International Immunology

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International Immunology, Vol. 12, No. 1, 51-55, January 2000
© 2000 Japanese Society for Immunology

AILIM/ICOS: a novel lymphocyte adhesion molecule

Takuya Tamatani, Katsunari Tezuka and Naomi Hanzawa-Higuchi

Pharmaceutical Frontier Research Laboratories, JT Inc., 1-13-2 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan

Correspondence to: T. Tamatani

	Abstract

Top Abstract Introduction Methods Results and discussion References

 
The adhesive interaction between T cells and antigen-presenting cells is required for the formation of the immunological synapse. Inducible co-stimulator (ICOS) is a third member of the CD28 family of co-stimulatory molecules. Here we describe a novel lymphocyte adhesion molecule, of relative molecular mass 47,000, designated AILIM, that is a rat homolog of ICOS. Rat AILIM was constitutively expressed on thymocytes and was induced on naive T cells after activation. Human thymoma cells bound to purified AILIM. Furthermore, cells transfected with the AILIM gene aggregated in an AILIM-dependent manner. These results suggest a novel function for AILIM/ICOS as an adhesion molecule, which plays an important role in T cell activation.

Keywords: adhesion molecule, AILIM, CD28, co-stimulator, ICOS

	Introduction

Top Abstract Introduction Methods Results and discussion References

 
Efficient T cell activation requires the formation of a specialized contact area between T cells and antigen-presenting cells termed the immunological synapse (1,2). This area consists of a TCR co-stimulators including adhesion molecule(s). Although the interaction of CD28 with the ligands CD80 (B7-1) (3) and CD86 (B7-2) (4,5) has been thought to be a major co-stimulatory pathway, the development of thymocytes is normal in CD28-deficient (6) and CD80-deficient (7) mice, and T cell selection in normal mice is not blocked by inhibiting the CD28 pathway (8,9), suggesting that alternative co-stimulatory pathways may exist.

Recently, Hutloff et al. reported a third member of the CD28 family, ICOS (inducible co-stimulator) (10). This molecule is induced on naive T cells after activation and can act as a co-stimulator in T cell activation. Engagement of ICOS does not up-regulate the production of IL-2, but induces the synthesis of IL-10. Additionally, this molecule is highly expressed on tonsillar T cells of the germinal center, suggesting that ICOS may play an important role in the terminal differentiation of B cells.

In this report, we have identified a novel adhesion molecule, AILIM, that belongs to CD28 gene family. A comparison of the predicted amino acid sequence demonstrated that AILIM is a rat homolog of human ICOS. Here, we describe the function and molecular characteristics of rat AILIM/ICOS.

	Methods

Top Abstract Introduction Methods Results and discussion References

 
Cells preparation and mAb
Rat thymoma cell line FTL43 cells (11) and human thymoma cell line HPB-ALL were grown in RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin and 10% FCS. Concanavalin A blasts were generated by incubating rat spleen cells with 2 µg/ml concanavalin A (Sigma, St Louis, MO) for 3 days. mAb used were anti-rat CD80 (3H5; mouse IgG1) (12) and anti-rat CD86 (24F; mouse IgG1) (12).

Generation of mAb
Female BALB/c mice were immunized every 5–7 days with a footpad injection of 2–5x10⁶ viable FTL43 cells. Complete Freund's adjuvant was included in the first inoculum. The booster injection was made in the footpad 2 days before the fusion. Popliteal lymph node cells were fused with mouse myeloma cells, PAI, using PEG 4000 and hybridomas were screened by their ability to induce or inhibit FTL-43 cell aggregation. All experiments followed the institutional guidelines for the ethical treatment of animals.

Immunoprecipitation
Cell-surface proteins were biotinylated by using NHS-biotin (Pierce, Rockford, IL) and solubilized in lysis buffer containing 1% NP-40. The lysates were immunoprecipitated with mAb-conjugated Protein G–Sepharose beads. The precipitates were eluted with elution buffer (0.17% SDS, 0.2 M sodium phosphate, pH 8.0 and 1.25% NP-40) and boiled for 5 min. Some precipitates were treated with N-glycanase (20 U/ml) for 24 h at 37°C. Immunoprecipitates were boiled in Laemmli sample buffer with or without 5% 2-mercaptoethanol and subjected to SDS–PAGE (10–20% gradient gels). The gels were blotted onto PVDF membranes. The membranes were soaked in blocking reagents and incubated with peroxidase-conjugated avidin of the Vectastain Elite ABC kit (Vector, Burlingame, CA). Biotinylated proteins were visualized with an enhanced chemiluminescence (ECL) detection system (Amersham, Little Chalfont, UK).

Transfectants
For construction of a rat AILIM expression vector, a cDNA fragment of rat AILIM (GeneBank accession no. AB023134) was cloned into pEFneo vector. This expression vector was transfected into HPB-ALL cells by electroporation using a Gene Pulser (BioRad, Hercules, CA).

