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Originally published online as doi:10.1189/jlb.0103045 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;74:40-48.)
© 2003 by Society for Leukocyte Biology

Composition of MHC class II-enriched lipid microdomains is modified during maturation of primary dendritic cells

Niclas Setterblad*, Corinne Roucard*, Claire Bocaccio{dagger}, Jean-Pierre Abastado{dagger}, Dominique Charron* and Nuala Mooney*

* INSERM U396 and
{dagger} IDM (Immuno-Designed Molecules), Institut Biomédical des Cordeliers, Paris, France

Correspondence: Nuala Mooney, INSERM U396, Institut Biomédical des Cordeliers, 15, rue de l’école de médecine, 75006 Paris, France. E-mail: nuala.mooney{at}bhdc.jussieu.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) are the most potent antigen presenting cells. Major histocompatibility complex (MHC) class II molecule expression changes with maturation; immature DCs concentrate MHC class II molecules intracellularly, whereas maturation increases surface expression of MHC class II and costimulatory molecules to optimize antigen presentation. Signal transduction via MHC class II molecules localized in lipid microdomains has been described in B lymphocytes and in the THP-1 monocyte cell line. We have characterized MHC class II molecules throughout human DC maturation with particular attention to their localization in lipid-rich microdomains. Only immature DCs expressed empty MHC class II molecules, and maturation increased the level of peptide-bound heterodimers. Ligand binding to surface human leukocyte antigen (HLA)-DR induced rapid internalization in immature DCs. The proportion of cell-surface detergent-insoluble glycosphingolipid-enriched microdomain-clustered HLA-DR was higher in immature DCs despite the higher surface expression of HLA-DR in mature DCs. Constituents of HLA-DR containing microdomains included the src kinase Lyn and the cytoskeletal protein tubulin in immature DCs. Maturation modified the composition of the HLA-DR-containing microdomains to include protein kinase C (PKC)-{delta}, Lyn, and the cytoskeletal protein actin, accompanied by the loss of tubulin. Signaling via HLA-DR redistributed HLA-DR and -DM and PKC-{delta} as well as enriching the actin content of mature DC microdomains. The increased expression of HLA-DR as a result of DC maturation was therefore accompanied by modification of the spatial organization of HLA-DR. Such regulation could contribute to the distinct responses induced by ligand binding to MHC class II molecules in immature versus mature DCs.

Key Words: HLA • rafts • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) have an unequaled ability for antigen presentation, which is most clearly demonstrated by their capacity to activate naïve T cells [1 ]. Immature DCs are recruited to the site of infection in response to proinflammatory stimuli. Maturation takes place in the course of migration, thereby allowing DCs to reach the germinal centers as fully competent antigen presenting cells (APCs), where they encounter and activate specific T lymphocytes [2 ]. The immature DC is specialized in antigen capture, whereas the mature DC is the most efficient APC [3 4 5 ]. Immature DCs are characterized by the abundance of major histocompatibility complex (MHC) class II molecules localized in intracellular compartments, the rapid turnover of MHC class II [6 , 7 ], and their greater ability for endocytosis [8 ]. A different distribution of proteases involved in antigen processing is observed in mature DCs [9 , 10 ] together with a higher level of cell-surface MHC class II expression, a longer half-life of MHC class II molecules, and a down-regulated capacity to internalize antigen [3 , 11 ]. A further distinction is the expression of empty MHC class II molecules at the surface of immature DCs [12 ]. These empty molecules have been proposed to play a role in antigen capture and processing at the cell surface in collaboration with the nonclassical MHC class II molecule human leukocyte antigen (HLA)-DM, which is also detected at the surface of immature DCs [13 , 14 ]. Studies in human and murine DCs have shown that immature cells can rapidly endocytose surface MHC class II proteins, whereas mature DCs have lost this ability [3 , 11 ].

