Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses
© Kasinrerk et al; licensee BioMed Central Ltd. 2011
Received: 1 June 2011
Accepted: 13 August 2011
Published: 13 August 2011
CD99, a leukocyte surface glycoprotein, is broadly expressed in many cell types. On the cell surface, CD99 is expressed as two distinct isoforms, a long form and a short form. CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T cell activation. However, the molecular mechanisms by which CD99 participates in such processes are unclear. As CD99 contains a short cytoplasmic tail, it is unlikely that CD99 itself takes part in its multi-functions. Association of CD99 with other membrane proteins has been suggested to be necessary for exerting its functions.
In this study, we analyzed the association of CD99 with other cell surface molecules involved in T cell activation. We demonstrate the association of MHC class I, MHC class II and tetraspanin CD81 with CD99 molecules on the cell surface. Association of CD99 with its partners was observed for both isoforms. In addition, we determined that CD99 is a lipid raft-associated membrane protein and is recruited into the immunologic synapse during T cell activation. The implication of CD99 on T cell activation was investigated. Inhibition of anti-CD3 induced T cell proliferation by an anti-CD99 monoclonal antibody was observed.
We provide evidence that CD99 directly interact and form the complex with the MHC class I and II, and tetraspanin CD81, and is functionally linked to the formation of the immunologic synapse. Upon T cell activation, CD99 engagement can inhibit T cell proliferation. We speculate that the CD99-MHC-CD81 complex is a tetraspanin web that plays an important role in T cell activation.
Upon T cell activation, T cell stimulation is initiated when a T cell receptor (TCR) encounters specific antigen peptide-MHC complexes expressed on the surface of antigen presenting cells (APCs). The interaction of various co-stimulatory molecules expressed on T cells and APCs is, in addition, involved in the induction of proper T cell responses. These interactions induce the formation of an immunological synapse (IS) at the cell-cell junction between T cells and APCs, resulting in the reorganization of the related cell membrane signaling molecules in a concerted fashion [1, 2]. The IS is proposed to function as a platform for signal transduction and cytoskeleton reorganization, which is essential for the determination of TCR sensitivity and responsiveness. Several co-stimulatory molecules have been shown to translocate into the IS and are crucial in determining antigen-specific T cell activation and tolerance [2, 3]. CD99 has been recently demonstrated to function as a co-stimulatory molecule in T cell activation . Co-ligation of CD99 and CD3 molecules leads to the translocation of TCR complexes into the IS and enhances TCR signaling events.
CD99 is a type 1 transmembrane glycoprotein encoded by the MIC2 gene, and shares no significant homology with any known protein family [5–9]. The CD99 molecule contains an extracellular domain, followed by a transmembrane domain and a short 36-amino acid intracytoplasmic domain . CD99 is broadly distributed among many cell types, both hematopoietic and non-hematopoietic cells [10–14]. Although the functional role of CD99 is not yet fully understood, it has been implicated in multiple cellular events. CD99 has been described as a T-cell co-stimulator and regulator of cytokine production [4, 15]. Engagement of CD99 with agonistic antibodies induced apoptosis of immune cells and tumor cells [14, 16, 17]. CD99 ligation was also demonstrated to induce expression of adhesion molecules, including ELAM-1, VCAM-1 and ICAM-1, which are associated with leukocyte adhesion and transendothelial migration [13, 14, 18–24]. Furthermore, CD99 engagement has been reported to induce the expression of TCR, MHC class I and MHC class II by accelerated mobilization of these molecules from the Golgi compartment to the plasma membrane . Requirement of CD99 expression in IFN-γ induced MHC class I expression has also been observed . Without CD99, upon IFN-γ stimulation, MHC class I molecules became accumulated within the Golgi apparatus .
Signaling pathways triggered by CD99 have been elucidated in several studies. Stimulation of CD99 with agonistic antibodies enhanced the expression of several T cell activation markers on anti-CD3-activating T cells, elevation of intracellular Ca2+ and the tyrosine phosphorylation of cellular proteins [15, 27]. We have demonstrated that protein kinase C inhibitor, sphingosine and a protein tyrosine kinase inhibitor, genistein, blocked cell aggregation induced by CD99 engagement . It has also been reported that CD99 ligation induced differential activation of three mitogen-activated protein kinase (MAPK) members, ERK, JNK and p38 MAPK . Activation of src kinase and focal adhesion kinase (FAK) by CD99 molecules has also been demonstrated . Although several lines of evidence indicate the involvement of CD99 in cell signaling, with its short cytoplasmic tail, it is unlikely that CD99 itself takes part in signaling events. In the cellular context, association of CD99 with other membrane proteins has been suggested to be necessary for exerting its functions.
