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Involvement of CD166 in the Activation of Human T Cells by Tumor Cells Sensitized with Nonpeptide Antigens¹

Yu Kato^*, Yoshimasa Tanaka^*,, Mikihito Hayashi^*, Katsuya Okawa and Nagahiro Minato^2,*

^* Department of Immunology and Cell Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan, and Horizontal Medical Research Organization, Kyoto University, Sakyo-Ku, Japan

	Abstract

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We previously reported that human V2V2-T cells were activated by many human tumor cell lines treated with pamidronate (PAM) in a TCR-dependent manner. In the present study, we indicated that a synthetic pyrophosphomonoester Ag, 2-methy-3-butenyl-1-pyrophosphate, could directly "sensitize" the tumor cells to activate T cells independently of the host metabolism, while the sensitizing effect of PAM was reported to be dependent on the pharmacological activity. Some exceptional tumor cells that failed to be sensitized by PAM were incapable of activating T cells by the treatment with 2-methy-3-butenyl-1-pyrophosphate either, suggesting a requirement of host factor(s) for the effective T cell activation in addition to the nonpeptide Ags. By screening mAbs against a large panel of tumor cell lines, we found that the expression of CD166 closely paralleled the capacity of activating T cells upon PAM treatment. The transfection of a CD166-negative tumor cell line with CD166 cDNA caused a marked enhancement of the capacity to activate T cells following PAM treatment. On the contrary, down-regulation of the CD166 expression in a CD166-bearing tumor cell line by short hairpin RNA resulted in a significant reduction of PAM-induced T cell-stimulatory activity. T cells expressed CD6, a receptor of CD166, and CD6 and CD166 were recruited together to the center of synapse between T cells and PAM-treated tumor cells, colocalizing with TCR/CD3. The results suggested that the engagement of CD6 with CD166 on tumor cells played an important role in the T cell activation by the tumor cells loaded with nonpeptide Ags either endogenously or exogenously.

	Introduction

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Human V2V2 (also termed as V9V2) TCR-bearing T cells represent the major T cells in the circulation and are activated by microbial nonpeptide Ags such as isopentenyl pyrophosphate (IPP)³ and alkyl amines to proliferate and produce various cytokines, including IFN- and TNF- (1). It is suggested that the T cells play an important role in infection such as tuberculosis in human and primates (2). More recently, synthetic nitrogen-containing bisphosphonate compounds, such as pamidronate (PAM), have been also shown to be capable of activating the same subset of human T cells (3). The activation of human T cells by these nonpeptide compounds is crucially dependent on the V2V2-TCR (4, 5), and rodent T cells lack the activity because of the absence of a particular germline segment of TCR (5). We previously reported that the activation of T cells by PAM in vitro required the presence of accessory cells such as macrophages, while pyrophosphomonoesters could activate the T cells in the absence of accessory cells, suggesting that distinct activation mechanisms might be involved (6). We further indicated that a wide variety of human tumor cell lines pretreated with PAM could efficiently activate T cells to proliferate and produce IFN- in a TCR-dependent manner (7). PAM inhibits the activity of farnesyl pyrophosphate (FPP) synthase (8), and it was reported that the T cell activation by the PAM-treated target cells might be due to the accumulation of endogenous TCR-Ags such as geranyl PP and IPP in the isoprenoid pathway (9). These results raised an intriguing possibility that V2V2-T cells could recognize the cell-associated endogenous nonpeptide Ags and thus might play a role in tumor immunity in human. The exact mechanisms of human T cell activation by PAM-treated tumor cells, however, remain to be elucidated.

