytokines, proliferation, or cell death (not shown). TIM-3 has been shown to be a positive regulator of antigen presenting cells [26], [27], [24], so we cannot fully rule out the possibility that the TIM-3 antibodies are agonistic, and thus trigger TIM-3 on CD4+ T cells resulting in enhanced cytokine secretion. We believe that this is unlikely, since extensive experimental evidence from multiple studies suggests that TIM-3 acts as a negative, rather than positive, regulator of CD4+ T cells [8], [10], [9], [12]. Indeed, we have utilized 2E2 to block Galectin-9-induced cytokine secretion by human dendritic cells [24]. Also, knockdown of TIM-3 in activated CD4+ T cells using siRNA leads to increased IFNγ production [28], in agreement with our antibody results. Thus it appears that the TIM-3 antibodies block the interaction of TIM-3 with either Galectin-9, or other unknown ligand(s), which results in a release from negative regulation and consequent increase in cytokine production.

Interestingly we found neither protection from, nor induction of, cell death in CD4+ T cells stimulated in the presence of TIM-3 antibodies. In addition, we found that mRNAs of IFNγ, IL-6, and IL-17A were increased in the presence of TIM-3 antibodies, suggesting human TIM-3 negatively regulates cytokine production at the level of transcription, rather than in a cell-death-dependent manner. However, we did not find major differences in expression of T-bet, GATA3, and RORγC, the key transcription factors involved in Th1, Th2, and Th17 differentiation. This is perhaps not surprising as we used total CD4 cells that include naïve and central memory T cells for our functional studies. Thus these data likely reflect memory T cell responses after 48 hours of stimulation as opposed to differentiation of naïve T cells.

In our in vitro system it appears that TIM-3 functions via T-T cell interactions in that TIM-3 may be binding to a ligand expressed on T cells themselves. We depleted regulatory T cells from the total CD4+ population by FACS sorting and observed no loss of cytokine induction resulting from TIM-3 antibody treatment of activated cells (data not shown), suggesting Tregs are not involved in TIM-3-mediated inhibition of cytokines in vitro. We hypothesize that TIM-3 expressed on activated T cells interacts with Galectin-9 or other unknown ligand(s) in either an autocrine or paracrine fashion to inhibit cytokine production. This negative feedback loop may serve as a switch to “turn off” IFNγ and Th1/Th17 responses that could mediate immunopathology if left unchecked.

Collectively, these data suggest TIM-3 is an important regulator of Th1 and Th17 cytokines in human CD4+ T cells. It will be of great interest to use these antibodies in the context of many human inflammatory diseases, where the frequency of TIM-3-expressing T cells can be analyzed and their function in the context of human disease further evaluated.

Materials and Methods

Subjects

Blood was obtained after informed consent from normal healthy donors. Pancreatic draining lymph nodes from normal organ donors were obtained from Dr. Bernhard Hering, Department of Surgery, Diabetes Institute for Immunology and Transplantation, University of Minnesota or the nPOD network of the Juvenile Diabetes Research Foundation International with appropriate Institutional Board Reviews. Lymph nodes were processed to single cell suspensions by mincing and forcing through cell stainers (CoStar). For the six lymph nodes utilized here, two subjects were female (ages 45 and 51 years) and 4 were male (ages 32, 27, 20 and 26 years). Cells were cryopreserved at 20x106/ml in 10% dimethylsulfoxide/90% fetal bovine serum and held in liquid nitrogen until use. Cells were washed in PBS and stained for TIM-3, CD25, and CD4 as described. Three of the lymph node samples were stimulated with plate bound anti-CD3 (OKT3) and anti-CD28 (3D10), both at 1μg/ml, for 48 hours and then stained for TIM-3 (2E2 antibody) CD25, and CD4 (BD Biosciences).

