1 Department of Microbiology and Immunology (M/C 790), University of Illinois at Chicago, Room E-709, Building 935, 835 South Wolcott Avenue, Chicago, IL 60612, USA
2 Rush University Medical Center, 1650 West Harrison Street, Chicago, IL 60612, USA
3 Unité de Biologie des Régulations Immunitaires, INSERM E 0352, Institut Pasteur, Paris 75724, France
Correspondence to: B. S. Prabhakar; E-mail: bprabhak{at}uic.edu
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Abstract |
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Keywords: co-stimulation, dendritic cells, T lymphocytes, tolerance/suppression/anergy
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Introduction |
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ICAM-3 belongs to the Ig superfamily and is structurally similar to ICAM-1 and ICAM-2 but is highly expressed on all resting T cells and thus can facilitate initial interaction between a T cell and an APC. The ICAM-3 is re-distributed to the interface of T cells and DCs regardless of whether the interaction is antigen independent or dependent (2). Furthermore, ICAM-3-, and not LFA-1- or ICAM-1-, blocking antibodies can inhibit antigen-independent T cellAPC conjugate formation, while both LFA-1- and ICAM-3-blocking antibodies can prevent antigen-dependent conjugate formation. These and other studies showed that ICAM-3 could play an important role in the initial T cellAPC interaction. Anti-ICAM-3 antibody treatment of Jurkat T cells can elevate the levels of intracellular calcium and cause tyrosine phosphorylation (4) and T lymphoblast homotypic aggregation in an LFA-1ICAM-1-dependent manner (5). Further, under conditions of sub-optimal TCR stimulation, anti-ICAM-3 antibody can up-regulate CD69 and CD25 expression (6, 7) and cause increased cell proliferation and IL-2 production (8). These studies indicate that ICAM-3 can provide critical signaling required for T cell stimulation. However, these studies used stimulating or blocking anti-ICAM-3 antibodies, instead of natural ligands.
In vivo, DC-SIGN is expressed at significant levels on subsets of APCs that include immature (CD83) DCs, mature DCs, specialized macrophages in the lungs and placenta and a subset of DC precursors in the blood. The DC-SIGN is a type II membrane protein that can bind HIV gp120 protein (9) and sequester HIV (10) and other pathogens until they are presented to T cells. Further, DC-SIGN has been shown to bind ICAM-2, which is highly expressed on endothelial cells and thus may mediate DC trafficking (11).
Geijtenbeek et al. showed that blood-derived monocytes use LFA-1 to bind ICAM-3 on a T cell but monocyte-derived DCs, which express both LFA-1 and DC-SIGN, bind ICAM-3 mostly through DC-SIGN (3). Further, they demonstrated that an anti-DC-SIGN-blocking antibody could decrease allostimulation of human T cells by monocyte-derived DCs. These studies suggested that both DC-SIGN and LFA-1 could facilitate interactions between T cells and monocyte-derived DCs. However, whether DC-SIGN can co-stimulate ICAM-3-bearing cells or only facilitate initial T cell and APC adhesion is not known. A clearer understanding of its function would provide further insights into the mechanisms involved in T cell stimulation.
In this study, we cloned and expressed DC-SIGN, a natural ligand for ICAM-3, and a splice variant of dendritic cell-specific intercellular cell adhesion molecule-grabbing non-integrin (sDC-SIGN) and tested for their ability to provide co-stimulatory signals to human T cells.
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Methods |
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PBMC and CD4+ T cell isolation
PBMCs were isolated using lympholyte-H reagent (Cedarlane, Vancouver, Canada) following the manufacturer's protocol. Briefly, 14 ml of peripheral blood from a healthy donor was mixed with 20 ml of PBS and was under layered with 9 ml of lympholyte-H. After centrifugation at 750 x g for 20 min at room temperature, the buffy layer was harvested and washed with PBS. PBMCs were used directly for co-stimulation assays and monocyte differentiation or further processed to isolate CD4+ T cells.
CD4+ T cells were isolated by negative selection using CD4+ T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA, USA), following manufacturer's protocol. Non-CD4+ T cells that were bound to beads were subjected to an automacs separation using the deplete program. Over 95% of the live enriched cells were CD4+.