We indicated that none of the tumor cells from other species besides human was capable of activating human T cells at all upon the optimal treatment with PAM (10). Considering that the pharmacological effect of PAM is not restricted to human cells, the results suggested that the species-specific cellular interaction might be needed for the activation of T cells other than the accumulation and expression of putative endogenous Ags in the target tumor cells. It was reported that the expression of MHC class I-related chain A on the target cells enhanced their TCR-dependent effector function likely through the interaction with NKG2D on T cells (11). We also reported that LFA-1/ICAM-1-mediated cell adhesion played a role in the activation of T cells by PAM-treated tumor cells (10). However, relatively limited types of tumor cells express these molecules, and thus, they may not represent the dominant host factors required for the activation of T cells by a quite broad range of human tumor cell lines upon PAM treatment (7). In the present study, we attempted to identify the molecules involved in the T cell activation by the majority of the tumor cells loaded with nonpeptide Ags. We first show that a pyrophosphomonoester Ag (PP Ag) can directly "sensitize" the tumor cells extracellularly to activate T cells in the absence of the pharmacological effect. We then provide evidence that CD166 (also called activated leukocyte-cell adhesion molecule) broadly expressed on the human tumor cell lines is a candidate molecule involved in T cell activation and suggest that the engagement of CD6 on T cells with CD166 may play a significant role in T cell activation by the tumor cells loaded with nonpeptide Ags, either endogenously or extracellularly.

	Materials and Methods

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Nonpeptide Ags

A PP Ag, 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP), was synthesized as previously described (12), and PAM and zoledronate (ZOL) were purchased from Nippon Kayaku.

Tumor cell lines and T cell line

Human tumor cell lines were purchased from Health Science Research Resources Bank (Osaka, Japan) and were maintained in the complete RPMI 1640 medium supplemented with 10% FCS. A T cell line was prepared as previously described (7) and maintained in the Yssel’s medium supplemented with 10% human AB serum and 100 U/ml recombinant human IL-2. For "sensitization," the tumor cells were incubated with 100 µM PAM or 100 µM 2M3B1PP at 4 or 37°C for 2 h and washed three times with RPMI 1640 medium supplemented with 0.25% human serum albumin before the coculture with T cells. In some experiments, the tumor cells were fixed with 1% paraformaldehyde (PFA) at room temperature for 15 min, followed by the sensitization. Human T cells (5 x 10⁵/well) were cocultured with such treated tumor cells (5.0 x 10⁵/well) at 37°C.

Flow cytometric analysis and intracellular staining

Multicolor flow cytometric analysis was done by using FACSCalibur (BD Biosciences) as before (7). The Abs included PE-conjugated anti-CD6 mAb (BD Pharmingen), FITC-conjugated anti-TCR V2 mAb (Immunotech), anti-ICAM-1 mAb (BD Pharmingen), and anti-CD166 mAb (Ancell). Intracellular staining for IFN- was done as before (7). Briefly, T cells were incubated with brefeldin A (10 µg/ml), washed three times with PBS, and stained with FITC-conjugated anti-TCR-V2 mAb for 20 min. The cells were then fixed with 1% PFA, permeabilized with 0.5% saponin, and stained with PE-conjugated anti-IFN- mAb (BD Pharmingen), followed by FACS analysis.

Proliferation assay

Tumor cells pretreated with 100 µM PAM for 2 h were incubated with 100 µg/ml mitomycin C at 37°C. The cells were washed three times with modified RPMI 1640 medium supplemented with 0.25% human serum albumin. T cells (1.0 x 10⁵) were cultured in the absence or presence of varying numbers of mitomycin C-treated tumor cells for 48 h, pulsed with 2 µCi of [³H]thymidine during the last 12 h, and the incorporation of radioactivity was counted by a scintillation counter.

Immunoblotting and immunoprecipitation

Tumor cells were lysed with TNE lysis buffer (50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 10 mM EDTA, 1% Triton X-100, phosphatase inhibitors, and protease inhibitor mixture) and immunoblotted with Abs, including anti-Rap1 mAb (Santa Cruz Biotechnology), anti-unprenylated Rap1A mAb (Santa Cruz Biotechnology), GAPDH (Santa Cruz Biotechnology), and newly developed mouse mAbs as described before (13). For immunoprecipitation, the tumor cell lysates precleared with protein A-Sepharose beads (Amersham Biosciences) were incubated with 3 µg of a newly established mAb (872-1) and precipitated with protein A-Sepharose beads. CD166-Fc fusion protein was purchased from R&D Systems.