On the cell surface, CD99 is expressed as two distinct isoforms depending on the alternative splicing of the encoding gene . The long form (type I) contains 185 amino acid residues and its mobility in SDS-PAGE corresponds to an apparent molecular weight (MW) of 32 kDa. The short form (Type II; 161 residues, apparent MW of 28 kDa) harbors a deletion in the cytoplasmic segment. The CD99 isoforms are differentially expressed in a cell type-specific manner among hematopoietic cells and cell lines [16, 22]. The CD99 isoform expression was shown to dictate distinct functional events [27, 30, 31]. Expression of the long form in CD99-deficient Jurkat T cell line is sufficient to promote CD99-induced cell adhesion, whereas co-expression of the two isoforms is required to trigger T cell death . In addition, on B cells, the short form of CD99 inhibited homotypic adhesion, while the activation of the CD99 long form promoted cell-cell adhesion. The opposing effects of CD99 isoforms on homotypic B cell aggregation were shown to be due to their opposing functions in controlling the expression of the cell adhesion molecule, LFA-1 .
Although the CD99 molecule has been described as a multi-functional cell surface molecule, it contains a short intracellular domain . Interaction with other cell surface molecules is, therefore, assumed to be necessary in order to regulate its multiple functions. In this study, we explored the possibility that CD99 may form a microdomain with other proteins on the cell surface. We demonstrate here the association of MHC class I, MHC class II and a tetraspanin CD81, with both CD99 isoforms. Upon T cell activation, translocation of CD99 into IS and inhibition of T cell proliferation by anti-CD99 monoclonal antibody (mAb) were observed, indicating an important role for CD99 in T cell activation.
Association of CD99 with various membrane proteins
Association of CD99 with MHC class I and MHC class II
To confirm the observed association of CD99 with MHC molecules, reciprocal co-immunoprecipitation experiments were carried out. In accordance with the above results, CD99 was observed in the immunoprecipitates using anti-MHC class II mAb (Figure 3B). However, CD99 could not be detected in the immunoprecipitates using anti-MHC class I mAb (data not shown). This may be because of the low affinity of the employed anti-MHC class I mAb itself, resulting in it being inappropriate for immunoprecipitation. Taken together, our findings suggest that CD99 form complexes with MHC class I and MHC class II molecules.
Association of CD99-MHC complexes with tetraspanin CD81
Colocalization of CD99 with MHC class I, MHC class II and tetraspanin CD81
To demonstrate whether the association of CD99 molecules with its interacting partners is also observed in peripheral blood cells, colocalization of CD99 with MHC and CD81 molecules on PBMCs' membrane were determined by confocal microscopic analysis. As shown in Figure 5B, colocalization of CD99-MHC class I, CD99-MHC class II and CD99-CD81 were observed. The colocalizations were observed only in cells expressing CD99 and MHC or CD81. In IgG control, anti-CD54 mAb was used instead of anti-MHC class I, class II or CD81 and found no colocalization between the CD99 and CD54 molecules indicating no cross-reactivity between conjugates and the primary antibodies used. These results indicated that association of CD99 with MHC and CD81 is physically occurred in peripheral blood cells.
Association of CD99 short and long forms with MHC class I, MHC class II and CD81
We performed immunoprecipitations from the lysates of cell lines expressing selectively the two CD99 isoforms using anti-CD99 mAb and analyzed the immunoprecipitates by Western immunoblotting. As shown in Figure 7A and 7B, MHC class I, MHC class II, and CD81 could be detected in the immunoprecipitates obtained from cells expressing both the CD99 short and long forms. No immunoreactive band could be observed in the control immunoprecipitate of CD99 negative Ramos cell lysates. As anticipated, the typical lipid raft containing molecule NTAL was not detected in the CD99 immunoprecipitated proteins (Figure 7B). Collectively, the results suggest that the association of MHC class I, MHC class II and CD81 with CD99 can be observed in both CD99 isoforms.
