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Vol. 16, Issue 7, 3314-3322, July 2005
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* Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain;
Instituto de Farmacología y Toxicología, CSIC-UCM, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain; and
Vita-Salute San Raffaele University School of Medicine, 20132 Milano, Italy
Submitted January 4, 2005;
Revised March 29, 2005;
Accepted April 22, 2005
Monitoring Editor: Mark Ginsberg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The interaction of T lymphocytes with APC involves different and sequential cellular events. Initially, the T-cell adheres transiently to the APC, scanning its surface for the presence of specific peptide-MHC complexes, which is independent of Ag recognition. This creates an initial contact interface that evolves into an Ag-dependent interaction and is accompanied by the reorganization of different molecules at T-cell-APC interface (Grakoui et al., 1999; Montoya et al., 2002b). Ag, adhesion, and costimulatory receptors as well as signaling intermediates are organized in supramolecular activation clusters (SMACs), comprising a central SMAC involved in Ag-dependent responses, in which the TCR, CD28, CD3, CD2, protein kinase C-
, Lck, and Fyn molecules have been located surrounded by a peripheral SMAC containing adhesion-related components, such as the LFA-1 integrin and talin (Monks et al., 1998; Grakoui et al., 1999).
Initial T-cell-DC adhesion-mediated scanning facilitates recognition of Ag-MHC complexes by the TCR. Several pairs of adhesion molecules are involved in the initial adhesive interactions such as CD2/LFA-3, LFA-1/ICAM-1,-3, or ICAM-3/DC-SIGN. LFA-1 ligands include ICAM-1,-2,-3, and JAM-1 (Montoya et al., 2002b; Ostermann et al., 2002). ICAM-1 and ICAM-3 provide costimulatory signals to T lymphocytes (Van Seventer et al., 1990; Campanero et al., 1993; Juan et al., 1994), and both have been localized at the T-cell-APC interface (Grakoui et al., 1999; Montoya et al., 2002a). Although LFA-1 binds to ICAM-3 with low affinity compared with ICAM-1 (Bleijs et al., 2000), blocking antibodies directed against ICAM-3 have been shown to inhibit T-cell proliferation in DC-stimulated mixed leukocyte reactions (Starling et al., 1995). These facts together with the high expression of ICAM-3 on naïve T lymphocytes support its role in initial transient contacts between T lymphocytes and APC (Montoya et al., 2002a).
Although DCs are the most important APC, relatively little is known on the nature of the immune synapse (IS) formed between T-cells and DCs. An active role of DC actin cytoskeleton during IS formation has been reported (Al-Alwan et al., 2003). The relative density of a given MHC-peptide complex on DC surface is quite low. Thus, cooperative mechanisms to enhance Ag presentation based on changes of surface density of MHC-II molecules may exist (Banchereau and Steinman, 1998).
We describe herein the organization and dynamics of adhesion and MHC class II molecules at the initial steps of DC interaction with T lymphocytes, as well as the role of ICAMs on the induction of MHC-II clustering at the DC-T lymphocyte contact area. Such clustering is induced by triggering of the LFA-1 integrin on mature DC and requires signaling involved in actin cytoskeleton reorganization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). At day 6, maturation of DCs was induced by LPS (10 ng/ml, Sigma Chemical Co., St. Louis, MO). CD4+ T-cell clone S3085B (S3) were generated as described (Montoya et al., 2002a). PPL-1, a Jurkat cell line variant deficient for ICAM-3 surface expression was provided by F. Lozano (Hospital Clinic I Provincial de Barcelona; Lozano et al., 1993), and PPL-1 stably transfected with ICAM-3 (PPL1-ICAM-3) was generated by J. M. Serrador (Servicio de Inmunología, Hospital de La Princesa, Madrid, Spain). Human CD4+ CD45RA+ CD45RO– naive Th-cells were isolated from human PBMC using negative selection CD4/CD45RO columns kit (R&D Systems, Minneapolis, MN).