MHC class II proteins have been widely documented as signal-transducing molecules, and their engagement leads to activation of the serine/threonine kinase protein kinase C (PKC) as well as tyrosine kinases [15 , 16 ]. Studies demonstrating the localization of various immunoreceptors, such as the B cell receptor (BCR) and the T cell receptor (TCR), in discrete membrane microdomains have led to a revision of the notion of their random distribution throughout the plasma membrane (see ref. [17 ] for review). The site of interaction between T lymphocytes and APCs is specifically enriched in signaling proteins localized in lipid membrane microdomains or detergent-insoluble glycolipid-enriched complexes (DIGs), also known as glycolipid-enriched membrane microdomains or lipid rafts [18 , 19 ]. The proportion of total cellular protein localizing in lipid-rich microdomains has been determined as only 2% [20 ]. Detection of a given protein in such microdomains therefore indicates a highly specific partitioning.

DCs are used for immunotherapy because of their outstanding ability to activate naïve T cells [21 ], but it remains unclear whether immature or mature DCs are more appropriate. Immature DCs have the advantage of longer survival, although tolerance induction has been reported in some cases [22 , 23 ]. Immature and mature DCs have well-documented differences in their abilities to present antigen and to generate antitumoral-immune responses in vitro and in vivo [24 ]. Further, they have markedly different responses to signal transduction via MHC class II molecules. Mature DCs undergo apoptosis, whereas immature DCs are relatively resistant to MHC class II-mediated apoptosis [25 , 26 ].

The localization of MHC class II molecules within DIGs in human monocytes determines their ability to transmit signals after ligand binding [27 ], and DIGs have been implicated in antigen presentation by B lymphocytes in conditions of limited peptide availability [28 ]. Tetraspan-containing MHC class II complexes have also been attributed a key role in antigen presentation on the basis of the relatively homogeneous nature of the peptides associated with MHC class II within such complexes [29 ]. As HLA-DR-containing microdomains have not been characterized in human DCs, we examined the expression and conformation of MHC class II molecules in immature versus mature DCs prepared according to a protocol developed for the clinical use of DCs in immunotherapy [30 ]. We have exploited the detergent insolubility of lipid-enriched microdomains to isolate and examine the constituents colocalizing with HLA-DR in immature and mature DC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DC preparation and cell treatment
Immature DCs were prepared as described previously [30 ]. Briefly, immature DCs were differentiated from peripheral blood mononuclear cells of healthy donors with granulocyte macrophage-colony stimulating factor and interleukin-13. Cells were kept immature or further matured for 48 h with monoclonal anti-CD40 antibody (clone J285, American Type Culture Collection, Manassas, VA) and Poly-IC (Sigma Chemical Co., St. Louis, MO). DCs accounted for 80–90% of the total population of HLA-DR-expressing cells in the immature DC preparations and for 70–80% of the total HLA-DR population in the mature DC preparations. Where indicated, cells were stimulated with HLA-DR monoclonal antibody (mAb) L243 [fluorescein isothiocyanate (FITC)-labeled or not] at 37°C for 15 min. Control cells were incubated under the same conditions with an immunoglobulin G (IgG)2a isotype control (FITC-labeled or not). Each experiment presented in this study was performed independently on three different donors.

Flow cytometry analysis
Cells (105) were washed in phosphate-buffered saline (PBS) and preincubated in 5% fetal calf serum (FCS), 100 µg/ml human {gamma}-globulins, and 0.02% azide in PBS. Cells were incubated with directly labeled mAb for 30 min in PBS, 1% bovine serum albumin (BSA), and azide before washing and analysis on a FACScan flow cytometer (CellQuesttm, Becton Dickinson, San Jose, CA). Immature and mature DCs were characterized with the following antibodies: anti-CD14-PECy5, anti-CD80-FITC, anti-CD83-FITC, anti-CD86-FITC, and anti-HLA-DR-FITC (Becton Dickinson).

