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The Journal of Immunology, 2006, 176: 1411-1420.
Copyright © 2006 by The American Association of Immunologists

Soluble Form of T Cell Ig Mucin 3 Is an Inhibitory Molecule in T Cell-Mediated Immune Response1

Hui Geng, Gui-Mei Zhang2, Dong Li, Hui Zhang, Ye Yuan, Han-Gang Zhu, Han Xiao, Ling-Fei Han and Zuo-Hua Feng2

Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, The People’s Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell Ig mucin 3 (Tim-3) has been found to play an important role in Th1-mediated auto- and alloimmune responses, but the function of soluble form of Tim-3 (sTim-3) remains to be elucidated. In this study, we report the inhibitory effect of sTim-3 on T cell-mediated immune response. In this study, sTim-3 mRNA was found, among different tissues and organs, only in splenic cells, and the activation of splenocytes resulted in up-regulated production of both sTim-3 mRNA and protein. We constructed a eukaryotic expression plasmid, psTim-3, which expresses functional murine sTim-3. In C57BL/6 mice inoculated with B16F1 melanoma cells, the growth of tumor was facilitated by the expression of this plasmid in vivo. Furthermore, sTim-3 inhibited the responses of T cells to Ag-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation and the production of cytokines IL-2 and IFN-{gamma} in vitro. In tumor rejection model, sTim-3 significantly impaired T cell antitumor immunity, evidenced by decreased antitumor CTL activity and reduced amount of tumor-infiltrating lymphocytes in tumor. Real-time PCR analysis of gene expression in tumor microenvironment revealed the decreased expression of Th1 cytokine genes and the unchanged profile of the genes related to T regulatory cell function, suggesting that the inhibitory effect of sTim-3 on the generation of Ag-specific T cells in vivo is dominated by T effector cells rather than T regulatory cells. Our studies thus define sTim-3 as an immunoregulatory molecule that may be involved in the negative regulation of T cell-mediated immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The search for cell surface markers that can distinguish Th1 from Th2 cells has led to the identification of a new gene family: T cell Ig- and mucin domain-containing molecule (TIM)3 family. The genes in this family are expressed in T cells and encode the proteins containing an Ig V-like domain and a mucin-like domain. To date, the TIM family consists of eight members in mice and three in humans, which might have important immunological function and biological effects (1, 2, 3, 4, 5). Among the members in this family, T cell Ig mucin 3 (Tim-3), which was identified as a negative regulator of immune responses, recently attracted much attention because of its important function in the regulation of immune-mediated disease (6, 7, 8). Previous study on Tim-3 indicated the importance of Tim-3 ligand (Tim-3L) pathway in the generation of T regulatory cells (Treg cells) and the limited expansion of Th1 cell populations. Treatment with anti-Tim-3 mAb exacerbated the Th1-dependent autoimmune disease experimental autoimmune encephalomyelitis (6). In addition, the administration of a soluble fusion protein, Tim-3-Ig, abrogated the induction of peripheral tolerance. Based on these observations, Tim-3 was supposed to limit the expansion of Th1 cell populations and contribute to the induction of tolerance in effector Th1 cells (7, 8).

It was found that Tim-3 mRNA could be alternatively spliced to produce two mRNA molecules. The longer one directs the synthesis of Tim-3 (i.e., full-length Tim-3), a type I transmembrane protein that is preferentially expressed on differentiated Th1 cells and has been identified as a cell surface marker distinguishing between Th1 and Th2 cells. The shorter one, without the region encoding the mucin domain and transmembrane domain, was supposed to direct the synthesis of a splice variant of Tim-3. It was supposed that the isoform of Tim-3 translated from the shorter Tim-3 mRNA should be a soluble molecule (sTim-3), which, however, has not been identified yet.

Many costimulatory molecules and immunoregulatory receptors, such as CD86, Fas, and CTLA-4, have native soluble molecules, and these soluble variants are important in controlling immune response as well as in susceptibility and resistance to autoimmune disease (9, 10, 11). The discovery of sTim-3 raised the issue of its function in regulating immune responses. Based on the observation of Tim-3-Ig fusion proteins causing hyperproliferation of Th1 cells and inhibiting the generation of Treg cells, sTim-3 was speculated to block the inhibitory effect of Tim-3, and promote the expansion and differentiation of Th1 cell population (7, 8). However, it has not been proved whether sTim-3-Ig really mimicked the function of native sTim-3. To date, it is still not clear when sTim-3 is made during lymphocyte differentiation and what its function might be. In this study, we provided the evidence for the expression pattern of sTim-3 mRNA and the existence of sTim-3 as a soluble molecule produced by the activated splenic lymphocytes. We constructed a recombinant expression plasmid encoding murine sTim-3, and investigated the effect of the expressed product on T cell response in vivo and in vitro. sTim-3 could inhibit T cell response to Ag-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation in vitro. The expression of the plasmid encoding sTim-3 in vivo significantly facilitated the growth of tumor by impairing T cell-mediated antitumor immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and cell lines

Female C57BL/6 and BALB/c mice, 6–8 wk old, were purchased from the Center of Experimental Animals of Chinese Academy of Medical Science and Center of Medical Experimental Animals of Hubei Province, respectively. The animals were maintained in our facilities under pathogen-free conditions. All studies involving mice were approved by the Huazhong University of Science and Technology Animal Care and Use Committee. Chinese hamster ovary cell line (CHO), murine melanoma B16F1 cell line, and murine H22 cell line were purchased from the China Center for Type Culture Collection, and cultured in DMEM supplemented with 10% FCS, 10 mM HEPES, 2 mM L-glutamine, 100 mg/ml penicillin, and 100 U/ml streptomycin. All cell culture reagents were obtained from Invitrogen Life Technologies.