Antibodies and Reagents
We used the following mouse anti-human mAbs: DCIS1/21 (anti-MHC-II; Mittelbrunn et al., 2002), HP2/19 (anti-ICAM-3), Hu5/3 (anti-ICAM-1), TP1/40 (anti-
L integrin chain), D3/9 (anti-CD45), anti-CD14, T3b (anti-CD3), anti-CD81 (I33.22), FN1 (tetraspanin associated anti-MHC-II), 448 (anti-TCR 
chain or CD247), anti- tubulin, TS1/11 (blocking anti-L integrin chain), and HP2/1 (anti-VLA-4). MR1 monoclonal antibody (mAb; anti-DC-SIGN) was from Dr. A. L. Corbí (Centro deInvestigaciones Biológicas, Madrid, Spain), and the blocking anti-DC-SIGN (AZN-D1) was from by Dr. Y. van Kooyk (University Medical Center, Amsterdam, The Netherlands). An additional blocking anti-DC-SIGN mAb was obtained from R&D (clone 120507). Blocking anti-CD11d (240I) was from Dr. D. E. Staunton (ICOS, Bothell, WA). pAb against phosphorylated (Tyr 174) Vav1 was kindly provided by Dr. X. R. Bustelo (CIC, Salamanca, Spain). pAb against total Vav1 was purchased from Upstate Biotechnology (Lake Placid, NY). Antibody against Rac1 was from Transduction Laboratories (Becton Dickinson, Mountain View, CA). ICAM-1-Fc, consisting of the entire extracellular portion of human ICAM-1 fused to human IgG Fc was generated and, ICAM-3-Fc was kindly provided by Dr. M. Robinson (Celltech, Cambridge, UK). Recombinant human fibronectin, poly(L)lysine (PLL), and SEB were from Sigma. Human recombinant IL-2 (Hoffmann-La Roche Nutley, NJ) was from the NIH AIDS Research and Reference Reagent program. CM-TMR (chloromethylbezoylaminotetrametil-Rhodamine) and CMAC (choromethyl derivative of aminocoumarin) fluorescent cell trackers were from Molecular Probes (Eugene, OR). The antagonist of LFA-1,(R)-5 (4-bromobenzyl)-3 (3,5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (BIRT 377) was kindly provided by T. A. Kelly (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT). CS3 (control peptide) was kindly provided by Dr. A. García-Pardo (Centro deInvestigaciones Biológicas, Madrid, Spain). D(+) mannose, Latrunculin A (LatA), taxol, and glycophorin were from Sigma.
Conjugate Formation and Immunofluorescence Assays
S3 T-cells were loaded with the blue fluorescent cell tracker CMAC (5 µM for 30 min at 37°C), washed, and resuspended in Hanks' balanced salt solution (HBSS) supplemented with fetal bovine serum 2%. Human mature DC (5 x 104), preloaded with SEB (1 µg/ml for 30 min at 37°C), were mixed with S3 cells (1.5 x 106), centrifuged, and incubated for 15 min at 37°C. Then, cells were allowed to settle on coverslips for 30 min at 37°C. Cellular conjugates using PPL-1, PPL-1-ICAM-3 Jurkat T-cells, or naive T-cells in absence of SEB, were also performed. Cell conjugates were fixed for 5 min in 4% formaldehyde-phosphate-buffered saline (PBS) and stained with the indicated antibodies, using a goat anti-mouse Alexa 488 highly cross-adsorbed IgG as secondary antibody. For double immunostaining, conjugates were blocked for 1 h with mouse serum and then incubated with a biotinylated anti-ICAM-1 (MEM-111) or an anti chain (448) mAbs, using streptavidin rhodamine red-X or a rabbit anti-mouse Rho-X as secondary reagents, respectively. Clustering of molecules at cell-cell contacts was analyzed by Leica TCS-SP confocal laser scanning unit equipped with Ar and He-Ne laser beams and attached to a Leica DMIRBE inverted epifluorescence microscope or by DMR photomicroscopy using Leica QFISH 1.0 software when indicated (Mannheim, Germany). Cell conjugates forming IS were identified by the presence of a TCR- chain cluster.