Immunostaining

Tumor cells either treated or untreated with 100 µM PAM were incubated with human T cells for 30 min. The cell mixtures were fixed with 3% PFA at room temperature for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and then blocked with 3% BSA for 15 min. They were double stained with anti-CD6 mAb/Cy3-conjugated anti-goat IgG (Invitrogen Life Technologies) vs anti-TCR-V2 mAb/Alexa Fluor 488-conjugated anti-mouse IgG, or anti-CD166 mAb/Alexa Fluor 555-conjugated anti-mouse IgG vs FITC-conjugated anti-CD3 mAb. 4',6'-Diamidino-2-phenylindole was used for nuclear staining. The cells were analyzed by fluorescence microscopy (Carl Zeiss). The proportions of T cells forming the synaptic and nonsynaptic conjugates with tumor cells as judged by the recruitment of CD3 into the center of adhesion sites were calculated by counting a total of a hundred T cells. Three independent determinations were performed.

Establishment of mAbs

BALB/c mice were immunized four times with EJ-1 cells, and the spleen cells were fused with SP2/0 cells by a standard polyethylene glycol method. The hybridoma supernatants were screened for the reactivity against a panel of 21 human tumor cell lines by using FACSCalibur.

Mass spectroscopic analysis

LK-2 cells (total 5.0 x 10⁸ cells) were incubated in hypotonic buffer (10 mM HEPES-KOH (pH 7.4), 5 mM KCl, and 2 mM MgCl₂), followed by homogenization in a Dounce homogenizer. After ultracentrifugation at 100,000 x g for 1 h at 4°C, the pellet was extracted with TNE lysis buffer. The extract was immunoprecipitated as described above, run on SDS-PAGE, and the gels were stained with the silver staining method. The specifically immunoprecipitated band at 105 kDa was cut into four pieces and excised separately from the gel. After in-gel digestion with trypsin (Promega) in a buffer containing 50 mM ammonium bicarbonate (pH 8.0) and 2% acetonitrile at 37°C overnight, molecular mass analyses of the tryptic peptides were conducted by MALDI-TOF mass spectroscopy (MS) using an Ultraflex TOF/TOF (Bruker Daltonics).

cDNA and short hairpin RNA (shRNA) transfection

CD166 cDNA was cloned by RT-PCR from the total RNA of VMRC-RCW tumor cells using the primers; 5'-CGTCAGTGGCCCACCAAGAA-3' and 5'-TCTCTGGACAACTAGGACAG-3', and subcloned into pcDNA3.1(+) vector (Invitrogen Life Technologies). The pcDNA-CD166 or empty pcDNA vector was transfected into K562 cells by electroporation at 230 V for 65 ms using ECM830 (BTX). After 2 days, the cells were cultured in the selection medium containing 1 mg/ml G418 (Nacalaitesque) in 96-well round-bottom plates. The expression level of CD166 was confirmed by using FACSCalibur. shRNA vector was constructed by inserting the relevant oligonucleotides into ApaI and EcoRI sites of pCl-neo Silencer vector (Toray Industries). The shRNA target sequence for CD166 was 5'-GCATATGGAGATACCATTATC-3'. LK-2 tumor cells were transfected with the shRNA vector with Lipofectamine 2000 reagent (Invitrogen Life Technologies) and incubated in the presence of 1 mg/ml G418. The cells expressing the reduced expression level of CD166 were sorted using FACSVantage. Total RNA of the sorted transfectants was prepared using ISOGEN (Nippon Gene), and the CD166 transcript was examined by RT-PCR using SuperScript III reverse transcription kit (Invitrogen Life Technologies) according to the manufacturer’s protocol. Primers were 5'-CAATATCACATGGTACAGGA-3' and 5' TGCTTGAACACCTTGACT-3' for CD166, and 5'- GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3' for the control GAPDH.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Human tumor cells loaded with nonpeptide Ags may require the additional host cell component(s) for inducing effective T cell activation