Cell aggregation assay
Cells were suspended at a concentration of 2.5–5x10⁶/ml. To flat-bottom 96-well microtiter plates, 50 µl of mAb and 50 µl of cells were added. Cells were incubated for 1 h at 37°C and the degree of cell aggregation was determined with an inverted phase-contrast microscope.

Purification of rat AILIM antigen
FTL43 cells (1x10¹⁰ cells) were solubilized in 200 ml of lysis buffer (10 mM Tris–HCl, pH 7.4, 0.15 M NaCl, 0.2 mM PMSF and 1% NP-40) at 4°C for 30 min. Insoluble material was removed by centrifugation. The lysate was preabsorbed with non-specific mAb coupled Protein G–Sepharose (Pharmacia). The cell lysate was incubated with 1 ml of mAb JTT.1-coupled Protein G–Sepharose beads at 4°C overnight with constant agitation. Beads were washed with several volumes of lysis buffer and PBS. The bound rat AILIM antigens were eluted with 0.2 M glycine buffer (pH 2.8). Eluates were immediately neutralized by the addition of 1/3 volume of 1 M Tris–HCl (pH 8.0), and fractions containing the eluted AILIM (1 ml) were identified by silver staining of SDS–PAGE and then pooled.

Binding of human thymoma cells to AILIM-coated plate
Wells of 96-well ELISA microplates (Corning, Corning, NY) were treated with 50 µl of purified rat AILIM antigens (5-fold diluted with PBS)/well for 24 h at 4°C. The wells were washed and blocked with 100 ml of 3% BSA/PBS for 4 h. After washing, the wells were treated with mAb (20 µg/ml, 50 µl/well) for 30 min. HPB-ALL cells were added to each well in a final volume of 100 µl (5x10⁴ cells/well) and incubated at 37°C for 1 h. Subsequently, the wells were filled with medium and the plates were inverted for 15 min to remove non-adherent cells. Supernatants were removed and the degree of cell binding was determined with an inverted phase-contrast microscope.

	Results and discussion

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We generated mAb to molecules expressed in the rat thymoma cell line FTL43 and identified a mAb (JTT.1; mouse IgG1) which markedly induced FTL43 cell aggregation (Fig. 1A). This aggregation was induced by the JTT.1 Fab fragment (Fig. 1A) and was not induced at 4°C (data not shown), suggesting that the aggregation was not induced by a cross-bridge of antigens recognized with the mAb. mAb against rat CD80 and CD86 were unable to block the aggregation (Fig. 1A). To examine the mechanism of the aggregation, we generated an aggregation-blocking mAb by immunizing FTL43 cells and identified a mAb (JTT.2; mouse IgG1). The mAb completely blocked JTT.1-induced FTL43 cell aggregation (Fig. 1A).

View larger version (73K): [in this window] [in a new window]   Fig. 1. Characterization of AILIM using mAb. (A) Induction and inhibition of rat thymoma cell aggregation by mAb against AILIM. Phase contrast photomicrographs of rat thymoma FTL43 cells aggregating in the presence of no mAb (cont.), mAb JTT.1 (5 µg/ml), JTT.1 Fab (5 µg/ml), JTT.1 Fab + JTT.2 (10 µg/ml), JTT.1 Fab + anti-rat CD80 (10 µg/ml) or JTT.1 Fab + anti-rat CD86 (10 µg/ml). (B) Immunoprecipitation of rat AILIM. Lysates of biotinylated FTL43 cells were immunoprecipitated with isotype-matched control mAb (lanes 1 and 4) or mAb JTT.1 (lanes 2, 3, 5 and 6) and subjected to SDS–PAGE under reducing conditions (lanes 1–3) or non-reducing conditions (lanes 4–6). In lanes 3 and 6, antigens were treated with N-glycanase. (C) Expression of AILIM on rat leukocytes. Each cell population was stained with mAb JTT.1 or JTT.2, followed by staining with FITC-conjugated anti-mouse IgG (Cappel, Durham, NC). The stained cells were assayed for fluorescence intensity on an Epics Elite flow cytometer (Coulter Electronics, Hialeah, FL). We analyzed a total of 10,000 viable cells for each sample. The results shown here are representative of three independent experiments.

 
Immunoprecipitates obtained from FTL43 cells with mAb JTT.1 were analyzed by SDS–PAGE (Fig. 1B). The precipitates migrated as a single band of 47 kDa under non-reducing conditions, and as two bands of 24 and 28 kDa under reducing conditions. N-glycanase treatment of the precipitates revealed a single band of 20 kDa, suggesting that the antigen may be a disulfide-linked homodimer with the two molecules differing only in the way that they are glycosylated. Identical results were obtained with mAb JTT.2.