Generation and screening of TIM-3 monoclonal antibodies

Monoclonal antibodies were generated from a fusion with draining lymph node cells obtained from TIM-3-deficient mice immunized with hTIM-3-Ig. Mice were immunized subcutaneously and in the foot pad a total of 6 times on days 0, 3, 7, 10, 13, and 17 with 50µg of protein, alternating the use of Freund's complete adjuvant and PBS with each successive immunization. On day 20, lymph nodes were collected from different sites and fused with SP2/0 cells using methodologies that have previously been described [29]. We used ELISA and flow cytometric analysis using human TIM-3 transfectants to screen culture supernatants for reactivity to hTIM-3-Ig.

Affinity measurements of TIM-3 antibodies

The binding affinities of three antibodies specific for TIM-3 were determined by Telos Pharmaceuticals, LLC using full-length human TIM-3/IgG1 Fc fusion protein and a Biacore T100 instrument. A CM5 chip with a rabbit-anti-mouse IgG Fc capture surface was used at 37° C while antibody samples were maintained at 8° C during the experiments. Binding data from a reference flow cell, which contained captured mouse IgG from buffer only analyte samples, was used to eliminate background binding of the fusion protein to the chip surface and electronic noise.

Cell isolation and T cell polarization

Purified populations of total CD4+, naïve and central memory CD4+, CD8+ cells were isolated by negative selection from peripheral blood mononuclear cells (PBMCs) from healthy subjects using magnetic bead isolation (Miltenyi Biotech), or by FACS sorting. Cell purities were typically between 90-98%. All blood samples were obtained in compliance with the BWH institutional review board protocol. Naïve CD4+ T cells were stimulated in serum-free X-VIVO 15 medium (Lonza) with plate-bound anti-CD3/CD28 monoclonal antibodies (mAb) (OKT3 and 3D10 clones, 1μg/ml) for 7 days in the presence of 10 ng/ml IL-12 and 1 μg/ml anti-IL-4 mAb to generate Th1 cells, 50 ng/ml IL-4 and 2 μg/ml anti-IL-12 to generate Th2 cells, or 50 ng/ml IL-21 and 20 ng/ml TGFß (antibodies and cytokines obtained from R&D Systems, except IL-21 which was obtained from Cell Sciences).

Flow cytometry

Intracytoplasmic staining for IFN-γ and IL-4 (BD Pharmingen) or IL-17A (eBioscience) in ex vivo CD4+ T cells and polarized Th1 cells was performed using the Cytofix/Cytoperm kit (BD Pharmingen) after 5 hours of stimulation with PMA/ionomycin/Golgistop. TIM-3 mAbs were labeled with R-Phycoerythrin (Dojindo Molecular Technologies) and added to cells prior to fixation and permeabilization. Ex vivo intracytoplasmic stains were also confirmed using PE-conjugated anti-TIM-3 and IFNγ-FITC monoclonal antibodies with comparable results. All staining and washes were performed in PBS containing 1% human AB serum (Lonza). Data was collected on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Treestar).

RT-PCR

Quantitative mRNA analysis of TIM-3 was performed using TIM-3-specific primers and probes and methodologies we have previously described [28]. Probes for IFNγ, IL-17A, IL-6, Tbet, RORγC, and GATA3 were obtained from Applied Biosystems. Values have been normalized to GAPDH or ß-2 Microglobulin (Applied Biosystems).

Functional assays

Ex vivo CD4+ T cells (5x104/well) were stimulated with graded doses of plate-bound anti-CD3/anti-CD28 mAb in the presence of 5 μg/ml of soluble anti-TIM-3 antibodies or isotype control, or alternatively in the presence of human TIM-3-Ig or control IgG-Fc (R & D Systems). Supernatants were collected after 48 hours for cytokine measurement by ELISA or Cytometric Bead Array (BD Biosciences) and tritiated [3H]thymidine was added to measure proliferation as we have previously described [28]. Cell death was measured by propidium iodide/annexin V staining (BD Biosciences).