Monocyte-derived DCs
Isolated PBMCs (510 x 106 cells per well) were plated into six-well dishes in RPMI containing 5% FCS and incubated at 37°C for 2 h. Wells were gently washed three times with PBS to eliminate non-adherent cells and the remaining cells were cultured in RPMI supplemented with granulocyte macrophage colony-stimulating factor (GM-CSF; 800 U ml1; PeproTech, Rockyhill, NJ, USA) and IL-4 (400 U ml1, PeproTech). Cultures were replenished with fresh GM-CSF- and IL-4-supplemented medium every 3 days. To induce differentiation, monocyte-derived immature DCs were treated with LPS (10 µg ml1, L2654), human IFN- (1000 U ml1, I-2396) or human tumor necrosis factor
(10 µg ml1, T-0157) (SigmaAldrich) for 24 h.
DC-SIGN and sDC-SIGN cloning and expression in CHO cells
cDNAs encoding DC-SIGN and sDC-SIGN were obtained by reverse transcriptionPCR using RNA isolated from monocyte-derived immature DCs as a template and F1-AGAGTGGGGTGACATGAGTG and B1-GAAGTTCTGCTACGCAGGAG primers (3). Amplified products were cloned, sequenced and sub-cloned into the pIRES-2 eGFP vector (Clontech, Palo Alto, CA, USA), which was used to establish permanently transfected cells. Briefly, CHO, DAP-3 or 293 T cells were seeded (2 x 105 cells per well in a six-well plate) the day before transfection. Two micrograms of DNA was mixed with 8 µl of Superfect reagent (Qiagen, Valencia, CA, USA) at room temperature for 5 min in 150 µl of serum-free medium and added to cells in a total volume of 1 ml. After 2 h of incubation, cells were washed twice with PBS and allowed to grow for 2 days. Permanently transfected cells were established by culturing cells in the presence of G418 (1 mg ml1) for at least 1 week. Cells expressing high levels of protein were obtained by sorting on the basis of green fluorescent protein (GFP) expression.
Co-stimulation assays
Human PBMCs or CD4+ T cells were mixed with irradiated CHO cells (8000 rads of gamma irradiation) at a ratio of 3.5 : 1 in 96-well flat-bottom plates and grown in 5% RPMI at 37°C and 5% CO2. Two separate anti-CD3 antibodies were used for T cell stimulation. For the first set of experiments, flat-bottom 96-well plates were coated with 2 µg ml1 (sub-optimal) or 30 µg ml1 (large dose) of anti-CD3 antibody (UCHT1, Pharmingen), incubated overnight at 4°C and gently washed once with PBS before cell mixtures were added. For the second set of experiments, soluble stimulating anti-CD3 antibody (10 ng ml1 of HIT3a, Pharmingen) or phorbol myristate acetate (PMA) (4 ng ml1) was added to the cell mixtures. Cells and culture supernatants harvested at 24 h were used to determine the levels of expression of CD69 and CD25 and cytokines. Cells harvested on day 3 or 4 were used to determine the cell division.
Cell division
Cells were washed twice with PBS and then incubated in the dark (107 cells ml1) with 0.250 µM of 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE; Molecular Probes, Eugene, OR, USA) in PBS at 37°C for 10 min. To quench the reaction, cells were diluted with PBS containing 10% FCS and incubated for an additional 20 min. These cells were washed with PBS containing 2% FCS and used in the co-stimulation assays. Cell division was determined by the extent of CFSE dilution detected using a flow cytometer (FACSCalibur; BD, San Jose, CA, USA) followed by data analysis using Win MDI 2.8 software (http://facs.scripps.edu/software.html). Live lymphocyte-gated cells were assessed for forward scatter (FSC) and green (FL1) fluorescence properties. Quadrants were set up using live CFSE-loaded untreated T cells as negative controls for cell size (FSC) and cell division (FL1 channel for CFSE).
Cell staining
Lymphocytes were re-suspended separately from triplicate wells gently so as to harvest only the non-adherent lymphocytes. CHO cells inadvertently carried over were gated out by size exclusion. Lymphocytes were incubated for 10 min on ice with Fc-block reagent (Pharmingen) in PBS + 2% FCS. All subsequent staining was carried out on ice for 30 min in the dark using 50 µl per reaction of antibodies (5 µg ml1) in PBS + 2% FCS. The CD4+ lymphocytes were stained with FITC-CD69 (Pharmingen) or CD25 (Pharmingen) and then with 4',6-diamidino-2-phenylindole (DAPI) (SigmaAldrich), Cy-chrome CD45RA and CD45RO-ECD. CCR7 staining was performed using a mouse anti-CCR7 antibody (Pharmingen) followed by the addition of biotinylated rat anti-mouse IgM (R6-60.2, Pharmingen) and streptavidinPE (Pharmingen). Cells were washed once with 200 µl of PBS + 2% FCS. DAPI-negative cells were analyzed by FACS to determine the expression levels of various markers. The results were analyzed using Win MDI software (http://facs.scripps.edu/software.html).