Recruitment of CD99 into the immunological synapse (IS)
Immune cell signaling pathways are at least partially mediated by lipid raft-associated proteins. During T cell activation, lipid rafts are accumulated in the IS and function as the platform for signal molecules . We thus attempted to confirm whether, in the Jurkat T cell line, CD99 is a lipid raft-associated membrane protein. As shown in Figure 8B, in the presence of mild lipid raft-preserving detergent 1% Brij-98, both short and long forms of CD99 were found in the raft and non-raft fractions. In contrast, in the presence of the raft disrupting detergent 1% LM, CD99 was mostly found in the non-raft fractions. The lipid raft containing protein Lck was included in the experiment as a positive control and was observed in the lipid raft fractions. These results indicate that CD99 molecules are recruited to the IS and a fraction of CD99 is associated with lipid rafts.
Inhibition of T cell proliferation by engagement of surface CD99
Cell surface molecules containing a short cytoplasmic tail usually form complexes with other molecules in order to modulate their signal transduction and functions. Identification of the interacting partners of cell surface molecules may lead to a better understanding of cellular function and immune responses. In the present study, we have identified several interacting partners of a multi-functional membrane protein, CD99. We observed that on the cell surface, CD99 molecules are associated with MHC class I, MHC class II and tetraspanin CD81. The CD99 molecule has been described by several studies as a signaling molecule. Engagement of CD99 induces signal transduction, resulting in the regulation of T cell activation, cell adhesion, cell migration, and cell death. However, CD99 itself contains a short intracellular domain without signaling motifs on the cytoplasmic tail except a site for PKCα phosphorylation [9, 30]. Signal transduction induced by CD99 is, therefore, incompletely understood. Since we found, in this study, that CD99 form complexes with other signal mediated molecules, i.e., MHC class I, MHC class II and tetraspanin CD81, this may help to clarify the precise signaling mechanism of the CD99 molecule and provide better understanding of its functional roles.
MHC molecules are cell surface proteins that play a critical role in antigen presentation. CD99 has been reported to regulate the expression of MHC class I and class II [25, 26, 32]. Up-regulation of MHC class I and class II by CD99 results from accelerated intracytoplasmic transportation of MHC molecules to the plasma membrane rather than de novo synthesis of these molecules [25, 32]. Deceased expression of CD99 resulted in the retention of MHC class I molecules in the Golgi compartment by affecting the transportation of the MHC molecules in the trans-Golgi network [26, 32]. Moreover, colocalization of CD99 and MHC class I molecules is clearly demonstrated both in the Golgi apparatus and at the cell surface . Strikingly, it was demonstrated that the CD99 and MHC class I association occurs at the transmenbrane domain. Valines located in the transmembrane region of CD99 are required for the binding to MHC molecules, likely in relation with their hydrophobicity . In agreement with the previous reports, in this study, we have shown that, after transportation, the CD99 molecules form complexes with their transported MHC molecules and are co-expressed as microdomains on the cell surface.
In addition to the MHC molecules, we demonstrated the association of CD99 with a tetraspanin, CD81. The tetraspanins are cell surface proteins that are broadly expressed in many cell types. Data from biochemical studies or knockout mice suggest that the tetraspanins play a major role in membrane biology [33, 35]. One of the most striking features of tetraspanins is their ability to form a network of multi-molecular complexes, known as the tetraspanin web, between each individual tetraspanin and other surface proteins. Within the immune cells, all cells express tetraspanins, which provide a scaffold that facilitates the spatial and temporal engagement of their associated proteins. Tetraspanins and their associated proteins modulate several intercellular immune interactions, including adhesion, migration, synapse formation, as well as assisting in intracellular interactions as organizers of membrane-signaling complexes. They are also involved in intracellular protein transport, endocytosis, and exocytosis, and function as chaperones or stabilizers of lineage-specific molecules. CD81, a member of the tetraspanins, has been reported to be involved in an astonishing range of physiological responses [33, 35, 40]. Association of CD81 with various surface molecules, including MHC class II, has previously been reported. As our results demonstrated the association of CD99 with CD81 and MHC molecules, we speculate that the CD99-MHC-CD81 complex is a tetraspanin web and plays an important role in the immune response.