Abs, cytokines, and reagents

Goat anti-mouse Tim-3 Ab was purchased from R&D Systems; hamster anti-mouse-CD3 Ab was obtained from eBioscience. HRP-labeled donkey anti-goat IgG Ab, HRP-labeled rabbit anti-mouse IgG Ab, and biotinylated mouse anti-hamster IgG Ab were purchased from Proteintech Group. Anti-mouse CD3 (145-2C11) and anti-CD28 mAb (37.51) mAbs used for stimulating T cells were obtained from eBioscience. Murine GM-CSF, IL-4, IL-2, and ELISA kits for the assay of murine IFN-{gamma}, IL-2, and IL-4 were purchased from eBioscience. Restriction enzymes HindIII, BamHI, BglII, and XbaI were purchased from Promega. Protein marker (SM0671) was purchased from Fermantas.

Analysis of gene expression by conventional RT-PCR and real-time PCR

Total RNA was extracted using the TRIzol reagent (Invitrogen Life Technologies), and samples were incubated with RNase-free DNase I (Promega) at 37°C for 30 min to avoid amplification/detection of contaminating genomic DNA. RNA concentration was measured spectrophotometrically, and equal amounts of RNAs were reverse transcribed.

Conventional RT-PCR was performed to confirm the existence of sTim-3 mRNA vs Tim-3 mRNA. The cDNAs were amplified at 95°C for denaturing, 54°C for annealing, and 72°C for extension. Conventional RT-PCR primers for mouse Tim-3 were 5'-TCCCTACACAGAGCTGTC-3' and 5'-CAGAAATGAAGGCGAGCC-3'. Primers were designed according to the sequences in 5' untranslated region and 3' untranslated region, which are identical in both Tim-3 and sTim-3 mRNAs. The expected products are 932 bp for Tim-3 mRNA and 680 bp for sTim-3 mRNA. Primers for mouse beta-actin were 5'-ATGGGTCAGAAGGACTCCTATG-3' and 5'-ATCTCCTGCTCGAAGTCTAGAG-3'. The PCR products were separated by electrophoresis on 1.5% agarose gel and stained with ethidium bromide.

Real-time quantitative PCR was performed in an ABI PRISM 7700 sequence detection system using the 5'-nuclease method (TaqMan). Primers and TaqMan probes were designed using the primer design software Primer Express (Applied Biosystems), except those for beta-actin, which were available commercially. To determine the expression of Tim-3 mRNA and sTim-3 mRNA, we used two sets of primers and probes. One set was designed according to the sequence in mucin-like domain, and the expected amplification product was from Tim-3 mRNA. Another set included the primer spanning the splice junction of sTim-3 mRNA, and the expected amplification product was from sTim-3 mRNA. The sequence of all primers and probes used in this study is shown in Table I. A total of 20 ng of each of cDNA samples, except for 10 ng of beta-actin cDNA, was mixed with primers and TaqMan Universal PCR Master Mix in a total volume of 25 µl, as described in the manufacturer’s directions (protocol 4304449; Applied Biosystems). The PCR was conducted using the following parameters: 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 1 min. Quantification of the expression of genes was performed using the comparative CT method (Sequence Detector User Bulletin 2; Applied Biosystems). The expression level of each mRNA was normalized to beta-actin mRNA and expressed as n-fold difference relative to the control (calibrator). All PCR assays were performed in duplicate, and results are represented by the mean values ± SEM.


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  		Table I. Real-time quantitative PCR primers and probes

 
Spleen cells stimulated with heat shock protein (HSP) 70-peptide complex

To examine the expression of sTim-3 mRNA vs Tim-3 mRNA after activation, spleen cells were stimulated with HSP70-peptide complex, as described previously (12, 13). Briefly, B16F1 melanoma peptides and HSP70, at the concentrations of 75 and 250 µg/ml, respectively, were mixed and incubated at 37°C for 2 h in the presence of 1 mM ADP and 1 mM MgCl2. Freshly isolated splenocytes were cultured at a concentration of 1 x 107 cells/ml in RPMI 1640 medium supplemented with 20 U/ml IL-2 in six-well culture plate in the presence of 0.75 µg/ml HSP70-peptide complex. The cells were passaged and restimulated with HSP70-peptide complex every 3 days for three times (totally stimulated for four times). At each time of passages, half of spleen cells were harvested for analysis with RT-PCR.

Construction of expression plasmid vectors

To construct the expression plasmids of Tim-3 and sTim-3, cDNA fragment encoding mouse Tim-3 was prepared by RT-PCR from C57BL/6 spleen cells. The PCR primers were 5'-GGGAACCGAGAAGCTTAAAGCTATCCCTACACAG-3' (HindIII) corresponding to nt 1–34 and 5'-CAGAACCTCGGATCCGGTTGCCAAGTGACATA-3' (BamHI) complementary to nt 989–1020 of murine Tim-3 cDNA. Two cDNAs with different sizes were digested and inserted into compatible enzyme restriction sites of pcDNA3.1 (Invitrogen Life Technologies). The constructed plasmids were designated as pTim-3 and psTim-3, respectively. For the construction of the plasmids expressing fusion proteins Tim-3-GFP (pTim-3-GFP) and sTim-3-GFP (psTim-3-GFP), both having GFP fragment at C terminus, another antisense primer, 5'-AACCAGAAGATCTCGGATGGCTGCTGGCTGTTG-3' (Bgl II), was used for the amplification of Tim-3 cDNA. Primers for the amplification of GFP fragment from pTracer plasmid (Invitrogen Life Technologies) were: sense, 5'-GGTTGAAGATCTTGGCTAGCAAAGGAGAAG-3' (BglII) and antisense, 5'-GGCACTGGTTCTAGATCAATCCATGCCATGTGT-3' (XbaI). The two amplified fragments were digested by restriction enzymes HindIII and BglII, BglII and XbaI, respectively, and recombinated with pcDNA3.1 digested with HindIII and XbaI. All cDNAs inserted into the constructed plasmids were confirmed by DNA sequencing.