We first compared PAM and a synthetic PP Ag, 2M3B1PP, in the ability to sensitize the tumor cells for T cell activation. Although EJ-1 bladder tumor cells preincubated with PAM at 37°C for 2 h potently stimulated the T cells to produce IFN-, those preincubated with PAM at 4°C or fixed with PFA before the PAM treatment showed markedly reduced T cell-stimulatory activity (Fig. 1A). This was consistent with the requirement of the pharmacological effect of PAM for the sensitization. In quite a contrast, the tumor cells incubated with 2M3B1PP at 4°C or those prefixed with PFA, followed by the incubation with 2M3B1PP, showed comparable or even enhanced T cell-stimulatory activity as compared with those treated at 37°C (Fig. 1A). The results suggested that, unlike PAM, extracellular binding of 2M3B1PP to the tumor cell per se might be sufficient to sensitize the tumor cells. 2M3B1PP, however, still failed to sensitize a gastric cancer cell line, MKN45, that was incapable of being sensitized with PAM, while it could sensitize another tumor cell line, LK-2 (Fig. 1B). We then compared the pharmacological activity of PAM to inhibit FPP synthase in LK-2 with that in MKN45 by examining the accumulation of unprenylated Rap1A. Treatment of MKN45 cells with PAM, ZOL, or isobutyl amine (IBA) resulted in the accumulation of unprenylated Rap1A comparably to LK-2 cells (Fig. 1C), and thus it was unlikely that the lack of PAM-sensitization in MKN45 cells was due to the defective pharmacological effect. Consistent with the direct extracellular sensitization of tumor cells by 2M3B1PP, the treatment with 2M3B1PP caused no accumulation of unprenylated Rap1A at all in both types of tumor cells (Fig. 1C). These results collectively suggested that certain host cell factor(s) was required for the effective activation of T cells by tumor cells other than the nonpeptide Ags, irrespective of whether the Ags were derived endogenously or exogenously, and that unsensitizable tumor cell lines might intrinsically lack such factor(s).

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Requirement of host factor(s) in addition to nonpeptide Ags for the stimulation of T cell activation by tumor cells. A, Viable or PFA-prefixed EJ-1 tumor cells were incubated with medium at 37°C, or with 2M3B1PP (100 µM) or PAM (100 µM) at 37 or 4°C for 2 h. These tumor cells (5 x 105/well) were cocultured with T cells (5 x 105/well) for 4 h, and the recovered cells were two-color stained with anti-TCR V2 and ant-IFN- mAbs. The proportions of intracellular IFN-+ T cells are indicated. B, LK-2 (lung cancer) and MKN45 (gastric cancer) cell lines treated with medium, PAM, or 2M3B1PP at 37°C for 2 h were cocultured with T cells, and the proportions of intracellular IFN-+ T cells were determined as above. C, LK-2 and MKN45 cells were treated with medium, PAM (100 µM), ZOL (10 µM), 2M3B1PP (100 µM), or IBA (5 mM) at 37°C for 12 h, and the cell lysates were immunoblotted using the Abs specific for unprenylated Rap1A, Rap1, and GAPDH. The concentrations of reagents were the nontoxic optimal doses for obtaining the sensitizing effects.

Identification of CD166 as a candidate host factor involved in the T cell activation upon PAM treatment