CD99 is expressed as two distinct isoforms, a long 32 kDa form (type I) and a short 28 kDa form (type II) resulting from an alternative splicing process of the product of the encoding gene . In the immune cells, CD99 are expressed in both short and long forms [16, 22]. Both isoforms have their functional roles, and differential expression can lead to distinct functional outcomes [27, 30, 31]. The truncation of the cytoplasmic domain of the CD99 short form may result in a loss of interaction with signaling molecules recognized by the cytoplasmic domain of the long form. It has been demonstrated that the cytoplasmic domain of the long form contains two putative phosphorylation sites, a serine at amino acid residue 168 and a threonine at amino acid residue 181. These potential phosphorylation sites may be important for intracellular signaling events and/or extracellular molecular interactions. Moreover, the S168 of CD99 long form has been reported to be a site for PKCα phosphorylation and is required for the oncosuppressor function . It has also been postulated that truncation of the cytoplasmic domain of the CD99 short form causes an alteration of the three-dimensional structure, leading to different binding sites for its ligand. However, it is still unknown whether CD99 mediated signaling pathways are modulated by the differential expression of CD99 isoforms [16, 22] or whether each CD99 isoform promotes different sets of signaling pathways . To address the mechanisms of differing functions of the two CD99 isoforms, we investigated whether the association of CD99 with its partner molecules depended on the distinct isoform. We, however, found that both CD99 isoforms interacted with MHC and CD81 molecules. It is tempting to assume that the association of CD99 with MHC and tetraspanin CD81 may bring both CD99 isoforms into the tetraspanin web and into close proximity with the intracellular membrane signaling proteins. The molecular mechanism by which CD99 mediated signaling occurs is likely to reflect the presence of signaling molecules, such as kinases [42, 43] and phosphatases , in the tetraspanin microdomain. This is, however, only speculation and requires further investigation.
The IS is a dynamic structure formed between T cells and antigen presenting cells, and is characterized by lipid and protein segregation, signaling compartmentalization, and bidirectional information exchange through soluble and membrane-bound transmitters . The IS is the site where signals are delivered by the T cell receptors, adhesion molecules, as well as co-stimulatory and co-inhibitory receptors. The IS is divided into distinct regions: a central-supramolecular activation cluster (c-SMAC), a peripheral-(p-) SMAC, and a distal-(d-) SMAC . It has been demonstrated that the c-SMAC mediates antigen recognition and subsequent T cell activation, whereas the p-SMAC supports T cell-APC conjugation and maintains the architecture of the IS. Several molecules, including MHC and tetraspanin CD81, have been shown to translocate into the IS during T cell activation [33, 35, 46]. Upon T cell activation, CD81 is redistributed to the contact area of T cell-APC conjugates of both T cells and APCs. Colocalization of CD81 with CD3 at the SMAC of T cells and of CD81 with MHC class II of APCs has been observed in which they function as cell surface co-stimulatory molecules. As CD99 form complexes with MHC and CD81 molecules, we investigated whether CD99 is translocated into the IS upon TCR triggering. We demonstrated that CD99 is a lipid raft-associated protein and is recruited into the IS, as has been observed for its associated molecules. Engagement of CD99 with agonistic antibody inhibits T cell activation. To our knowledge, this is the first demonstration of the accumulation of CD99 within the IS upon T cell activation and the association of CD99 with the proteins of the SMAC. We speculate that CD99-MHC-tetraspanin CD81 complexes may play an important role in T cell activation.
In the present study, we demonstrate that CD99 is associated with MHC class I, MHC class II and a tetraspanin, CD81. The association was observed in both CD99 long and short isoforms. Our data collectively show that upon T cell activation, CD99 is translocated into the IS and involved in regulation of T cell proliferation. The CD99-MHC-CD81 complexes may play an important role in immune responses.