Cell transfection

CHO cells were transfected with indicated plasmid with Dosper liposomal transfection reagent, according to the manufacturer’s protocol (Boehringer Mannheim). In brief, CHO cells were grown to 60–80% confluence in 24-well plates and then transfected with 1.0 µg/well indicated plasmid mixed with Dosper liposomal transfection reagent. The transfected cells were selected in complete medium containing 1 mg/ml G418 (Invitrogen Life Technologies), and were subsequently cloned by limiting dilution. Expression of pTim-3-GFP and psTim-3-GFP was observed using fluorescence microscope. The supernatants of the cells transfected with psTim-3 were collected for additional experiments.

Western blot analysis

For the detection of sTim-3 produced by the activated splenocytes, 72-h culture supernatants were concentrated using Freeze Dry System (Labconco). For the detection of sTim-3 produced by the expression of psTim-3 in vivo, livers from the mice were excised 48 h after transfection and digested with collagenase and EDTA; the collected hepatic cells were cultured at a concentration of 5 x 106 cells/ml; and 48-h culture supernatants were concentrated using the Freeze Dry system. The method of standard Western blot for detection of protein has been previously described (10, 14). Briefly, the proteins were separated by SDS-PAGE, followed by transfer onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with 3% BSA in Tween 20 plus TBS, and probed with the anti-mouse Tim-3 Ab at a concentration of 0.2 µg/ml. After incubation with the secondary anti-goat Ab conjugated with HRP, membranes were extensively washed and the bound Abs were detected using the ECL Western Blotting Detection System (Amersham Biosciences), according to the manufacturer’s instructions. To analyze the existence of anti-Tim-3 Ab in the sera of mice, 48-h supernatants of psTim-3-transfected cells were loaded to the gel by using a comb without tooth, and the blot was prepared by normal procedures. The membrane was carefully cut into strips. Each of the strips was incubated with a different dilution of sera from mice, and then went through the following normal procedures.

Isolation of T cells by MACS MultiSort

T cells were purified from spleen cells by magnetic cell sorting using a MiniMACS device. The separation procedure was conducted according to the manufacturer’s instructions (Miltenyi Biotec). In brief, spleen cells at the concentration of 107 cells/100 µl were incubated with T cell isolation kit for 15 min at 6–10°C. Spleen cell suspension was resuspended in degassed buffer and poured into the column reservoir. Labeled cells were retained within the magnetized matrix of the column, whereas nonlabeled cells passed through and were collected as the nonmagnetic fraction. To retrieve the magnetic fraction, the column was removed from the separator, and 1 ml of degassed buffer was added to the reservoir. The cells were flushed out of the column with the aid of a plunger.

Preparation of Ag-loaded dendritic cells (DC)

Bone marrow-derived DC was prepared, as previously described (15, 16). In brief, bone marrow cells were flushed from the femurs and tibias of mice under aseptic conditions, depleted of RBC, and then cultured at a concentration of 1 x 106 cells/ml in complete RPMI 1640 medium supplemented with 10 ng/ml GM-CSF and 10 ng/ml IL-4. On day 6, nonadherent and loosely adherent cells were harvested and separated by 14.5% metrizamide complete medium gradients. DC was collected by gentle pipette aspiration and washed twice with complete medium, and then cultured in the presence of 0.75 µg/ml HSP-B16F1 peptide complex (prepared by the same method described above). After 18 h of incubation, Ag-loaded DC was harvested, irradiated (3000 rad), and resuspended in HBSS for additional experiments.

Functional assay of sTim-3 in vitro

MACS MultiSort-purified T cells were cultured at a concentration of 2 x 106 cells/ml in the presence of Ag-loaded DC (at a responder to stimulator ratio of 20:1) or a combination of anti-CD3 (coated, 5 µg/ml) and anti-CD28 (5 µg/ml) in complete RPMI 1640 medium containing serial dilutions of the supernatants of CHO cells transfected with psTim-3 (17, 18, 19). For blockage assay, the supernatants of psTim-3-transfected cells were pretreated with anti-mouse Tim-3 (10 µg/ml). To determine the proliferation of T cells, 1.0 µCi of [3H]thymidine was added on day 4, the cells were cultured continuously for 18 h, and then the incorporation of [3H]thymidine was measured in a MicroBeta TriLux liquid scintillation counter (Wallac). To detect the production of cytokines, the supernatants were collected, and the concentrations of IFN-{gamma}, IL-2, and IL-4 were determined by sandwich ELISA, according to manufacturer’s instructions.

FACS analysis

The purified T cells or B16F1 melanoma cells were washed with PBS and incubated with supernatants of CHO cells transfected with psTim-3-GFP for 1 h at 37°C. In blocking experiment, supernatants of psTim-3-GFP-transfected cells were pretreated with anti-mouse Tim-3 before the incubation with splenic T cells. After washing with PBS, the cells were used for flow cytometric analysis. Parameters were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed with CellQuest software (BD Biosciences).