In an attempt to identify the cell surface components on tumor cells involved in the activation of T cells, we generated mAbs against an EJ-1 tumor cell line, one of the most efficiently sensitizable cell lines, and screened across a panel of 21 human tumor cell lines, which included 13 lines capable of potently stimulating the T cells by PAM treatment (group A), 5 lines that weakly stimulated T cells (group B), and 3 lines incapable of doing so (group C) (Table I). After several rounds of screening by FACS analysis, we established three candidate mAbs among >5000 clones, 24-6, 819-31, and 872-1. The three mAbs showed the identical reactivity profiles to the panel of tumor cell lines in that they reacted to all the 13 lines of group A with no exception and 4 of 5 lines in group B, while they reacted to only 1 of the 3 lines in group C. Since the reactivity profile of the mAbs showed a fairly good, if not complete, correlation with the capacity of activating T cells upon PAM treatment, we proceeded to the identification of the Ag. Immunoblotting of the total as well as 872-1 mAb-immunoprecipitated ACHN tumor cell lysates with 24-6 mAb revealed a broad band at 105 kDa suggestive of a highly glycosylated protein, whereas no specific band was detected at all in the lysate of MRK-nu-1 cells (Fig. 2A). We then identified the cryptic peptide sequences of the immunoprecipitated bands by time of flight (TOF)/MS analysis. Since the specific signal was detected as a broad band, the region was cut into four pieces from top to bottom, and each part was digested with trypsin, followed by the analysis on MALDI-TOF/MS. The results indicated that the peptide sequences corresponding to CD166 were commonly detected in all the four sample pieces (Fig. 2B). To confirm the result, we examined the reactivity of 24-6 mAb to the CD166/Fc chimera protein by immunoblotting. As shown in Fig. 2C, 24-6 mAb clearly reacted to the CD166/Fc chimera protein, indicating that the newly established mAbs recognized CD166 expressed on the tumor cell surface. Consistently, FACS analysis of the panel of tumor cell lines with an anti-CD166 mAb showed the identical pattern to the three mAbs (Table I), indicating that all the tumor cell lines in groups A and B exhibited the CD166 expression, except for a line, K562. It was noted that K562 cells strongly expressed ICAM-1, which we previously reported to play a role in the activation of T cells by PAM-sensitized tumor cells (10).

View larger version (28K): [in this window] [in a new window]   FIGURE 2. CD166 as a candidate Ag selectively expressed on the tumor cells capable of activating T cells upon PAM treatment. A, Total lysate of ACHN (renal cancer) cells of group A and MRK-nu-1 (breast cancer) cells of group C was immunoprecipitated with mAb 872-1, followed by immunoblotting with mAb 24-6. B, The membrane fragment-enriched cell lysate of LK-2 tumor cells were immunoprecipitated with mAb 872-1 and silver stained. The specific broad band at 105 kDa was cut into four pieces, and each sample was subjected to the in-gel digestion with trypsin followed by the MALDI-TOF/MS analysis. The identified proteins from the tryptic peptides in each gel are indicated. C, A CD166-Fc fusion protein was loaded at 1, 10, and 100 µg per lane and immunoblotted with mAb 24-6.

CD166 is functionally involved in the activation of T cells by tumor cells treated with PAM