Antibodies, reagents and cell lines
Anti-CD99 mAbs clones MT99/3 (IgG2a) and MT99/1 (IgM) were produced in our laboratory [13, 14]. Anti-CD54 mAb MEM-111 (IgG2a), anti-MHC class I mAb HC10 (IgG1), anti-MHC class II mAb MEM-136 (IgG1), anti-CD81 mAb M38 (IgG1), anti-NTAL mAb NAP-07 (IgG1), anti-Lck mAb Lck-01 (IgG1) and anti-CD45 mAb MEM-55 (IgG1) were kindly provided by Prof. Vaclav Horejsi (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic). Isotype matched control mAbs, PB-1 (anti-hemoglobin Bart's; IgG1) and 13 M (anti-bacteriophage protein; IgG2a) were generated in our laboratory. Isotype matched control mAbs 4G2 (anti-dengue viral protein; IgG2a) and were obtained from Dr. Prida Malasit (Division of Medical Molecular Biology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand). Fluorescein isothiocyanate (FITC)- and Phycoerythrin (PE)-conjugated sheep F(ab')2 anti-mouse immunoglobulin antibodies were purchased from Chemicon Australia Pty Ltd (Victoria, Australia) and Beckman Coulter (Marseille, France), respectively. Alexa Fluor 488-labeled goat anti-mouse IgG antibodies, Alexa Fluor 568-labeled goat anti-mouse IgM antibodies, CellTrace™ Far Red DDAO-SE and Hoechst 33258 dye were obtained from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulin antibodies and HRP-conjugated streptavidin were obtained from Dako (Glostrup, Denmark).
Sulfo-NHS-LC-Biotin and detergent Brij-58 were obtained from Pierce (Rockford, IL, USA). Laurylmaltoside (n-dodecyl-β-D-maltoside; LM) was obtained from Calbiochem/Merck (Darmstadt, Germany). Detergent Brij-98, protease inhibitors (phenylmethylsulfonyl fluoride (PMSF), pepstatin A, aprotinin), TRITC-phalloidin and Staphylococcal enterotoxin B (SEB) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Several cell lines including Ramos (human Burkitt's lymphoma cell line), Raji (human Burkitt's lymphoma cell line), Jurkat (human T cell lymphoblast-like cell line), Molt4 (human acute lymphoblastic leukemia cell line), U937 (human leukemic monocyte lymphoma cell line) and THP-1 (human acute monocytic leukemia cell line) were used in this study. All cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) (Gibco, Grand Island, NY), 40 μg/ml gentamycin and 2.5 μg/ml amphotericin B in a humidified atmosphere of 5%CO2 incubator at 37°C.
Co-immunoprecipitation of cell surface molecules
Jurkat cells were labeled with 5 mM Sulfo-NHS-LC-Biotin at 4°C for 1 hr. The biotinylation was quenched by washing once with 1 mM glycine in PBS and twice with PBS. The biotinylated cells (5 × 107 cells) were solubilized for 30 min on ice in 1 ml lysis buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 5 mM iodoacetamide, and protease inhibitors) containing either detergent 1% Brij-58 or 1% LM. The cell suspension was clarified by centrifugation at 10,000 × g for 30 min at 4°C. The clarified cell lysates were precleared with protein G Sepharose beads (Pierce) coated with mouse immunoglobulins. The precleared lysates were then incubated with 10 μg of purified mAbs for 2 hr at 4°C. Protein G Sepharose beads were added and the mixtures were rotated overnight at 4°C. After incubation, beads were washed five times, and proteins were dissociated from the Protein G beads by addition of SDS-reducing sample buffer (62.5 mM Tris-HCl pH 6.8, 5% β-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 10% glycerol, and 0.01% bromophenol blue) and boiled for 5 min. The precipitated proteins were then resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to a nitrocellulose membrane (Pall Corp., East Hill, NY, USA). The membrane was blocked overnight in PBS containing 5% BSA at 4°C. The blocked membrane was incubated for 1 hr at room temperature with HRP-conjugated streptavidin. The reactive protein bands were visualized by the chemiluminescence detection system (Pierce).