Expression of Recombinant DNA Constructs
Chimeric MHC-II-GFP kindly provided by Dr. J. Neefjes (The Netherlands Cancer Institute), CD44-, ICAM-3-, ICAM-1- and L-GFP were used for dynamic assays. Mature DCs at 7 d (2 x 106 cells) were transiently transfected by nucleoporation with an Amaxa Nucleofector (Amaxa, Cologne, Germany) according to the manufacturer's instructions. After electroporation, DCs were cultured for 16 h on six-well plates in the presence of IL-4 and GM-CSF and then were used for dynamic assays.
Time-lapse Fluorescence Confocal Microscopy
Coverslips were coated with fibronectin (20 µg/ml) for 2 h at 37°C and blocked with PBS containing bovine serum albumin (BSA) 0.1% for 30 min at 37°C. Then, coverslips were washed with HBSS and mounted in Attofluor open chambers (Molecular Probes) and placed on the microscope stage. DCs (8 x 104) preloaded with SEB were allowed to adhere on these chambers for 30 min. CD4 T-cell clone (4 x 105) previously incubated with 5 µg/ml CM-TMR were added to the chambers containing adhered DCs and maintained at 37°C in a 5% CO2 atmosphere. Confocal images were acquired using Leica TCS-SP confocal laser scanning unit equipped with Ar and He-Ne laser beams and attached to a Leica DMIRBE inverted epi-fluorescence microscope. We used the 63x objective and processed and assembled the images into movies using Leica confocal software.
Ligand-coated Microspheres
Styrene divinilbenzene latex beads (6 µm, Sigma) were coated with ICAM-1-Fc, ICAM-3-Fc, or both as follows. Latex beads were resuspended in adhesion buffer (NaCO3, NaHCO3) and incubated with an anti-human IgG mAb (Fc specific, Sigma), for 2 h at RT. Beads were washed twice and incubated with the Fc-chimerical proteins (10 µg/ml) in 0.4 ml of adhesion buffer overnight at 4°C. After washing with PBS, ligand-coated beads were resuspended in BSA 1% wt/vol and stored at 4°C. The presence of ICAM-1 and ICAM-3 recombinant proteins on microspheres was corroborated by flow cytometry with specific mAbs. For control assays, latex beads were coated with BSA 1%, anti-human IgG mAb (Fc specific), or glycophorin (10 µg/ml, Sigma).
DC-microspheres Conjugates and Cluster Analysis
Immature or mature DC cells (5 x 105) were mixed with latex beads (1 x 106) precoated with ICAM-1 and ICAM-3, incubated for 15 min at 37°C in HBSS 2% BSA, and plated on fibronectin-coated slides. Slides were fixed, immunostained for MHC-II, CD81, CD86, FN1, LFA-1, CD45, or actin and analyzed by confocal microscopy. In some experiments, DC were pretreated with 1 µM latrunculin A or 10 µM Taxol for 20 min at 37°C. When indicated, DCs were preincubated with two blocking anti-DC-SIGN mAbs (AZN-DI and clone 120507), D(+) mannose (100 mM), blocking anti-LFA-1 L (TS1/11) 10 µg/ml, blocking anti-CD11d (240I) 40 µg/ml, anti-VLA-4 (10 µg/ml), BIRT377 (20 µM), or CS3 (control peptide) for 15 min at 37°C. Clustering of molecules at the contact of DC with latex microspheres was analyzed by Leica TCS-SP confocal laser scanning unit. Clustering was defined as an increment of fluorescence intensity at the contact area compared with the rest of the DC membrane using Leica confocal software.