Confocal analysis of HLA-DR localization and internalization
For analysis of total HLA-DR molecule localization, immature and mature DCs were allowed to attach to poly-L-lysine-coated slides for 15 min at 37°C. Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 20 min, incubated in NH4Cl 50 mM for 10 min, and permeabilized before incubation with L243-FITC mAb for 45 min, washing, and mounting with Vectashield (Vector Laboratories, Burlingame, CA). For analysis of HLA-DR internalization, immature and mature DCs were allowed to adhere to poly-L-lysine-coated slides for 15 min at 37°C and were then incubated on ice in complete RPMI medium with 100 µg/ml human {gamma}-globulins, followed by L243-FITC (10 µg/ml) for 30 min at 4°C. Cells were washed and kept on ice or washed before incubation at 37°C for 15 min. Cells were fixed in 4% PFA in PBS for 20 min and incubated in NH4Cl 50 mM for 10 min. After washing in PBS, slides were mounted with Vectashield before analysis on a Zeiss LSM 510 confocal microscope (Zeiss, Jena, Germany).

Detection of empty MHC class II molecules
A recombinant soluble human invariant chain (Iisol; amino acid sequence 72–216 of human invariant chain p31) was produced as described [31 ] with some modifications. Briefly, Iisol was produced in Escherichia coli strain BL21 DE3 pLysS and was purified under nondenaturing conditions on a Nickel affinity column. After purification, Iisol was dialyzed against PBS, frozen, and stored at -80°C. The trypsin inhibitor (TI; Sigma Chemical Co.), which has a similar molecular weight as Iisol, was used to detect nonspecific binding as described previously [31 ]. Iisol and TI were directly labeled with FITC (Molecular Probes, Eugene, OR) for 2 h, dialyzed against PBS, and stored at 4°C before use in flow cytometry. Cells (105) were washed in PBS and preincubated in 5% FCS, 100 µg/ml human {gamma}-globulins, and 0.02% azide in PBS. Cells were incubated with TI-FITC or Iisol-FITC (50 µg/ml) for 1 h in PBS, 1% BSA, and azide before washing and analysis on a FACScan flow cytometer (CellQuesttm, Becton Dickinson).

DIG preparation
Cells were washed twice in cold PBS and resuspended in 0.4 ml ice-cold MBS (25 mM 2-[N-morpholino]ethanesulfonic acid (MES), 150 mM NaCl, pH 6.5), 0.5% Triton X-100, 1 mM Na3VO4, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mg/ml aprotinin. Following a 15-min lysis on ice, the lysates were mixed with 0.6 ml 85% sucrose (w/v) in MBS (final concentration, 50% sucrose) and were deposited at the bottom of a SW55ti centrifuge tube. The sample was then overlaid with 1 ml 40% sucrose, 1 ml 30% sucrose, and 1 ml 5% sucrose in MBS containing 1 mM Na3VO4, 2 mM EDTA, 1 mM PMSF, and 1 µg/ml aprotinin and was centrifuged for 14 h at 200,000 g at 4°C. Ten fractions were obtained by collecting 400 µl fractions from the bottom of the tube to the top. The visible band at the 5/30% sucrose interface containing the DIGs corresponded to Fraction 8.

Fluorometric analysis of MHC class II cell-surface expression in lipid microdomains
Cells were directly labeled with L243-FITC on ice for 15 min at a concentration of 10 µg/ml and lysed as described above. Sucrose gradients were harvested in 10 fractions (400 µl each). A VersaFluor fluorometer (Biorad, Hercules, CA) was used to quantify the total fluorescence in each fraction. Each fraction (200 µl) was diluted in 1.8 ml MBS buffer, and the fluorescence was measured in arbitrary fluorescence units. FITC-labeled mouse isotype control IgG2a (Becton Dickinson) was used in parallel to FITC-L243 to detect nonspecific binding.