ELISA for cytokines
Supernatants, harvested from co-stimulation assays performed in triplicate, were frozen at 80°C until assayed. A volume of 50 µl of supernatant was used to assay for IL-2, IL-4 and IFN- as outlined in the manufacturer's instructions (IL-4 and IFN-
: eBioscience, San Diego, CA, USA; IL-2: OptEIA human IL-2 kit, Pharmingen). ACH8122 and biotinylated AHC7129 (Biosource, Camarillo, CA, USA) antibodies were used to capture and detect IL-12, respectively. MAXI-SORP ELISA plates (439 454; Nunc, Rochester, NY, USA) were coated with ACH8122 antibody in (50 µl per well) carbonate buffer (0.1 M Na2CO4/NaHCO4, pH = 9.0) overnight at 4°C. Next day plates were washed three times with PBS + 0.05% Tween-20 and then blocked for 1 h with 100 µl PBS + 10% FCS at room temperature. Plates were washed extensively between the following steps. Fifty microliters per well of culture supernatant was added in triplicate and incubated for 2 h, biotinylated AHC7129 antibody was added and incubated for 1 h and streptavidinHRP was added and incubated for 30 min; the reaction was developed using 50 µl of tetramethylbenzidine (TMB) substrate (BD) for 15 min and stopped with 25 µl of 1 M HCl. Plates were read at 450 nm using a Bio-Rad 550 plate reader (Bio-Rad, Hercules, CA, USA) and then analyzed using Excel software (Microsoft, Redmond, WA, USA).
DC-SIGN ELISA
DAP-3 and 293T cells stably expressing DC-SIGN or sDC-SIGN were grown to confluence in six-well dishes containing 3 ml of medium. Culture supernatants were tested in triplicate for the presence of soluble DC-SIGN using a sandwich ELISA. A polyclonal rabbit antiserum against the C-terminal 20 AAs of DC-SIGN was used as the capture antibody. For detection, monoclonal anti-DC-SIGN antibody (MAB1621, R&D Systems) was used at 1 µg ml1 in PBS + 10% FCS followed by the addition of HRP-labeled goat anti-mouse IgG2a polyclonal antibody (Caltag, Burlingame, CA, USA) and TMB substrate as outlined above. DAP-3 sDC-SIGN cell lysate was used as a positive control.
Western blot
293T, sDC-SIGN-293T and DC-SIGN-293T cells (0.8 x 106) were washed and re-suspended in 30 µl of 1 x boiling protein sample buffer supplemented with ß-mercaptoethanol. Samples were boiled for 5 min and subjected to SDSP on a 10% gel (Bio-Rad) using a commercial electrophoresis buffer (161-0732, Bio-Rad). Proteins were then transferred to a nitrocellulose membrane (162-0115, Bio-Rad) using a mini-gel transfer apparatus (Bio-Rad) overnight at 4°C at 30 V in transfer buffer (3 g Tris l1, 14.4 g glycine l1 and 20% methanol). The nitrocellulose membrane was blocked with 2% skim milk in PBS (milk/PBS) for 2 h at room temperature. Rabbit anti-DC-SIGN antibody was added (20 µg ml1 in 1% milk/PBS) and incubated for 2 h at room temperature with gentle rocking. The membrane was washed three times with PBS + 0.05% Tween-20 for 5 min each. The secondary HRP-labeled anti-rabbit (Caltag) antibody was added at 1/500 in 1% milk/PBS and washed as before. Western blotting detection system (RPN2132; Amersham Bioscience, Piscataway, NJ, USA) was used following the manufacturer's instructions.