We then investigated the functional involvement of CD166 in the activation of T cells by the PAM-treated tumor cells. Since our extensive attempt to obtain stable CD166 transfectants of the CD166^– tumor lines in group C failed, we generated the stable CD166 transfectants using K562 cells, which also lacked CD166 expression (Fig. 3A). The sorted CD166 transfectants of K562 cells (K562/CD166) and those transfected with an empty vector (K562/cont) were incubated in the absence or presence of PAM for 2 h at 37°C, washed, treated with mitomycin C, and cocultured with the T cells. The PAM-treated K562/CD166 cells exhibited markedly enhanced activity to stimulate T cells, compared with the similarly treated K562/cont cells in terms of both proliferative responses and IFN- production, whereas the untreated transfectants failed to induce detectable T cell activation (Fig. 3B). Using three independently cloned K562/CD166 cells, essentially the same results were obtained (data not shown). We next investigated whether the endogenous CD166 was involved in the activation of T cells. For this purpose, we used LK-2 lung tumor cells, which expressed significant CD166 in the absence of detectable ICAM-1. LK-2 cells were transfected with a CD166 shRNA vector and selected with G418 (LK-2/shCD166). LK-2/shCD166 cells exhibited significantly, if not completely, reduced CD166 transcript as well as the surface expression as compared with the control cells transfected with an empty vector (Fig. 3C). When cocultured with T cells, the PAM-treated LK-2/shCD166 cells induced significantly compromised proliferation of T cells as compared with LK-2/cont cells (Fig. 3D). Taken together, these results indicated that CD166 broadly expressed on the tumor cells played a role in activating T cells upon PAM treatment.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. CD166 is functionally involved in the activation of T cell by the PAM-treated tumor cells. A, K562 cells were transfected with pcDNA-CD166 (K562/CD166) or empty vector (K562/cont) and cultured in the selection medium. Twelve days later, the cells were recovered and analyzed for the expression of CD166 by FACS. The mean fluorescence intensities (MFI) are indicated. The line indicates the MFI of the isotype-matched control IgG staining. B, K562/cont and K562/CD166 cells were treated with medium or PAM (100 µM) and cocultured with T cells. The proliferation of T cells was assessed 48 h later (left), and the proportions of IFN-+ T cells were determined 12 h later (right). The SEs of triplicate cultures are indicated for the former. C, LK-2 cells were transfected with pCl-neo Silencer vector containing shRNA target primer for CD166 (LK-2/shCD166) or vector alone (LK-2/cont) and cultured in the selection medium. Two weeks later, the cells were harvested, and the CD166 low fraction was sorted by using FACS Vantage for the LK-2/shCD166 cells. Total RNAs were extracted, and CD166 as well as control GAPDH transcripts were detected by RT-PCR (left). Surface expression of CD166 was determined by FACS analysis, the MFI being indicated (right). The line indicates the MFI of the isotype-matched control IgG staining. D, LK-2/cont and LK-2/shCD166 cells were treated with medium or PAM (100 µM), cocultured with T cells, and the proliferation of T cells was determined. The SEs of triplicate cultures are indicated.

T cells constitutively express CD6, a receptor of CD166, and both CD6 and CD166 colocalize with CD3/TCR at the central sites of synapses between T cells and PAM-treated tumor cells

It was shown that CD166 functioned as a ligand for CD6, and therefore, we examined the expression of CD6 on human T cells. FACS analysis revealed that both freshly isolated primary and in vitro activated V2⁺ T cells strongly expressed CD6 (Fig. 4A). We then investigated the possible involvement of CD6 and CD166 in the interaction between T cells and PAM-treated tumor cells. T cells and LK-2 or MKN45 tumor cells either untreated or pretreated with PAM were incubated for 30 min at 37°C and then fixed followed by immunostaining. While 15% of the T cells formed the conjugates with untreated LK-2 cells, only a negligible proportion (<2%) of them showed the synaptic interaction associated with the recruitment of TCR/CD3 complex into the adhesion sites (Figs. 4B and 5A). On the other hand, significant proportion (20%) of the T cells exhibited the synaptic interaction with PAM-treated LK-2 cells with the nonsynaptic interaction being increased only slightly (Figs. 4B and 5B). In contrast, few synaptic conjugate formations were observed between T cells and PAM-treated MKN45 cells, albeit rather abundant nonsynaptic conjugates (Fig. 4B). The same experiments were done using K562/cont and K562/CD166 cells. Consistent with the high expression of ICAM-1, 30% of T cells formed the nonsynaptic conjugates with both PAM-treated K562/cont and K562/CD166 cells. However, the proportion of T cells forming the synaptic conjugates was significantly greater in the latter than in the former (20 vs 8%), paralleling the enhanced activation of T cells (Fig. 4C). These results strongly suggested that the synaptic conjugate formation was required for the activation of T cells by tumor cells. In the nonsynaptic conjugates with untreated tumor cells, the T cells showed diffuse expression of CD6, and also the LK-2 tumor cells revealed diffuse and thus quite faint staining of CD166 with no evidence for the CD6/CD166 interaction (Fig. 5A). In the synaptic conjugates with PAM-treated LK-2 cells, however, CD6 of T cells was specifically recruited to the center of synaptic sites colocalizing with TCR/CD3 (Fig. 5B). Furthermore, it was noted that CD166 on the PAM-treated LK-2 cells exhibited a patchy expression pattern and a portion of it also colocalized with CD3 at the synaptic sites (Fig. 5B). The results strongly suggested that the engagement of CD6 with CD166 took place at the synaptic sites and played a part in the TCR/CD3-mediated activation of T cells by PAM-treated tumor cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Expression of CD6, a receptor of CD166, on T cells and correlation of the synaptic conjugate formation with the activation of T cells by PAM-treated tumor cells. A, Freshly isolated normal PBMC (upper) and those cultured with 2M3B1PP and IL-2 for 2 wk (lower) were two-color stained with anti-CD6 and anti-TCR V2 mAb. B and C, LK-2, MNK45, K562/cont, and K562/CD166 cells treated with medium or PAM (100 µM) were incubated with T cells for 30 min, fixed, and double stained with anti-V2 TCR and anti-CD6 mAbs. A total of 100 T cells was examined under the fluorescence microscopy, and the proportions of T cells forming the nonsynaptic conjugates with the tumor cells with no apparent redistribution of TCR () and the synaptic conjugates (), in which TCR was recruited into the center of adhesion sites, were determined. The averages of three independent experiments are indicated.