Determination of Active Vav1 and Rac1
DC (0.5 x 105) were resuspended in RPMI 1640 medium (Flow Laboratories, Irvine, United Kingdom) containing 0.1% BSA and incubated with microspheres coated with ICAM-1-Fc, ICAM-3-Fc, or both. Microspheres coated with anti-Fc or glycophorin were used as controls. After incubation, cells were washed twice with ice-cold HBSS and lysed at 4°C in buffer containing 50 mM Tris, pH 7.4, 100 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, and COMPLETE cocktail inhibitor tablets (Roche Boehringer Mannheim, Indianapolis, IN). The protein content in the cell lysates was measured using protein detection kit (Bio-Rad, Hercules, CA). Samples were separated in 7% SDS-PAGE in the case of Vav and transferred to nitrocellulose membranes. For pulldown experiments GST-PAK-CD, which binds to active Rac, donated by J. Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands), was prepared as described (Sander et al., 1999). Equal amounts of protein were incubated with glutathione-agarose beads coupled to GST-PAK-CD for 60 min at 4°C and then washed four times in lysis buffer, resuspended in Laemmli buffer, separated in 15% SDS-PAGE, and transferred to nitrocellulose membranes. Western blot was performed using an antibody against Rac1 or phosphorylated (Tyr 174) Vav1, followed by an HRP-conjugated anti-mouse or anti-rabbit serum. For Vav1 total detection nitrocellulose membranes were stripped with Restore Western Blot Stripping buffer (Pierce, Rockford, IL), blocked, and then incubated with anti-Vav. Detection of chemiluminescence was performed using SuperSignal Pico detection kit from Pierce. For statistical purposes, gels of active and total Rac1 or Vav1 were subjected to densitometric analysis and normalized with respect to the loading control. Arbitrary units obtained for incubation point were then referred to the value of cells treated with Fc-coated microspheres to obtain fold induction.
Statistical Analysis
Data obtained were compared by the nonparametrical Mann-Whitney U-test with an alpha value of 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Such clustering at the T-cell side of the IS prompted us to investigate the distribution of their two main counterreceptors, DC-SIGN and LFA-1. First, we studied the localization of LFA-1, ICAM-3, ICAM-1, and DC-SIGN in conjugates formed between SEB specific CD4+ T lymphocytes (S3 T-cells) and SEB-loaded DC (Figure 1A). To recognize mature IS, only those conjugates in which T-cells showed the specific clustering of CD3 or CD3-associated chain were analyzed. ICAM-3, ICAM-1, and LFA-1 were detected at the contact area, forming a clear peripheral ring (Figure 1, A and B). Although DC-SIGN has been described as an important counterreceptor for ICAM-3 (Geijtenbeek et al., 2000), it did not concentrate at the interface of DC-T-cell contacts (Figure 1A). CD11d is another integrin described as counterreceptor for ICAM-3 (Van der Vieren et al., 1995), this molecule is weakly expressed on human monocyte derived-DC and failed to redistribute at the IS (unpublished data).
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The dynamic behavior of LFA-1, in addition to that of ICAMs, was studied in primary DC transiently transfected with GFP constructs by time-lapse fluorescence confocal microscopy during DC interactions with SEB-specific T lymphocytes (Figure 1C). LFA-1 redistributed to the contact area; the first clusters were found 4 min after the cellular interaction (Figure 1C and Supplementary Video 1). These clusters were small at the beginning, but evolved into well-organized clusters, which redistributed to the peripheral ring upon conjugate stabilization. Both ICAM-3 and ICAM-1 also redistributed rapidly to the DC contact area with the T lymphocyte (Figure 1C). ICAM-3 was the first molecule that localizes at the DC site of interaction after contact with T lymphocyte. We observed the presence of small transient clusters of ICAM-3 within the first 45 s (Figure 1C). On conjugate stabilization, ICAM-3 was accumulated at the outer zone of the cell-cell contact (zoom Figure 1C). ICAM-1 redistributed later to the contact area (Figure 1C). Like ICAM-3, we observed that upon conjugate stabilization ICAM-1 was distributed at the outer zone, forming a peripheral ring. As control, no relocation of CD44 was observed during the process of DC-T-cell conjugate formation (Supplementary Video 2).