In vivo gene delivery and tumor growth studies

Plasmid DNA was prepared and analyzed, as described (13). All plasmid preparations were free of detectable RNA, and endotoxin levels were <1.5 EU/µg. Spectrophotometric analysis revealed 260/280 nm ratios ≥1.80. Purity and conformation of the prepared plasmid DNA were confirmed by agarose gel electrophoresis. On day 0, 1 x 105 B16F1 cells in 100 µl of sterile 0.9% saline were inoculated in the right hind limbs of mice. On day 2, the mice received the injection of 100 µg of plasmid DNA using the hydrodynamics-based gene delivery technique (20, 21). Mice tolerated this treatment regimen well without obvious side effects after injection. Gene delivery was conducted every 3 days for four times in all. Tumor growth was inspected by Vernier caliper measurement every other day from day 6 after inoculation. Tumor volume was calculated according to the formula V = (a x b2)/2, with a as the larger diameter and b as the smaller diameter. The mouse survival rate was also recorded.

Cytotoxicity assay

Splenic T cells from tumor-bearing mice were cultured at 1 x 107 cells/ml and restimulated with Ag-loaded DC in RPMI 1640 medium supplemented with 20 U/ml IL-2 for 5 days. B16F1 target cells were labeled with Na51CrO4 (0.1 µCi/106 cells; Amersham Biosciences) at 37°C for 1 h. After extensive washing, target cells were incubated with effectors at different E:T ratios in triplicate at 37°C for 4 h, and 51Cr released (cpm) into the supernatants was measured in a gamma counter to calculate percentage of specific release. Specific lysis was determined as follows: percentage of specific release = 100 x (experimental release – spontaneous release)/(maximum release – spontaneous release). Spontaneous release was ≤20% of maximum release in all experiments.

Histology and immunohistochemistry

Tumor tissues were surgically excised on day 16 after inoculation and fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were stained with H&E. Immunohistochemistry was performed with the method of SP, as described (13, 22). The primary Ab was hamster anti-mouse CD3 Ab. Biotinylated Ab to hamster IgG was used as secondary Ab. The reaction product was visualized with the peroxidase-conjugated streptavidin system with 3,3-diaminobenzidine (Serva) as substrate. Images were analyzed by HMIAS-2000 analyzer, and the mean value of area-integrated OD was calculated.

Data analysis

Results were expressed as mean value ± SEM and interpreted by ANOVA-repeated measures test, and mouse survival rate was interpreted by Wilcoxon’s rank test. Differences were considered to be statistically significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression analysis of sTim-3

Although the existence of sTim-3 mRNA in spleen cells has been found, the distribution of sTim-3 mRNA in different organs or tissues was still unknown. To this end, sTim-3 mRNA in normal mice was examined by RT-PCR. Total RNA was isolated from bone marrow, thymus, spleen, and also from other organs, including liver, brain, heart, lung, pancreas, and kidney of C57BL/6 mice. RT-PCR results revealed that Tim-3 mRNA was existent in the cells from thymus, bone marrow, and spleen, whereas sTim-3 mRNA was existent only in splenic cells (Fig. 1A). Neither Tim-3 mRNA nor sTim-3 mRNA was detectable in nonlymphoid organs (data not shown).


Figure 1
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  		FIGURE 1. Expression pattern of sTim-3. A, Distribution of sTim-3 mRNA in the indicated organs or tissues was determined by conventional RT-PCR. Lanes 1 and 5, DNA markers (DL2000; TaKaRa Biotech); lane 2, splenocytes; lane 3, thymus; lane 4, bone marrow. B, Conventional RT-PCR analysis of sTim-3 mRNA and Tim-3 mRNA in freshly isolated splenocytes and activated splenocytes. Primers were the same as those in A. Splenocytes were stimulated with HSP70-peptide complex, as described in Materials and Methods. C, Quantification of Tim-3 mRNA and total Tim-3 transcripts in freshly isolated and in vitro activated splenocytes by real-time PCR with primers and probes described in Tab 1. Real-time PCR primers were tested, and no cross-reactivity was found (left). The expression level of both Tim-3 mRNA and sTim-3 mRNA was significantly increased after each round of stimulation; however, there appears to be a higher increase of sTim-3 product than that of Tim-3 product. D, Detection of sTim-3 in the culture supernatants of splenocytes after the indicated round of stimulation (lanes 1–5) and transfected CHO cells (lanes 6 and 7) by Western blot analysis. Lane 1, without stimulation; lane 2, round 1; lane 3, round 2; lane 4, round 3; lane 5, round 4; lane 6, transfected with pcDNA3.1; lane 7, transfected with psTim-3.

 
To investigate the expression pattern of sTim-3 and Tim-3 mRNA in immune response, we first examined the expression of these mRNAs in freshly isolated and activated splenocytes with conventional RT-PCR. Tim-3 mRNA was observed in freshly isolated splenocytes from 12 of 12 mice, whereas sTim-3 mRNA was observed in 10 of 12 healthy subjects tested. HSP70-peptide complex has been served as effective immunogens to stimulate T cells in vitro and in vivo (15, 16, 23, 24). The expression of both Tim-3 mRNA and sTim-3 mRNA was up-regulated along with several rounds of stimulation with HSP70-peptide complex in vitro (Fig. 1B). We then analyzed the expression of sTim-3 and Tim-3 further by real-time PCR. Under the conditions used for quantitative PCR, cDNA from either Tim-3 mRNA or sTim-3 mRNA could be amplified independently by using two sets of primers (Fig. 1C, left). Quantitative PCR revealed that both Tim-3 mRNA and sTim-3 mRNA were significantly increased after each round of stimulation, whereas there appeared to be a higher increase of sTim-3 product than that of Tim-3 product. These results indicated that the expression of Tim-3 gene was up-regulated after stimulation, and the alternative splicing of mRNA favored the production of sTim-3 mRNA after several rounds of stimulation.