View larger version (68K): [in this window] [in a new window]   FIGURE 5. CD6 and CD166 are recruited together into the center of synaptic sites between T cells and PAM-treated tumor cells and colocalize with TCR/CD3. LK-2 cells treated with medium (A) or 100 µM PAM (B) were cocultured with T cells for 30 min, fixed, and double stained with anti-V2 TCR vs anti-CD6 mAbs or anti-CD3 vs anti-CD166 mAbs. Nuclei were additionally stained with 4',6'-diamidino-2-phenylindole (blue). Arrowheads indicate the synaptic adhesion sites. *, T cells. Magnification, x1000.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In this study, we identified CD166 as a candidate of the putative host factors required for the T cell activation on the basis of the expression profile in a large panel of human tumor cell lines. Thus, CD166 was expressed in 17 of the 18 lines capable of activating T cells upon PAM treatment. On the other hand, two of three incompetent tumor cell lines lacked the expression of CD166. Functional involvement of CD166 in the activation of T cells was demonstrated by two independent experiments. First, the introduction of CD166 cDNA into K562 cells, which lacked the endogenous CD166 expression, markedly enhanced the activity to induce T cell proliferation only upon PAM treatment. K562 cells are typical NK cell targets expressing ICAM-1, and thus, it might be possible that the weak T cell activation by K562 cells reflected the TCR-independent "NK-like" activity of T cells. Consistently, PAM-treated K562 cells could efficiently form the conjugates with T cells in the absence of a significant level of synaptic interaction and TCR recruitment, whereas the proportion of synaptic conjugates was markedly increased by the introduction of CD166. In any case, the marked enhancement of sensitizing effect by CD166 in the target cells strongly expressing ICAM-1 suggested the nonredundant roles of the two adhesion molecules, CD166 and ICAM-1 (see below). Second, the down-regulation of endogenous CD166 expression in LK-2 tumor cells by means of shRNA resulted in the significantly compromised activity to stimulate the T cell proliferation upon PAM treatment. The reduction of CD166 expression by shRNA was incomplete, and accordingly, the decline in T cell-stimulatory activity was partial. Nonetheless, the results reinforced the functional involvement of CD166 on the tumor cells in the activation of T cells by PAM treatment. One of the tumor cell lines in the group C did express CD166, and the reason why the tumor cell line was still incapable of activating T cells remained to be seen. It may be possible that these tumor cells additionally express negative receptors for the TCR signaling (16). Alternatively, these rare tumor cells may have a defect in "presenting" the extracellular and intracellular nonpeptide Ags on the cell surface to be recognized by T cells, although the possible presentation mechanisms remain totally unknown at present.