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ICAMs Trigger MHC-II Clustering in DC
To investigate whether LFA-1 engagement influences MHC class II redistribution on membrane DC, we studied the reorganization of LFA-1 and MHC-II in DCs upon conjugation with latex microspheres coated with human recombinant ICAM-1-Fc, ICAM-3-Fc, or both. LFA-1 clustering was markedly enhanced at the area of cell contact with ICAM-1-latex microspheres, whereas ICAM-3 induced a lower clustering. Interestingly, ICAM-3-microspheres binding, induced formation of MHC-II clusters at the contact area with DC in a large percent of conjugates (Figure 3A). In contrast with LFA-1 clustering, ICAM-3 tended to be a more efficient inductor of MHC-II clustering than ICAM-1. We also found that microspheres coated with both ICAM-1 and ICAM-3 exerted an additive effect on MHC-II relocation (Figure 3A). The fluorescence intensity of MHC-II in the contact area with ICAMs was twofold higher than the rest of the DC membrane fluorescence (Table 1). This phenomenon was specific of MHC-II since the leukocyte-specific molecule CD45 was not clustered under the same experimental conditions (Figure 3, A and B, and Table 1).
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Interestingly, clustering of MHC class II molecules at the contact area seems to be intrinsic to mature DC, whereas immature DCs and other APCs analyzed such as Raji and LG-2 B cells were unable to redistribute MHC-II in the presence of ICAM-coated latex microspheres (Figure 3C, and unpublished data).
DC-SIGN and CD11d are other counterreceptors for ICAM-3 besides LFA-1 (Geijtenbeek et al., 2000). To assess the role of these molecules in MHC-II clustering, DCs were incubated with blocking reagents against LFA-1, DC-SIGN, and CD11d before the addition of ICAMs latex microspheres. BIRT 377 (Kelly et al., 1999; Woska et al., 2003), and the blocking anti-L chain mAb (TS1/11), were used to impair function of LFA-1; D(+) mannose, and two mAb (AZN-DI and clone 120507) were used to block DC-SIGN and blocking anti-CD11d (240I) to inhibit CD11d. Anti-VLA-4 mAb was used as control. BIRT 377 prevented clustering of LFA-1 induced by ICAM-1 or ICAM-1 + ICAM-3 (unpublished data). Remarkably, clustering of MHC-II induced by ICAM-3 or ICAM-1 + ICAM-3 was also significantly reduced by LFA-1 blockade (BIRT 377 and mAb TS1/11), whereas no effect was observed with inhibitors of DC-SIGN, CD11d, or VLA-4 (Figure 4 and unpublished data). These results point to LFA-1 as the major DC counterreceptor for ICAM-3 and ICAM-1 involved in MHC-II clustering.
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). It has been described that a subset of di- or oligomerized MHC-II molecules localizes to tetraspanin-based microdomains (Kropshofer et al., 2001). We have found that ICAM-3 is also able to induce the clustering of CD81 (unpublished data), a member of tetraspanin family. Moreover, we have detected MHC class II clustering using the FN1 mAb (unpublished data), which specifically recognize tetraspanin-associated MHC-II molecules (Drbal et al., 1999). To further confirm the role of ICAM-3 in MHC-II clustering, DC-T-cell conjugates were formed either with ICAM-3–deficient PPL1 T-cells or with PPL-1 cells stably transfected with ICAM-3 (PPL1-ICAM-3). Both PPL1 and PPL1-ICAM-3 displayed comparable levels of ICAM-1 (Figure 5A). MHC-II clustering was detected in a large percent of DCs in conjugates with PPL1-ICAM-3 compared with a lower proportion of PPL1 cells (Figure 5, B and C; 58.13% ± 9.9 vs. 24.8% ± 6.7). In addition, DCs in conjugates with PPL1-ICAM-3 cells also showed a clear relocation of the costimulatory molecule CD86 to the contact area (Figure 5, B and C), which postulates a role for ICAMs not only in MHC-II clustering but in relocation of costimulatory molecules for efficient Ag presentation. Thus, ICAM-3 binding to LFA-1 on DC rearranges the Ag presentation and costimulatory molecular machinery at the DC interface with the T-cell.