To prove the existence of the translated product of sTim-3 mRNA, the proteins in culture supernatants of splenocytes and CHO cells transfected with psTim-3 were analyzed with Western blot. The results showed that a ~24-kDa protein that binds specifically with anti-Tim-3 Ab was detected in the cell-free supernatants of the actived splenocytes, but not in the culture supernatants of freshly isolated splenocytes (Fig. 1D). The 24-kDa protein was also detected in the supernatants of CHO cells transfected with psTim-3. The molecular mass of sTim-3, based on the predicted protein size derived from the primary amino acid sequence, is 21,865. Given that there are N-linked glycosylation sites and O-linked glycosylation sites in IgV domain of sTim-3 molecule, the results suggested that sTim-3 molecule was glycosylated and expressed as a secreted form.

sTim-3 protein binds to its putative ligand(s)

To verify whether sTim-3 is capable of binding with its putative ligand(s), we constructed expression plasmids of sTim-3-GFP and Tim-3-GFP. CHO cells were transfected with psTim-3-GFP and pTim-3-GFP, respectively. Green fluorescence was found in CHO cells transfected with pTim-3-GFP, but scarcely in CHO cells transfected with psTim-3-GFP (Fig. 2A), indicating that sTim-3 is mostly secreted out, which further confirmed that sTim-3 is a soluble molecule. The binding of sTim-3-GFP to its putative ligand(s) on splenic T cells was determined by FACS analysis. The fluorescence intensity on T cells incubated with sTim-3-GFP was significantly increased as compared with control (Fig. 2B). When the supernatants of psTim-3-GFP-transfected cells were pretreated with anti-mouse Tim-3 before the incubation with splenic T cells, the fluorescence intensity decreased nearly to the control level. These results clearly indicated that sTim-3 could bind to putative Tim-3 ligand(s).


Figure 2
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  		FIGURE 2. Analysis of protein encoded by sTim-3 cDNA. A, Analysis of the expressed Tim-3-GFP and sTim-3-GFP in the transfected CHO cells by fluorescence microscope; green fluorescence was found in CHO cells transfected with pTim-3-GFP (left), but scarcely in CHO cells transfected with psTim-3-GFP (right). Images are representative of multiple microscopic fields observed in two individual experiments under same conditions. B, Analysis of the binding of sTim-3 to its putative ligand(s) on T cells by flow cytometric analysis. Curve 1, T cells alone; curve 2, T cells incubated with the expression product of psTim-3-GFP pretreated with anti-mouse Tim-3; curve 3, T cells incubated with the expression product of psTim-3-GFP.

 
Expression of psTim-3 in vivo facilitates tumor growth

On the basis of the regulatory effect of sTim-3-Ig fusion protein on immune response, sTim-3 was speculated to block the inhibitory effect of Tim-3, and promote expansion and differentiation of Th1 cell population (7, 8). So, we tried to observe the inhibitory effect of sTim-3 on the growth of tumor in vivo by enhancing the immune response. To test this, in vivo transfection with psTim-3 was performed by the hydrodynamics-based gene delivery technique. Given that the hydrodynamics-based gene delivery by i.v. administration of naked DNA is a simple and efficient method for the expression of the secretory protein in vivo, and the technique is gaining increasing favor for studying the function of secretory protein in vivo (20, 21), we first examined whether the transgene was efficiently taken up and expressed by hepatic cells. As expected, conventional RT-PCR results confirmed that sTim-3 mRNA was expressed by hepatic cells transfected with psTim-3, but not in control hepatic cells (Fig. 3A). Western blot analysis revealed that the expressed product of psTim-3, a secretory molecule, was existent in supernatants of hepatic cells and in serum of mice treated with psTim-3 (Fig. 3B). Importantly, we found no obvious toxicity caused by the injection of psTim-3 plasmid at dosage up to 200 µg/mouse. Extensive pathological examination and comparison of the mice that received different injections showed no evidence of obvious toxicities such as weight loss and the change of glutamic pyruvic transaminase and creatine levels in serum (data not shown).


Figure 3
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  		FIGURE 3. sTim-3 facilitates the growth of tumor in vivo. A, Conventional RT-PCR analysis of the expression of sTim-3 in liver cells. sTim-3 mRNA was only detected in hepatic cells transfected with psTim-3. Lane 1, Hepatocytes of normal mice; lane 2, hepatocytes of mice treated with saline; lane 3, hepatocytes of mice treated with pcDNA3.1; lane 4, hepatocytes of mice treated with psTim-3. B, Western blot analysis of sTim-3 expression. Cell-free culture supernatants of hepatic cells and sera samples were collected and analyzed with Western blot, as described in Materials and Methods. Lanes 1-4, Culture supernatants of hepatocytes from normal mice, and mice treated with saline, pcDNA3.1, and psTim-3; lanes 5–8, sera from normal mice, and mice treated with saline, pcDNA3.1, and psTim-3. C and D, C57BL/6 mice were inoculated with 1 x 105 B16F1 cells (C), and BALB/c mice were inoculated with 1 x 105 H22 cells (D). The mice were treated by i.v. injection with psTim-3, pcDNA3.1, or saline. Tumor volume (left) and survival rate (right) were monitored. Data from two identically performed experiments were combined. E, B16F1 melanoma cells were incubated with sTim-3-GFP and analyzed by FACS. Fluorescence intensity on the examined B16F1 melanoma cells was not increased compared with control. *, Significant difference between groups (p < 0.05).