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Results |
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Since DC-SIGN is a physiological ligand for ICAM-3, a classical co-stimulation assay was used to test for the ability of DC-SIGN and sDC-SIGN to provide co-stimulation. DC-SIGN and sDC-SIGN were sub-cloned into the pIRES-GFP2 vector and sorted on the basis of equivalent levels of GFP expression. DC-SIGN expression levels in stably transfected CHO cells used in the co-stimulation assays are shown in Fig. 3. Human PBMCs were stimulated with a sub-optimal dose of plate-bound UCHT1 anti-human CD3 (2 µg ml1) antibodies in the presence of CHO cells expressing either DC-SIGN or sDC-SIGN or B7 molecules, CD80 and CD86 (shown in Fig. 3). As a test for T cell stimulation we assayed the supernatants for the production of IL-2. Both DC-SIGN and sDC-SIGN could not co-stimulate IL-2 production, while B7 molecules were able to co-stimulate a 5-fold increase in IL-2 production relative to PBMCs stimulated with anti-CD3 alone (not shown). Interestingly, DC-SIGN, but not sDC-SIGN, consistently inhibited anti-CD3-induced IL-2 production by PBMCs, relative to controls (not shown). In order to better characterize the T cell response in this system, CD4+ T cells were isolated via negative selection to maintain them in resting state and used in subsequent assays.
We performed the co-stimulation assay using PMA (Fig. 4A), which activates tyrosine kinases and the high-affinity form of LFA-1. We confirmed that CD80 and CD86 could co-stimulate cytokine production from PMA-treated T cells. To a lesser extent, but consistently, DC-SIGN but not sDC-SIGN could up-regulate IL-2 production as compared with controls. The enhancement of IL-2 production by DC-SIGN in PMA-stimulated T cells represented >40% of the levels of IL-2 production by T cells when they were treated with PMA and ionomycin.
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Since DC-SIGN could provide limited co-stimulation to PMA-stimulated T cells, we wanted to determine whether a similar enhancement could be seen when T cells were stimulated with a non-limiting concentration of anti-CD3 (30 µg ml1) antibody. Relative to the control, there was a moderate but significant increase in the production of IL-2 and IFN- when T cells were stimulated in the presence of DC-SIGN (Fig. 4C).
We further explored the effects of DC-SIGN on weakly stimulated T cells. Previously, it was shown that anti-ICAM-3 treatment of anti-CD3-stimulated T cells could induce expression of activation markers CD69 and CD25. Therefore, we tested for the ability of DC-SIGN or sDC-SIGN to up-regulate these activation markers on T cells stimulated with sub-optimal anti-CD3 or PMA (Fig. 5). CD80- and CD86-expressing CHO cells induced higher levels of CD69 and CD25 expression on anti-CD3-stimulated T cells when compared with control CHO cells (CD69: 59 and 44%, respectively, compared with 23%; CD25: 17% each compared with 8% for the control). A modest suppression of CD69 up-regulation was seen in the presence of CHO cells expressing DC-SIGN relative to control CHO cells or cells expressing sDC-SIGN (9% compared with 23 and 22%, respectively).
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Next, we tested the abilities of DC-SIGN and sDC-SIGN to co-stimulate plate-bound anti-CD3- or PMA-induced division of Th. CD4+ T cells were loaded with CFSE dye that dilutes on cell division. T cells were analyzed by flow cytometry 3 days post-stimulation for levels of CFSE and forward scatter, which is a crude measure of cell size (Fig. 6). In the anti-CD3 stimulation assay, both CD80 and CD86 could induce blast formation (77 and 61% of T cells compared with 11% in the presence of GFP control) and cell proliferation (53 and 20%, respectively, compared with 1% for GFP control) within 3 days. However, GFP vector control, DC-SIGN and sDC-SIGN could not induce any cell division. By day 3, PMA-treated T cells underwent significant cell division in the presence of CD80 (44%) and CD86 (30%) relative to 5% in the presence of CHO-GFP. Cell division in the presence of DC-SIGN and sDC-SIGN control was 10 and 4%, respectively. Non-dividing CD4+ T cells co-stimulated with GFP, sDC-SIGN, DC-SIGN, CD80 and CD86, nonetheless showed blast formation in 55, 54, 67, 54 and 54% of cells, respectively.
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Different ratios of CHO-DC-SIGN to CHO-CD80 (10 : 1 and 1 : 1) were used to co-stimulate anti-CD3-treated Th. We tested for IL-2 secretion and activation marker up-regulation and compared them with T cells co-activated with the same ratios of CHO-vector and CHO-CD80 cells. When CHO-DC-SIGN : CHO-CD80 or CHO-GFP : CHO-CD80 cells were used at a ratio of 10 : 1, they induced 461 and 1049 pg ml1 of IL-2 and 550 and 1100 pg ml1 of IFN-, respectively. The effects of DC-SIGN were somewhat subdued when they were used at a 1 : 1 ratio (Fig. 7).