Western-blot analysis
Total cell lysates (TCL; 5 µg prepared in 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 1 mM EDTA, 1 mM PMSF, 10 mM NaF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 4 µg/ml aprotinin on ice for 30 min) or 10 µl fractions collected from sucrose density gradients were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. For SDS stability determination, TCL or sucrose gradient fractions were incubated in reducing Laemmli sample buffer for 30 min at room temperature before migration by SDS-PAGE. For Western blot analysis, samples of sucrose gradient fractions were incubated in reducing Laemmli sample buffer and denatured at 95°C for 10 min before SDS-PAGE migration. Proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Uppsala, Sweden) in Tris-glycine buffer. Membranes were blocked in 5% skimmed milk in PBS overnight at 4°C and incubated with the following primary antibodies: mouse mAb anti-HLA-DR{alpha} DA6-147; mouse mAb anti-HLA-DM{alpha} 5C1 [32 ] (kindly provided by John Trowsdale, Cambridge, UK); rabbit polyclonal anti-HLA-DOß (kindly provided by John Trowsdale); mouse mAb anti-CD45 (Upstate Biotechnology, Lake Placid, NY); mouse mAb antiactin (MAB1501, Chemicon, Temecula, CA); mouse mAb anti-tubulin (TUB2.1, Sigma Chemical Co.); and rabbit polyclonal antibodies anti-Lyn (sc-15) and anti-PKC-{delta} (sc-937) from Santa Cruz Biotechnology (Santa Cruz, CA) for 2 h at room temperature in 2.5% skimmed milk in PBS. They were then washed in 0.1% Triton X-100–PBS before incubation with secondary antibody directly labeled with horseradish peroxidase (HRP) and washed in 0.1% Triton X-100–PBS before detection by enhanced chemiluminescence (Amersham Biosciences). GM1 was detected by immunoblotting the membranes with HRP-labeled cholera toxin ß subunit (Sigma Chemical Co.) for 1 h at room temperature.

Quantification of autoradiographs
Autoradiographs were scanned with a Kodak scanner, and bands were quantified with the Scion Image software (Scion Corp., Frederick, MD) based on the National Institutes of Health Image software developed by Wayne Rasband.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of immature and mature DC
The maturation state of the DCs used in this study was confirmed by fluorescence-activated cell sorter (FACS) analysis. Immature DCs were CD14-positive, CD83-negative, and expressed HLA-DR. In contrast, mature DCs had increased expression of HLA-DR (Fig. 1A ), had lost expression of CD14, and had gained expression of CD83 (Fig. 1A) . CD80 and CD86 expression was also increased by maturation (data not shown).



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  		Figure 1. (A) Phenotypic characterization of immature and mature DCs. Freshly prepared immature (DCi) and mature DCs (DCm) from the same donor were stained with directly labeled antibodies against CD83, CD14, and HLA-DR (L243 mAb) before FACS analysis. (B) Intracellular localization of HLA-DR molecules in DCs. In DCi, HLA-DR expression is mainly intracellular, whereas it is localized at the cell surface in DCm. DCi and DCm from the same donor were allowed to adhere to poly-L-lysine-coated coverglasses, fixed and permeabilized. Cells were stained with the FITC-labeled anti-HLA-DR mAb L243. Images were acquired on a Zeiss confocal microscope. A 10-µm scale is indicated. (C) SDS stability of MHC class II complexes indicate a higher level of peptide association with HLA-DR in DCm. TCL of DCi and DCm from the same donor were quantified, and 5 µg protein was loaded on a 10% SDS-PAGE gel and immunoblotted to detect HLA-DR{alpha}. SDS-stable {alpha}ß complexes and free {alpha} chains (after boiling) are enriched in mature DCs. Molecular weights are indicated on the left. Actin was immunoblotted to confirm equivalent protein loading and transfer.

 
Confocal microscopy confirmed morphological differences between immature and mature DCs as well as the modification of HLA-DR localization upon maturation. In immature DCs, intracellular HLA-DR expression was detected, and cell membrane expression was relatively heterogeneous. In mature cells, the majority of HLA-DR was homogeneously distributed at the cell surface, and little intracellular expression was observed (Fig. 1B) .