Co-immunoprecipitation of un-labeled cell lysates and Western immunoblotting
The tested cell lines (5 × 107 cells) were pelleted and lysed with 1 ml lysis buffers containing either 1% Brij-58 or 1% LM for 30 min on ice. The lysates were clarified by centrifugation. The immunoprecipitations were performed according to the method described above. The precipitated proteins were resolved by SDS-PAGE under non-reducing conditions and transferred to a nitrocellulose membrane, followed by Western immunoblotting experiments. The nitrocellulose membranes were blocked with 5% BSA in PBS at room temperature for 1 hr. Then, the blocked membrane was incubated with mAbs for 1 hr at room temperature. After being washed five times with 0.1% Tween-20 in PBS, the membrane was incubated with HRP conjugated rabbit anti-mouse immunoglobulin antibodies for 1 hr. The specific protein bands were then visualized by the chemiluminescence detection system (Pierce).
Generation of CD99 expressing and CD99 non-expressing Ramos cells by immunomagnetic cell sorting
Ramos cells were stained with anti-CD99 mAb MT99/3 for 30 min on ice. Then, FITC-conjugated anti-mouse immunoglobulin antibodies were added and incubated for 30 min on ice. After incubation, cells were washed and incubated with anti-FITC MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Cells were washed and resuspended in MACS sorting buffer (0.5% BSA, 2 mM EDTA in PBS) and sorted with an AutoMACS cell sorter (Miltenyi Biotec). The obtained positive and negative cell fractions were collected and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 40 mg/ml gentamicin and 2.5 mg/ml amphotericin B in a humidified atmosphere of 5% CO2 at 37°C. CD99 expression on the sorted cells was verified by flow cytometric analysis.
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of healthy donors by Ficoll-Hypague density gradient centrifugation. PBMCs or Raji B cell line (1 × 107 cells/ml) was pre-incubated with 10% human serum (blood group AB) at 4°C for 30 min. For staining of the surface membrane proteins, 50 μl of the cell suspension were incubated with an equal volume of 20 μg/ml anti-MHC class I, anti-MHC class II, anti-CD81 mAbs, or isotype matched control PB-1 mAb (for Raji) or MEM-111 mAb (for PBMCs) at 4°C for 30 min. The cells were washed twice with PBS containing 1%BSA and 0.02% NaN3 (1% BSA-PBS-NaN3) and then incubated with Alexa Fluor 488-conjugated goat F(ab')2 anti-mouse IgG antibodies at 4°C for 30 min. After twice washing, the cells were then incubated with mouse immunoglobulins at 4°C for 30 min to neutralize the reactivity of the adding conjugates. Subsequently, CD99 molecules were stained using anti-CD99 mAb MT99/1 (IgM isotype) at 4°C for 30 min, followed by Alexa Fluor 568-conjugated goat F(ab')2 anti-mouse IgM antibodies at 4°C for 30 min. Finally, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and plated on poly-D-lysine coated slides. Cell nuclei were visualized with Hoechst 33258 dye. For evaluation of colocalization, cells were visualized and images were acquired using a confocal laser scanning microscope (LSM 700; Zeiss, Le Pecq, France).
Generation of Romos cell expressing CD99 short and long isoforms
Plasmid DNA encoding full-length CD99 cDNA  was used as a template for generation of cDNA encoding CD99 short and long forms by the polymerase chain reaction (PCR). The PCR products were inserted into the EcoR I site of the MSCV retroviral vectors, according to the manufacturer's instructions (Clonetech Laboratory, Mountain View, CA, USA). The obtained plasmid DNA were transformed into E. coli TOP10 (Invitrogen). The plasmid DNA were then isolated from the transformed E. coli by Qiagen chromatography columns (Qiagen, Hilden, Germany). The inserted genes in the constructed plasmids were checked by restriction fragment analysis using corresponding restriction enzymes and DNA sequencing.
To prepare retroviruses (RV) harboring plasmid carrying CD99 isoforms, the plasmid DNA were transfected into Phoenix-Ampho cells (Origene, Rockville, MD, USA) using Lipofectamine (Invitrogen). At 48 hr post-transfection, RV-containing supernatants were collected and clarified by centrifugation. The RV were then used to spin-infect (1200 × g, 90 min at 32°C) the Ramos CD99 negative cells in the presence of polybrene (10 μg/ml)(Sigma-Aldrich). Cells were allowed to expand in culture and sorted by immunomagnetic cell sorting to isolate the CD99 expressing cells. Stable transfectant clones with high CD99 expression were identified by flow cytometric analysis.