CD166 is a member of the Ig superfamily expressed on a variety of normal as well as tumor cells (17, 18, 19). While CD166 may mediate homotypic cell adhesion (20), it also functions as a ligand for CD6 receptor (17, 21). CD6 is a member of scavenger receptor cysteine-rich protein superfamily expressed on T cells and some B cells (22, 23) and was suggested to be involved in the thymocyte adhesion to thymic epithelial cells as well as T cell activation (24, 25, 26, 27, 28, 29). More recently, it was reported that the blockade of the engagement between CD6 on human T cells and CD166 on monocytes by soluble extracellular monomeric proteins significantly interfered with the Ag specific T cell activation, indicating that the CD6/CD166 interaction played a role in the optimal T cell activation by Ags (30, 31). Present results indicated that all the human V2V2 T cells, either primary or activated, also strongly exhibited the CD6 expression. T cells formed the synaptic conjugates with PAM-treated, but not untreated, tumor cells, in which TCR/CD3 was recruited specifically into the center of adhesion sites, reminiscent of the interaction of T cells with the specific Ag-loaded APC (32). It was further indicated that CD6 on T cells and CD166 on PAM-treated tumor cells were also recruited to the center of synaptic sites colocalizing with TCR/CD3. Since CD6 was shown to associate with Lck, Fyn, Zap-70, and Tec family kinases (28), the results strongly suggested that the extracellular engagement of CD6 by CD166 might be involved in the activation of T cells at the center of synaptic sites. T cells exhibited only marginal CD166 expression, and also the binding affinity of homotypic CD166 was reported to be much lower than that of CD166 and CD6 (30), and thus, the possible involvement of CD166 homotypic interaction would be marginal, if any. Based on the present results, we propose that LFA-1/ICAM-1 interaction plays a role in establishing the stable adhesion between T cells and tumor cells loaded with nonpeptide Ags, and the engagement of CD6 with CD166 on the target cells in the synaptic sites provides the costimulatory signal for TCR activation (Fig. 6). So far, our attempt to block the T cell activation with available anti-CD6 mAb was unsuccessful, and we are currently generating the soluble extracellular domain of CD6 for the blocking.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. A schematic model for the activation of human T cells by the tumor cells sensitized with nonpeptide Ags. Human T cells are activated by either exogenous PP Ags or putative endogenous PP Ags accumulated by the pharmacological activity of nitrogen-containing bisphosphonates such as PAM or alkyl amines such as IBA to inhibit FPP synthase. Although exact mechanisms for the display of PP Ag on the tumor cell surface remain unknown, we propose that the T cells directly recognize such presented Ags via TCR based on the previous reports (12 34 ). The TCR/PP Ag interaction initiates the formation of immunological synapses between T cells and the sensitized tumor cells in analogy with T cells. LFA-1/ICAMs interaction may play a dominant role in the stable cell adhesion and synapse formation (10 ). Present results indicate that both CD166 expressed on the tumor cells and CD6 on the T cells are together recruited into the center of immunological synapse, colocalizing with TCR/CD3 complex. We propose that CD6 engaged with CD166 at the synapse provides the costimulatory signal for TCR-mediated T cell activation.

	Disclosures

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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 a grant-in-aid for scientific research from the Ministry of Education, Science, Culture, Sports, and Technology.

² Address correspondence and reprint requests to Dr. Nagahiro Minato, Department of Immunology and Cell Biology, Graduate School of Biostudies, Kyoto University, Yoshidakonoe-Cho, Sakyo-Ku, Kyoto 606-8501, Japan. E-mail address: minato{at}imm.med.kyoto-u.ac.jp

³ Abbreviations used in this paper: IPP, isopentenyl pyrophosphate; PAM, pamidronate; FPP, farnesyl pyrophosphate; PP Ag, pyrophosphomonoester Ag; 2M3B1PP, 2-methy-3-butenyl-1-pyrophosphate; ZOL, zoledronate; PFA, paraformaldehyde; MS, mass spectroscopy; shRNA, short hairpin RNA; IBA, isobutyl amine; TOF, time of flight; MFI, mean fluorescence intensity.

Received for publication February 28, 2006. Accepted for publication May 4, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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