Acknowledgements

The authors would like to thank Deneen Kozoriz for help with cell sorting, and Dr. Misuomi Hirashima for providing us with anti-Galectin-9. WDH is supported by a postdoctoral fellowship from the National Multiple Sclerosis Society. This work was also supported by the American Cancer Society (DEA), National Institutes of Health (NS030863, NS045937), National Multiple Sclerosis Society (RG2571-E-10), and Javits Neuroscience Investigator Awards (DAH, VKK) and by the Juvenile Diabetes Research Foundation international and nPOD (S.C.K.).

References

1. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins J Immunol 1986. 1362348-2357. [PubMed]
2. Kalinski P, Moser M. Consensual immunity: success-driven development of T-helper-1 and T-helper-2 responses Nat Rev Immunol 2005. 5251-260. [PubMed]
3. Bettelli E, Korn T, Oukka M, Kuchroo VK. Induction and effector functions of T(H)17 cells Nature 2008. 4531051-1057. [PubMed]
4. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells Nature 2006. 441235-238. [PubMed]
5. Yang L, Anderson DE, Baecher-Allan C, Hastings WD, Bettelli E, Oukka M, Kuchroo VK, Hafler DA. IL-21 and TGF-beta are required for differentiation of human T(H)17 cells Nature 2008. 454350-352. [PMC free article] [PubMed]
6. Manel N, Unutmaz D, Littman DR. The differentiation of human T(H)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor RORgammat Nat Immunol 2008. 9641-649. [PMC free article] [PubMed]
7. Volpe E, Servant N, Zollinger R, Bogiatzi SI, Hupe P, Barillot E, Soumelis V. A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses Nat Immunol 2008. 9650-657. [PubMed]
8. Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury SJ, Zheng XX, Strom TB, Kuchroo VK. The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 2005. 61245-1252. [PubMed]
9. Sabatos CA, Chakravarti S, Cha E, Schubart A, Sanchez-Fueyo A, Zheng XX, Coyle AJ, Strom TB, Freeman GJ, Kuchroo VK. Interaction of Tim-3 and Tim-3 ligand regulates T helper type 1 responses and induction of peripheral tolerance Nat Immunol 2003. 41102-1110. [PubMed]
10. Sanchez-Fueyo A, Tian J, Picarella D, Domenig C, Zheng XX, Sabatos CA, Manlongat N, Bender O, Kamradt T, Kuchroo VK, Gutierrez-Ramos JC, Coyle AJ, Strom TB. Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance Nat Immunol 2003. 41093-1101. [PubMed]
11. Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, Manning S, Greenfield EA, Coyle AJ, Sobel RA, Freeman GJ, Kuchroo VK. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease Nature 2002. 415536-541. [PubMed]
12. Jones RB, Ndhlovu LC, Barbour JD, Sheth PM, Jha AR, Long BR, Wong JC, Satkunarajah M, Schweneker M, Chapman JM, Gyenes G, Vali B, Hyrcza MD, Yue FY, Kovacs C, Sassi A, Loutfy M, Halpenny R, Persad D, Spotts G, Hecht FM, Chun TW, McCune JM, Kaul R, Rini JM, Nixon DF, Ostrowski MA. Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection J Exp Med 2008. 2052763-2779. [PMC free article] [PubMed]
13. Yang L, Anderson DE, Kuchroo J, Hafler DA. Lack of TIM-3 immunoregulation in multiple sclerosis J Immunol 2008. 1804409-4414. [PubMed]
14. Modlin RL. Th1-Th2 paradigm: insights from leprosy J Invest Dermatol 1994. 102828-832. [PubMed]
15. Salgame P, Abrams JS, Clayberger C, Goldstein H, Convit J, Modlin RL, Bloom BR. Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones Science 1991. 254279-282. [PubMed]