 
To investigate the effect of sTim-3 on tumor growth in vivo, mice were inoculated with 1 x 105 B16F1 melanoma cells into the hind thigh muscle and then treated with in vivo transfection of psTim-3, pcDNA3.1, or injection of saline as control. Unexpectedly, the growth of B16F1 melanoma was not suppressed, but facilitated significantly in mice treated with psTim-3 as compared with control mice treated with pcDNA3.1 or saline (Fig. 3C). The survival of psTim-3-treated tumor-bearing mice was also significantly shorter than that of control mice. All mice in the treatment group had died within 28 days after tumor inoculation, whereas 80% of mice in the control group were still alive on day 28 (Fig. 3C). To examine whether the treatment with sTim-3 also facilitated the growth of other type of tumor, we treated the BALB/c mice inoculated with hepatocarcinoma H22 cells in the same way, and similar effect of sTim-3 on tumor growth was also observed (Fig. 3D), suggesting that the facilitation of sTim-3 on tumor growth is not specific on the indicated tumor cells.

To exclude a direct effect of sTim-3 on tumor cell growth, tumor cell proliferation assay was performed by trypan blue exclusion. The proliferation of B16F1 melanoma cells in vitro was not facilitated in the presence of supernatants from the psTim-3-transfected cells (data not shown). In addition, B16F1 melanoma cells did not express putative Tim-3 ligand(s) as assessed by FACS analysis using sTim-3-GFP (Fig. 3E). Thus, a direct effect of sTim-3 on tumor cells was excluded. Taken together, these results suggested that the impaired immune response might account for the effect of sTim-3 on tumor growth in vivo.

sTim-3 inhibits T cell response in vitro

To test the notion, based on the above results, that sTim-3 is possibly an inhibitory molecule in the control of cell-mediated immune response, we first investigated the effect of sTim-3 on T cell activation in vitro. When T cells were stimulated with Ag-loaded DC, the supernatants containing sTim-3 significantly impaired the proliferation of T cells in a dose-dependent manner (Fig. 4A). The inhibitory effect was abrogated if the expression product of psTim-3, before added into stimulation system, was pretreated with anti-mouse Tim-3 (Fig. 4A). When we examined the effect of sTim-3 on the activation of T cells by immobilized anti-CD3 mAb and soluble anti-CD28 mAb, similar inhibitory effect was also observed (Fig. 4B). To determine whether sTim-3 has an effect on the production of cytokines, the culture supernatants were collected 72 h after stimulation, and analyzed with ELISA for the production of IL-2, IFN-{gamma}, and IL-4. When T cells were stimulated with Ag-loaded DC, the production of IL-2 and IFN-{gamma} by T cells in the presence of sTim-3 was suppressed in a dose-dependent manner, but the production of IL-4 was little influenced by sTim-3 (Fig. 4A). Same effect was observed in anti-CD3 mAb plus anti-CD28 mAb costimulation system (Fig. 4B). In addition, anti-mouse Tim-3 could abrogate the inhibitory effect of sTim-3 on the production of IL-2 and IFN-{gamma} by the activated T cells. These findings, taken together with the above results, suggested that the expression product of psTim-3 was a functional molecule that had an inhibitory effect on T cell response.


Figure 4
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  		FIGURE 4. Effect of sTim-3 on T cell proliferation and cytokine production in vitro. T cells purified from spleen cells of C57BL/6 mice were stimulated in the presence of the supernatants of CHO/psTim-3 cells or CHO/pcDNA3.1 cells, as described in Materials and Methods. In blockage assay, the supernatants of CHO/psTim-3 cells were pretreated with anti-mouse Tim-3 (10 µg/ml) before the incubation with T cells. The effect of sTim-3 on T cell proliferation after stimulation with Ag-loaded DC (A) or with anti-CD3 mAb plus anti-CD28 mAb (B) was determined by 18-h [3H]thymidine uptake in triplicate wells. Supernatants were collected 72 h after stimulation, and the concentration of cytokines was determined by cytokine ELISA for IL-2, IFN-{gamma}, and IL-4. Data were expressed as mean ± SEM.

 
In vivo expression of psTim-3 impairs T cell antitumor immunity

To understand the potential mechanisms of sTim-3 responsible for promoting the development of tumor in vivo, we investigated the effect of sTim-3 on the generation of tumor-specific T cells. Splenic T cells were isolated from tumor-bearing mice with different treatments on day 16 after tumor inoculation and restimulated in vitro. With the stimulation of Ag-loaded DC, T cells from mice treated with pcDNA3.1 or saline showed a high proliferative response, but the response of T cells from mice treated with psTim-3 was significantly impaired (Fig. 5A). Consistent with this finding, the cytotoxicity of T cells to B16F1 cells in psTim-3-treated group was also significantly lower than that in pcDNA3.1 group or saline group (Fig. 5B), indicating that sTim-3 could impair T cell antitumor immune response in vivo.


Figure 5
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  		FIGURE 5. sTim-3 impairs generation of Ag-specific T cells in tumor rejection model. Splenic T cells were obtained from tumor-bearing mice with different treatments and restimulated in vitro with Ag-loaded DC. Proliferation of T cells was measured by [3H]thymidine incorporation (A), and the cytotoxicity of lymphocytes to B16F1 cells was tested by a standard 4-h 51Cr release assay (B). Data represent the average ± SEM from three independent experiments. C, Microscopic findings of tumor-infiltrating cells with different treatments. H&E staining (upper panels) and immunostaining for CD3 (lower panels) were performed on day 16 after inoculation of tumor cells. The amounts of both infiltrating cells (upper panels) and CD3+ cells in the infiltrating cells in psTim-3-treated group were significantly reduced as compared with those in cont rol groups. Images are representative of multiple microscopic fields observed in three mice per group. D, Analysis of anti-Tim-3 Ab in sera. Serum samples of psTim-3-treated mice were collected on day 16 after tumor inoculation, and anti-Tim-3 Ab in the sera was detected by Western blot.