A flow cytometric analysis revealed that JTT.1 antigens were constitutively expressed on FTL43 cells and thymocytes (Fig. 1C). Although naive lymphocytes expressed little or no JTT.1 antigens, the expression was strongly induced by concanavalin A stimulation. The antigen was not detected on neutrophils. The expression pattern was identical to that of JTT.2 (Fig. 1C). These results indicate that mAb JTT.1 and JTT.2 recognize the same molecule and the molecule itself may be an adhesion molecule. We designated the novel adhesion molecule as AILIM (activation-_inducible _lymphocyte immunomodulatory molecule).

We isolated cDNA clones encoding rat AILIM (GeneBank accession no. AB023134; manuscript in preparation). A comparison of the predicted amino acid sequence of AILIM with the GenBank database demonstrated that AILIM is a member of the CD28/CTLA-4 family. The amino acid sequence of rat AILIM was 67% identical to that of human ICOS, which was recently reported as an inducible T cell co-stimulator (10), suggesting that AILIM is the rat ICOS homolog.

To directly evaluate the involvement of AILIM in lymphocyte adhesion, we analyzed the binding of cells of the human thymoma cell line HPB-ALL, which do not express human AILIM, CD80 or CD86 (data not shown), to affinity-isolated rat AILIM antigens (Fig. 2A). We used mAb JTT.1-affinity chromatography to purify AILIM antigens from detergent lysates of FTL43 cells. HPB-ALL cells adhered efficiently to plate-bound AILIM. The binding was specifically inhibited with aggregation-blocking anti-AILIM mAb JTT.2. mAb JTT.1 was not required for this binding.

View larger version (59K): [in this window] [in a new window]   Fig. 2. (A) Binding of human thymoma cells to AILIM-coated wells. Phase contrast photomicrographs of HPB-ALL cell binding to BSA- (non-coated) or rat AILIM-coated wells in the presence of no mAb or mAb JTT.2 (10 µg/ml). (B) Flow cytometry analysis of HPB-ALL cells or AILIM-transfected HPB-ALL cells (HPB-AILIM). Cells were stained with isotype-matched control mAb 3.30.1 (cont.) (16), mAb JTT.1 or JTT.2, followed by staining with FITC-conjugated anti-mouse IgG. The stained cells were assayed as described in Fig. 1(C). (C) Aggregation of AILIM transfectants in an AILIM-dependent manner. Phase contrast photomicrographs of HPB-ALL cells or AILIM-transfected HPB-ALL cells (HPB-AILIM) aggregating in the presence of no mAb (cont.), mAb JTT.1 Fab (5 µg/ml) or JTT.1 Fab + JTT.2 (10 µg/ml). The results shown here are representative of three independent experiments.

 
To further examine the adhesion pathway, we transfected HPB-ALL cells with a full-length AILIM cDNA. Both JTT.1 and JTT.2 mAb specifically reacted with the transfectants (HPB-AILIM) (Fig. 2B). We analyzed the effects of aggregation-inducing mAb JTT.1 (Fig. 2C). The transfectants were aggregated with addition of JTT.1 Fab fragments, but non-transfectants were not. The aggregation was specifically blocked with mAb JTT.2. These results indicate that AILIM acts as an adhesion molecule. AILIM-dependent FTL43 cell aggregation induced by JTT.1 was not blocked with mAb against CD80 and CD86. These results suggest that AILIM binds to receptor(s) other than AILIM, CD80 and CD86. AILIM expressed in the certain lymphoid cells required the presence of mAb JTT.1 to induce cell aggregation, whereas purified AILIM supported HPB-ALL adhesion in the absence of the agonistic mAb, suggesting that AILIM expressed in lymphoid cells may be inactive in terms of ligand binding.

Human AILIM is known to act as a co-stimulator of T cell activation (10). We also observed that when AILIM was engaged by the specific mAb, it induced lymphocyte activation in rat and mouse as well as in human (manuscript in preparation). Together, these results indicate that AILIM is a third member of the CD28 family of co-stimulatory molecules and that it can act as an adhesion molecule. A major difference between CD28 and AILIM is their adhesion supporting ability. Although CD28 is thought to be a major co-stimulator, because of its low affinity toward the ligand(s) (13), an additional adhesion pathway, e.g. LAF-1–ICAM-1 interaction, is necessary for co-stimulation in the formation of the immunological synapse (14,15). The preceding results show that AILIM causes strong cell adhesion, suggesting that AILIM may play an important role in building the immunological synapse. Our results indicate that AILIM is a novel major regulator in T cell development and activation. An understanding of AILIM should yield new insights into the mechanisms of T cell activation. This molecule could be important as a target for therapeutic drugs designed to regulate lymphocyte function.

	Acknowledgments

 
We thank Drs. Y. Watanabe, Y. Saito, S. Toyoshima, S. Sakamoto and K. Suzuki for comments. The nucleotide sequence of the gene reported in this paper have been deposited in the GeneBank database with accession no. AB023134 (rat AILIM).

	Abbreviations

 

AILI Mactivation-inducible lymphocyte immunomodulatory molecule

ICOS inducible co-stimulator

	Notes

 
Transmitting editor: M. Miyasaka

Received 25 August 1999, accepted 29 September 1999.

	References

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