16. Kharkevitch DD, Seito D, Balch GC, Maeda T, Balch CM, Itoh K. Characterization of autologous tumor-specific T-helper 2 cells in tumor-infiltrating lymphocytes from a patient with metastatic melanoma Int J Cancer 1994. 58317-323. [PubMed]
17. Sheu BC, Lin RH, Lien HC, Ho HN, Hsu SM, Huang SC. Predominant Th2/Tc2 polarity of tumor-infiltrating lymphocytes in human cervical cancer J Immunol 2001. 1672972-2978. [PubMed]
18. Umetsu DT, DeKruyff RH. TH1 and TH2 CD4+ cells in human allergic diseases J Allergy Clin Immunol 1997. 1001-6. [PubMed]
19. Albanesi C, Cavani A, Girolomoni G. IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFN-gamma and TNF-alpha J Immunol 1999. 162494-502. [PubMed]
20. Aarvak T, Chabaud M, Miossec P, Natvig JB. IL-17 is produced by some proinflammatory Th1/Th0 cells but not by Th2 cells J Immunol 1999. 1621246-1251. [PubMed]
21. Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, Parente E, Fili L, Ferri S, Frosali F, Giudici F, Romagnani P, Parronchi P, Tonelli F, Maggi E, Romagnani S. Phenotypic and functional features of human Th17 cells J Exp Med 2007. 2041849-1861. [PMC free article] [PubMed]
22. Allen JE, Maizels RM. Th1-Th2: reliable paradigm or dangerous dogma? Immunol Today 199718387-392. [PubMed]
23. Rook G. Th1- or Th2-cell commitment during infectious disease: an oversimplification? Trends Immunol 2001. 22481-482. [PubMed]
24. Anderson AC, Anderson DE, Bregoli L, Hastings WD, Kassam N, Lei C, Chandwaskar R, Karman J, Su EW, Hirashima M, Bruce JN, Kane LP, Kuchroo VK, Hafler DA. Promotion of tissue inflammation by the immune receptor Tim-3 expressed on innate immune cells Science 2007. 3181141-1143. [PubMed]
25. de Beaucoudrey L, Puel A, Filipe-Santos O, Cobat A, Ghandil P, Chrabieh M, Feinberg J, von Bernuth H, Samarina A, Janniere L, Fieschi C, Stephan JL, Boileau C, Lyonnet S, Jondeau G, Cormier-Daire V, Le Merrer M, Hoarau C, Lebranchu Y, Lortholary O, Chandesris MO, Tron F, Gambineri E, Bianchi L, Rodriguez-Gallego C, Zitnik SE, Vasconcelos J, Guedes M, Vitor AB, Marodi L, Chapel H, Reid B, Roifman C, Nadal D, Reichenbach J, Caragol I, Garty BZ, Dogu F, Camcioglu Y, Gulle S, Sanal O, Fischer A, Abel L, Stockinger B, Picard C, Casanova JL. Mutations in STAT3 and IL12RB1 impair the development of human IL-17-producing T cells J Exp Med 2008. 2051543-1550. [PMC free article] [PubMed]
26. Nagahara K, Arikawa T, Oomizu S, Kontani K, Nobumoto A, Tateno H, Watanabe K, Niki T, Katoh S, Miyake M, Nagahata S, Hirabayashi J, Kuchroo VK, Yamauchi A, Hirashima M. Galectin-9 increases Tim-3+ dendritic cells and CD8+ T cells and enhances antitumor immunity via galectin-9-Tim-3 interactions J Immunol 2008. 1817660-7669. [PubMed]
27. Nakae S, Iikura M, Suto H, Akiba H, Umetsu DT, Dekruyff RH, Saito H, Galli SJ. TIM-1 and TIM-3 enhancement of Th2 cytokine production by mast cells Blood 2007. 1102565-2568. [PubMed]
28. Koguchi K, Anderson DE, Yang L, O'Connor KC, Kuchroo VK, Hafler DA. Dysregulated T cell expression of TIM3 in multiple sclerosis J Exp Med 2006. 2031413-1418. [PMC free article] [PubMed]
29. Ponath PD, Kassam N, Qin S. Monoclonal antibodies to chemokine receptors Methods Mol Biol 2000. 138231-242. [PubMed]