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). We found that ICAM-3 and ICAM-1 were able to induce actin reorganization at the contact area of DC with ICAM-3+ICAM-1–coated latex microspheres. Our results show not only that ICAMs binding triggers actin rearrangement on the interacting area of DC (Figure 6, A and B), but also that the disruption of actin cytoskeleton impairs MHC-II clustering. In this regard, clustering of MHC-II induced by ICAM-3 was blocked by Latrunculin A, an inhibitor of actin polymerization, but no significant effect was exerted by tubulin disrupting drugs such as taxol (Figure 6C). These data support the active role of the DC actin cytoskeleton during IS formation. In contrast, DC microtubular network and MTOC positioning remain unaffected by ICAM-mediated initial cellular interactions (Figure 6A). To assess whether the actin cytoskeleton-associated signaling machinery is triggered by ICAMs engagement on DC, we analyzed the activation of Vav1 and GTPase Rac, two key molecules in actin cytoskeleton rearrangement. Phosphorylation of Vav1 in Tyr 174, which is essential for Vav1 activation, was studied in DC stimulated with latex microspheres coated with ICAM-1, ICAM-3, or both. ICAM-3, and ICAM-1 to a lower extent, induced Vav1 phosphorylation at this residue with a kinetics profile that peaked at 10 min (Figure 6D). Consistent with Vav1 activation, Rac1 activation was also observed in DC treated with ICAMs (Figure 6E). The kinetics of Rac1 activation correlated with that of Vav1 phosphorylation, suggesting that the activation of Vav1 and Rac1 are causally related (Figure 6E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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During initial contacts, low-affinity adhesive interactions facilitate the exploration of the DCs surface by T-cells. ICAM-3 is expressed by all leukocytes, particularly by naive T lymphocytes, and is essential in the initial scanning of the APC surface by T-cells, clustering at the contact area during the first stages of the T-APC contact (Montoya et al., 2002a). Interestingly, ICAM-3 binding to LFA-1 on DCs induces the redistribution of MHC-II, which suggests the existence of an adhesion-dependent component in MHC-II lateral mobility on the membrane of the DC. Thus, our data support a key role for ICAM-3 and, to a lower extent ICAM-1, in the initial contact with DCs, mediating cell adhesion and facilitating the priming of T-cells by inducing an enhancement of the density of MHC-II and the costimulatory molecule CD86 on DC-T-cell contact area prior to TCR Ag recognition. These data are further supported by the relocation on DC of MHC class II at the contact area with naïve T-cells in the absence of Ag. Previously it has been described that immunological synapses are formed between DCs and polyclonal naive T-cells in the absence of antigen (Revy et al., 2001). The clustering of MHC class II induced by ICAMs is observed in mature but not immature DCs. In this regard, recent findings have described the presence of organized IS in conjugate of mature, but not immature, DCs with naive T lymphocytes (Benvenuti et al., 2004b).
Our data based on the use of both peptide and antibody blocking reagents demonstrate that LFA-1 is the ICAM-3 counterreceptor implicated in the induction of MHC class II clustering on DCs. It has been previously described that some B lymphoma APC supported CD4 clustering in the absence of antigen or during presentation of null peptides, and this CD4 recruitment was dependent on MHC class II, and LFA-1 on the APC (Zal et al., 2002). LFA-1 is relocalized to the contact area within the first 5 min during the process of IS formation, later than MHC-II molecules. This suggests that an organized cluster of LFA-1 is not required for its role in MHC class II clustering. Also, the apparent temporal dissociation between clustering of MHC-II and of LFA-1 would rule out the simplest explanation of the findings, i.e., that a cluster of MHC-II is induced by ICAM-3 through a lateral association with LFA-1.