 
H&E analysis of tissue sections showed that moderate infiltration of inflammatory cells was observed in the tumor of mice treated with saline or pcDNA3.1, but the infiltration of inflammatory cells was scarce in the tumor of mice treated with psTim-3 (Fig. 5C). The area-integrated OD of psTim-3 group (0.069 ± 0.03) was significantly lower than that of either saline group (0.223 ± 0.04) or pcDNA3.1 group (0.227 ± 0.04) (p < 0.01). Immunohistochemical studies revealed that there was a difference in the intensity of T cell infiltration between tumors from control mice and psTim-3-treated mice. The amount of CD3+ cells in psTim-3-treated group (19 ± 4 positive cells per high power field) was significantly reduced as compared with that in saline group (49 ± 12) or pcDNA3.1 group (51 ± 14) (p < 0.01), indicating that the amount of tumor-infiltrating T cells at tumor site in psTim-3-treated mice was significantly reduced. The decrease of tumor-infiltrating lymphocytes was not induced by anti-Tim-3 Ab, because the Ab was not detectable in the sera of mice collected on day 16 after tumor inoculation (Fig. 5D). These results further reinforced that sTim-3 had an inhibitory effect on T cell response.

Because both effector CD4+CD25– T cells and Treg CD4+CD25+ cells express a putative Tim-3 ligand (7, 8), the impairment of antitumor immunity by sTim-3 might be the result of either impairing the function of effector CD4+ T cells or activating the function of CD4+ Treg cells. To understand the underlying cellular mechanism contributing to the impaired antitumor immunity by sTim-3, we analyzed the expression of genes related to the function of tumor-infiltrating lymphocytes on days 4, 8, 12, and 16 after inoculation of tumor cells by real-time quantitative PCR. A striking feature of gene expression in sTim-3-treated mice, compared with control, was the down-regulated expression of IL-2, IFN-{gamma}, and TNF-beta mRNA from day 8 to 16, suggesting that the decreased expression of Th1 cytokines was involved in the impairment of cellular immunity (Fig. 6). But the expression of forkhead transcription factor 3 (Foxp3), IL-10, and TGF-beta mRNA was not influenced as compared with control (Fig. 6). Because Foxp3, IL-10, and TGF-beta are highly related to Treg cell phenotype and function (25, 26, 27, 28, 29), these results indicated that sTim-3 did not influence Treg cells. Taken together, these results suggested that sTim-3 did not influence Treg cells, but otherwise resulted in the profound immune unresponsiveness of CD4+ T effector cells.


Figure 6
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  		FIGURE 6. Effect of sTim-3 on the gene expression in tumor microenvironment. Total RNAs were isolated from half part of tumor tissues together with half part of vicinal muscle tissues of tumor-bearing mice on days 4, 8, 12, and 16 after inoculation of tumor cells. Relative mRNA levels of IL-2, IFN-{gamma}, TNF-beta, Foxp3, IL-10, and TGF-beta were measured with real-time quantitative PCR in five to eight mice from two experiments. The level of each mRNA was expressed as n-fold difference relative to the saline control on day 4 (*, p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The expression of sTim-3 mRNA, the existence of sTim-3 as a soluble protein, and its effect on T cell immune response were determined in this study. In initial experiments, we demonstrated that sTim-3 mRNA was expressed only in spleen cells, but not in bone marrow, thymus, and nonlymphoid organs, suggesting that sTim-3 is involved mainly in immune response and expressed in the lymphocytes that have experienced immune response. This hypothesis was further proved by the experiments in which the expression of sTim-3 mRNA in spleen cells could be up-regulated by repeated stimuli, suggesting that the expression of sTim-3 relied on the activation of lymphocytes and it might play a potentially important role in the regulation of immune response.

We next proceeded to examine the existence of sTim-3’s protein product and its ability to bind the putative ligand(s). In this study, we provided the evidence for the existence of sTim-3, which was produced as a soluble molecule from the activated lymphocytes. In addition, the transfection of cells with the expression vector carrying sTim-3 cDNA also resulted in the production of sTim-3 as a soluble molecule. Furthermore, the analysis with FACS confirmed that sTim-3 could bind to the putative Tim-3L on T cells. Although sTim-3 mRNA in freshly isolated splenocytes was detectable by RT-PCR, a much more sensitive approach, the protein of sTim-3 was not detectable by Western blot, suggesting that only a very low ratio of freshly isolated splenocytes produces sTim-3.

On the basis of the regulatory effect of sTim-3-Ig fusion protein on auto- and alloimmune responses, it was speculated that sTim-3 might act to block the inhibitory effect of Tim-3-Tim-3L interaction, and promote the expansion and differentiation of Th1 cell populations. But when we tried, on the basis of the above speculation, to augment the antitumor immune response by the expression of sTim-3 in vivo, we observed an unexpected result. sTim-3 did not inhibit, but facilitated the growth of tumor in vivo and shortened survival of tumor-bearing mice. The possibility of the direct effect of sTim-3 on the growth of tumor cells was ruled out because B16F1 melanoma cells did not express the putative Tim-3 ligand(s), and the growth of tumor cells in vitro was not influenced in the presence of sTim-3. Thus, we supposed that sTim-3 facilitated tumor growth in vivo by impairing antitumor immunity. The results in our following experiments confirmed that the effect of sTim-3 on immune response is not positive, but negative.

The results of our experiments in vitro showed that the response of T cells to Ag-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation was significantly suppressed in the presence of sTim-3, and the blockade of sTim-3 with anti-Tim-3 Ab recovered the response of T cells to these stimuli, supporting the notion that sTim-3 mediates the inhibitory effect in immune response. Besides the inhibition on the proliferation of T cells, sTim-3 mainly suppressed the production of Th1 cytokines, IL-2 and IFN-{gamma}, suggesting that sTim-3 could inhibit T cell response.