Flow cytometric analysis
Cells (1 × 107cells/ml) were pre-incubated with 10% human serum (blood group AB) at 4°C for 30 min to block nonspecific Fc-receptor-mediated binding of mAbs. For staining of the surface membrane proteins, 50 μl of the cell suspension were incubated with an equal volume of 20 μg/ml anti-CD99 mAb MT99/3 at 4°C for 30 min. The cells were washed twice with 1% BSA-PBS-NaN3 and then incubated with PE-conjugated sheep F(ab')2 anti-mouse immunoglobulin antibodies at 4°C for 30 min. Cells were washed and membrane fluorescence was analyzed by a flow cytometer (FACSCalibur; Becton Dickinson, Sunnyvale, CA, USA).
Immunological synapse and confocal image analysis
Raji B cells (1 × 107 cells/ml) were loaded with 1 μg/ml of staphylococcal enterotoxin B (SEB) (Sigma-Aldrich) for 15 min at 37°C. Jurkat T cells (1 × 107 cells/ml) were labeled with CellTrace™ Far Red DDAO-SE. After washing, an equal number of Jurkat cells were mixed with Raji cells and incubated at 37°C for 15 min. Cell mixtures were placed on poly-D-lysine coated slides, fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 5 min on ice. Cells were then incubated with anti-CD99 mAb MT99/3 for 30 min at room temperature. Alexa Fluor 488-labeled goat anti-mouse IgG secondary antibody and TRITC-phalloidin were added and incubated for 30 min. Cell nuclei were stained with Hoechst 33258 dye. The stained cells were analyzed and images were acquired using a confocal laser scanning microscope (LSM 700; Zeiss, Le Pecq, France).
Determination of CD99 in lipid rafts
Lipid rafts were isolated according to the method described elsewhere . Briefly, cells (1 × 108) were resuspended in 0.4 ml of ice-cold hypotonic buffer (10 mM HEPES pH 7.4, 42 mM KCl, 5 mM MgCl2, and protease inhibitors), incubated on ice for 15 min and then passed 10 times through the 30-gauge needle. The suspension was centrifuged for 5 min, 300 × g, at 2°C to remove nuclei. The supernatant was re-centrifuged for 10 min, 25,000 × g, at 2°C to sediment the membranes. Membranes were then lysed in 0.2 ml of lysis buffer containing 1% Brij-98 or 1% LM for 30 min on ice, and spun at 10,000 × g for 30 min. The clarified lysates (0.1 ml) were applied at the top of a 1 ml Sepharose 4B column (Sigma-Aldrich) and sequentially washed with 0.1 ml of the lysis buffer. The fractions (0.1 ml) were collected at 4°C and analyzed for the presence of CD99 by SDS-PAGE and Western immunoblotting.
PBMCs were isolated from heparinized whole blood of healthy donors by Ficoll-Hypague density gradient centrifugation. Cells (1 × 107 cells/ml in PBS) were incubated with 0.5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR, USA) at 37°C for 10 min. The CFSE staining was terminated by adding cold RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 40 mg/ml gentamicin, and 2.5 mg/ml amphotericin B (10% FBS-RPMI 1640). The cells were then washed twice and resuspended with 10% FBS-RPMI 1640. To determine the influence of anti-CD99 mAb on T cell activation, CFSE labelled PBMCs (5 × 105 cells) were cultured in 96-well plate with or without immobilized anti-CD3 mAb OKT3 (60 ng/ml) in the presence or absence of 20 μg/ml anti-CD99 mAb or isotype matched control mAb. The plate was cultured at 37°C in a 5% CO2 incubator for 5 days. Cells were then harvested and determined for cell proliferation by flow cytometry (Becton Dickinson).
This study was supported by the NSTDA Research Chair Grant, National Sciences and Technology Development Agency (Thailand). We also wish to thank the National Research University Project under Thailand's Office of the Higher Education Commission for financial support. SP, WL and KM are doctoral candidates in the Royal Golden Jubilee Ph.D. program. We thank Prof. Václav Hořejší (Laboratory of Molecular Immunology, Institute of Molecular Genetics AS CR, Prague, Czech Republic) for insightful discussions of the manuscript and providing laboratory facilities. We thank Dr. Dale E. Taneyhill for proofreading the manuscript.
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