ICAM-3–mediated MHC-II clustering may be related to the inclusion of the latter into lipid (rafts) or protein (tetraspanin-based) membrane microdomains (Anderson et al., 2000; Kropshofer et al., 2001; Hemler, 2003). We found that MHC-II molecules clustered by interaction with ICAM-coated microspheres were associated with tetraspanins as demonstrated by CD81 clustering and detection of MHC-II clusters with the mAb FN1. Although it has been reported that MHC-II molecules on B-cells seem to cluster into detergent-resistant membrane microdomains (Anderson et al., 2000), recent evidence indicates that rafts exhibit a random distribution on the T-cell during IS formation (Glebov and Nichols, 2004).
The enrichment of MHC-II at the synapse could be due at least in part to an enhanced polarized transport of intracellular MHC class II to the synapse. In this regard, it has been described that cytochalasin D treatment leads to a delayed appearance of stable forms of class II molecules and a reduced presentation efficiency of Ag determinant in B-cells (Barois et al., 1998). Boes et al. (2002) have observed that interaction of Ag-loaded DCs with Ag-specific CD4 T-cells induces the formation of long tubular class II MHC-positive compartments that polarize toward the interacting T-cell. Under our experimental conditions we did not observe those class II MHC compartments. These apparent discrepancies could be attributed to specific characteristics of T-cells and APCs used in the different experimental systems. Thus, in our work, DCs were loaded with SEB, which does not require endosomal processing, and it has been described that endosomal tubulation requires acquisition of peptide in endosomal compartments (Bertho et al., 2003). Our results show not only that ICAM-3 binding triggers actin rearrangement on the interacting area of DC, but also that the disruption of actin cytoskeleton impairs MHC-II clustering. These data that support the active role of the DC cytoskeleton during IS formation complement those previously described by other group (Al-Alwan et al., 2001, 2003) Our data clearly demonstrate that cytoskeletal reorganization is required for ICAM-driven MHC-II clustering.
ICAMs binding to DC triggers activation of Vav1 and GTPase Rac1, in accordance with the promotion of actin rearrangement. Vav1 regulates integrin clustering on the T-cell during Ag presentation (Krawczyk et al., 2002), being involved in TCR-
2 integrin cross-talk during this process (Ardouin et al., 2003). Despite being expressed on DC and B-cells, the role of Vav in supramolecular activation on the APC side of the IS has not yet been addressed. Recently, it has been described the role of both Rac1 and Rac2 in the initial phases of DC-T-cell interactions and in T-cell priming. Rac1/2–/– DCs have an impaired ability to interact with naïve T-cells, as well as in priming T-cells (Benvenuti et al., 2004a). Our data provide a mechanism to Rac activation in DCs by naïve T-cells at early phases of synapse formation, linking cytoskeletal rearrangements with clustering of plasma receptors such as LFA-1 and MHC-II molecules to the DC area in close contact with T-cells.
In summary, our results demonstrate that the initial binding mediated by ICAM-3 at the T-cell side, to LFA-1 on the DC side triggers the clustering of MHC class II and other costimulatory molecules toward DC synapse area, before Ag recognition by TCR. This provides a mechanism for a more efficacious presentation of peptide-MHC complexes by DCs.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: Ag, antigen; APC, Ag presenting cell; c-SMAC, central supramolecular activation complex; DC(s), dendritic cell(s); DIC, differential interference contrast; Gp, glycophorin; IS, immune synapse; p-SMAC, peripheral supramolecular activation complex; SEB, staphylococcal enterotoxin B.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Address correspondence to: Francisco Sánchez-Madrid (fsanchez.hlpr{at}salud.madrid.org).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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