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Discussion |
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Unlike stimulating anti-ICAM-3 HP2/19 antibody, DC-SIGN was unable to co-stimulate human CD4+ T cells treated with the same anti-CD3 antibody at 2 µg ml1 (not shown and Figs 47 ). It is interesting to note that both the HP2/19- anti-ICAM-3-stimulating antibody and LFA-1 bind the first Ig-like domain of ICAM-3 (15), while DC-SIGN binds the second Ig-like domain (16). In our co-stimulation assay system, DC-SIGN decreased the efficiency of anti-CD3-induced T cell stimulation with respect to CD69 up-regulation, IL-2 production (Figs 46
) and CD80-mediated T cell co-stimulation (Fig. 7). In contrast, DC-SIGN enhanced IL-2 production by T cells stimulated with PMA, which can substitute for TCR signaling and activate protein tyrosine kinases and induce the high-affinity form of LFA-1. Moreover, the observation that DC-SIGN can enhance IL-2 secretion in the presence of 30 µg ml1 of anti-CD3 suggested that a stronger stimulus (perhaps through increased tyrosine kinase activation) might be required to elicit the positive effects of DC-SIGN. These results revealed a complex role for DC-SIGN in the initial co-stimulation required for IL-2 production by T cells.
Low-affinity interactions between accessory molecules allow T cells to probe the surface of APCs. Actin-binding proteins ezrin and moesin interact with ICAM-3, helping to localize it to the uropod of migrating lymphocytes (17, 18). Subsequently, ICAM-3 quickly re-distributes to the point of contact in an antigen-independent manner (2). This then facilitates further interactions between the T cell and APC allowing the formation of an immunological synapse. This results in re-distribution and co-localization of ICAM-3 with ICAM-1 and LFA-1 in the outer edges of the immunological synapse. TCR-mediated events within the synapse further strengthen the T cellAPC interaction by inducing the high-affinity form of LFA-1 (19). Previous studies using in vitro co-stimulation assays demonstrated that ICAM-1 signaling induced by LFA-1 could enhance T cell stimulation (20, 21) and restore co-stimulation in CD28 knockout mice (22). Further, cells lacking both pathways were unable to respond to anti-CD3-induced T cell signaling and suggested that in the absence of B7-CD28 signaling, LFA-1ICAM-1 is the predominant co-stimulatory pathway.
Based on our current and others' findings (1922), we speculate that the DC-SIGN has two roles in stimulating a T cell. There is an initial and transient binding that occurs between APCs and T cells through DC-SIGN and ICAM-3, which allows for the engagement of TCR and the MHCpeptide complex. This initial TCR stimulation increases LFA-1 avidity for ICAM-1 (5) and decreases LFA-1ICAM-3 interaction. Since DC-SIGN can interact with ICAM-3, and not ICAM-1, it can induce signaling through ICAM-3 and enhance IL-2 production by T cells that receive a strong TCR signal.
In vivo, both CD83+ and CD83 APCs and specialized macrophages express DC-SIGN. Monocytes differentiated into immature DCs, which induce tolerance, express higher levels of DC-SIGN as compared with matured DCs (3). Based on our observation that DC-SIGN can lower the levels of IL-2 production by T cells on sub-optimal stimulation by anti-CD3, we speculate that DC-SIGN may help maintain tolerance by inhibiting IL-2 production by weakly stimulated T cells (23, 24). How DC-SIGN brings about these differential effects under sub-optimal versus optimal T cell signaling is not fully understood but might involve negative signaling through ICAM-3 (25), interference with LFA-1 binding to ICAM-3 (26) or freeing up LFA-1 from ICAM-3 to allow homotypic ICAM-1LFA-1 interaction (5). Further studies are required to delineate its mechanism of action both in vitro and in vivo.
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Abbreviations |
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APC | antigen-presenting cell |
CFSE | 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester |
DAPI | 4',6-diamidino-2-phenylindole |
DC | dendritic cell |
DC-SIGN | dendritic cell-specific intercellular cell adhesion molecule-3-grabbing non-integrin |
FSC | forward scatter |
GM-CSF | granulocyte macrophage colony- stimulating factor |
ICAM | intercellular cell adhesion molecule |
LFA | leukocyte function-associated antigen |
PMA | phorbol myristate acetate |
sDC-SIGN | a splice variant of dendritic cell-specific intercellular cell adhesion molecule-3-grabbing non-integrin |
TMB | tetramethylbenzidine |
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Notes |
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Received 20 July 2004, accepted 13 March 2005.
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References |
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