We then attempt to further understand the function of sTim-3 in vivo: we expressed sTim-3 by in vivo transfection of the expression plasmid and observed its effect on T cell antitumor response. The proliferation of T cells from sTim-3-treated mice was significantly reduced when they were restimulated by Ag-loaded DC in vitro, indicating that T cell antitumor immune response was inhibited by sTim-3 in vivo. Moreover, the antitumor CTL activity was significantly reduced by sTim-3, and the amount of T cells in tumor tissue was also significantly decreased. To sum up, these results further confirmed the inhibitory effect of sTim-3 on the generation of Ag-specific T cell immune response.

CD4+ T cells that express Tim-3L could have played a significant role in the suppression of antitumor immunity by sTim-3. CD4+ T effector cells and CD4+ Treg play distinctly different roles in regulating host immune response against cancer. CD4+ effector (helper) T cells are required for the priming and maintenance of CD8+ T cells, thus enhancing the overall immune response (29, 30, 31, 32). Paradoxically, CD4+ Treg cells can profoundly suppress host immune response (33, 34, 35, 36). Real-time RT-PCR analysis of gene expression in tumor microenvironment revealed the decreased expression of Th1 cytokines, which was in good agreement with the results that sTim-3 inhibited T cell proliferation and production of IL-2 and IFN-{gamma} in vitro. The reduced expression of IL-2, IFN-{gamma}, and TNF-beta in the existence of sTim-3 indicated that the activation of CD4+ T cells or the differentiation of naive CD4+ T cells into Th1 cells was suppressed. But the unimpaired expression of Foxp3, IL-10, and TGF-beta indicated that the development and function of CD4+ Treg cells were not influenced by sTim-3. Therefore, the inhibitory effect of sTim-3 on T cell response was due to CD4+ T effector cells rather than CD4+ Treg cells.

Although the precise cellular and molecular interactions involving sTim-3 and its ligand(s) remain to be fully elucidated, our data indicate that the interaction between sTim-3 and the putative Tim-3L might constitute a mechanism through which the activation of naive T cells by Ag is inhibited. The inhibitory effect of sTim-3 could be mainly involved in the initial activation and expansion of naive T cells. Given that the expression of sTim-3 was up-regulated along with the repeated stimulation, the physiological function of sTim-3 might be the prevention of activation of more naive T cells in later phase of immune response so to maintain the balance of immune response.

Tim-3 has been identified as a putative marker for Th1, but not Th2 cells, whereas its role in the positive and negative control of immune responses is just being elucidated (6, 7, 8, 37, 38). To date, the in vivo functions of Tim-3 have remained largely unknown. Our preliminary study on membrane-bound Tim-3 revealed that the expression of Tim-3 in vivo by transfection with expression plasmid in the established mouse B16F1 melanoma induced partially regression of tumor and enhanced T cell response (our unpublished data), suggesting that the effect of Tim-3 on antitumor immunity is a positive one. These results were consistent with a more recent study in which the administration of Tim-3+ Th1 cells facilitated the development of tumor-specific CTL, suggesting that Tim-3+ Th1 cells could be used to induce host antitumor response and inhibit the development and growth of tumor (37). These data offer a new insight into the mechanism through which Tim-3 regulates immune response of T cells, and add an additional level of complexity to the current description of the role of Tim-3 in immunoregulation. All of these data could reach a conclusion that will be quite different from that based on the observation of Tim-3-Ig fusion proteins causing hyperproliferation of Th1 cells and inhibiting the generation of Treg cells. This discrepancy could be possibly attributed to the different structure of Tim-3-Ig and other molecules on Th1 cells that could collaborate with Tim-3, but not sTim-3. It is possible that Tim-3, by collaboration with other molecules on the surface of Th1 cells, could be capable of promoting the activation of naive CD4+ T cells at the initial phase, and activating CD4+ Treg cells at the later phase of immune response. Tim-3-Ig might not have the biological function of either native sTim-3 or Tim-3 because of the absence of cytoplasmic domain and not being anchored on the cellular membrane, but it can influence, by binding to Tim-3L, the function of either native sTim-3 or Tim-3. The further investigation of the function of Tim-3, the potential collaboration of Tim-3 with other molecules on Th1, and the potential regulation of Tim-3 vs sTim-3 mRNA and protein will be needed to reveal the complicated regulatory effects of Tim-3/sTim-3 on immune response in vivo.


   Acknowledgments
 
We are grateful to Mu-Lan Yang, Zhi-Yong Gong, Yi-Nong Zhang, and Mei-Rong Zheng for technical assistance, and Prof. You-Bing Ruan for her help and suggestions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Development Program (973) for Key Basic Research (2002CB513100) of China and the National Natural Science Foundation of China (30471587). Back

2 Address correspondence and reprint requests to Prof. Zuo-Hua Feng or Prof. Gui-Mei Zhang, Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, The People’s Republic of China. E-mail address: fengzhg{at}public.wh.hb.cn;zhanggm58@163.com Back

3 Abbreviations used in this paper: TIM, T cell Ig- and mucin domain-containing molecule; CHO, Chinese hamster ovary; DC, dendritic cell; Foxp3, forkhead transcription factor 3; HSP, heat shock protein; sTim-3, soluble form of T cell Ig mucin 3; Tim-3, T cell Ig mucin 3; Tim-3L, Tim-3 ligand; Treg, T regulatory. Back

Received for publication July 6, 2005. Accepted for publication November 10, 2005.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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