Mature DCs express more peptide-bound HLA-DR than immature DCs
The resistance of a MHC class II molecule to SDS detergent at room temperature indicates that the heterodimer has been stabilized by peptide binding. MHC class II molecules, which have not bound peptide, dissociate into free {alpha} and ß chains when incubated in SDS-containing buffer [33 ]. TCL of mature or immature DCs from the same donor were incubated in SDS buffer before migration on SDS-PAGE, and {alpha}ß heterodimers as well as free {alpha} chains were detected by immunoblotting with an anti-HLA-DR{alpha} mAb (Fig. 1C) . Immature and mature cells expressed dimers. Mature DCs clearly expressed a higher level of peptide-stabilized HLA-DR{alpha}ß molecules (55-kDa band) and of the corresponding band of free {alpha} chains (30 kDa) after denaturation (Fig. 1C) . Quantification of the autoradiographs revealed that mature DCs contain twice the amount of stable {alpha}ß dimers detected in immature DCs (Table 1 ). The ratios of {alpha} chain expression in {alpha}ß dimers to the total amount of {alpha} chain are comparable in immature and mature cells (~50%) measured in three donors.


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  		Table 1. Quantification of HLA-DR{alpha}ß Dimers in Immature and Mature DCs

 
Characterization of HLA-DR internalization
Internalization of HLA-DR molecules is a characteristic of immature DCs. We examined internalization by incubating live DCs with a FITC-labeled anti-HLA-DR mAb. HLA-DR engagement results in rapid endocytosis of HLA-DR molecules in immature DCs after 15 min at 37°C (Fig. 2B ), whereas no internalization of HLA-DR molecules is observed in mature DCs (Fig. 2D) . Incubation of mature DCs with HLA-DR mAb-induced cell aggregation (Fig. 2D) as previously reported [34 , 35 ]. These data demonstrate that ligand binding to HLA-DR at the cell surface initiates signaling and distinguishes the immature from the mature DCs.



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  		Figure 2. Internalization of HLA-DR in immature DCs is induced by cell-surface HLA-DR ligand binding. Freshly prepared DCi and DCm from the same donor were allowed to adhere to poly-L-lysine-coated coverglasses and were then incubated with the L243-FITC mAb for 30 min at 4°C. Cells were directly fixed (A and C) or incubated for 15 min at 37°C to allow internalization before fixing (B and D). Images were acquired on a Zeiss confocal microscope with identical parameters. A 10-µm scale is indicated.

 
Immature and mature DCs express cell-surface HLA-DR localized in DIGs
The distribution of cell-surface HLA-DR molecules in DIGs from immature and mature DCs was analyzed. The HLA-DR molecules were labeled with L243-FITC antibody on ice before lysis to prevent endocytosis of the antibody. Cells were lysed in Triton X-100, and lysates were fractionated on a sucrose density gradient before measurement of fluorescence throughout the gradient. Fluorescence was expressed in arbitrary fluorescence units (Fig. 3A ). Nonspecific fluorescence detected with a FITC-labeled isotype control Ig was low (30–100 fluorescence units). The fluorescence profile obtained (Fig. 3A) reveals that the majority of the HLA-DR protein is found in the high-density fractions corresponding to detergent-soluble material (Fractions 1–4). However, Fraction 8, from immature and mature DC, is selectively enriched in surface-labeled HLA-DR molecules. Based on their flotation properties in the sucrose gradient, these fractions correspond to a lipid-rich partition. In mature DCs, cell-surface HLA-DR was also detected in fractions of intermediate density (Fractions 5–7). Maturation of DCs increases the total amount of cell-surface HLA-DR at least twofold (total fluorescence, 24,132, in mature DCs compared with 11,535 in immature DCs, measured in arbitrary fluorescence units), whereas the amounts of DIG-associated HLA-DR detected in Fractions 8 are comparable in mature and immature DCs (1239 and 1865 fluorescence units, respectively). The proportion of DIG-associated cell-surface HLA-DR in immature DCs is therefore threefold higher (15.2%) than in mature DCs (4.7%).



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  		Figure 3. (A) Cell-surface HLA-DR is constitutively present in DIGs in DCi and DCm, as revealed by fluorometric analysis of sucrose gradients. HLA-DR is expressed in Fraction 8, corresponding to the lipid raft-enriched fraction in immature and mature DCs. Cells were incubated on ice with L243-FITC before lysis with nonionic detergent and fractionation on a sucrose gradient. Fluorescence from each fraction was quantified on a fluorometer and expressed in arbitrary fluorescence units. (B) Analysis of SDS-stable HLA-DR complexes in DIG fractions (Fraction 8), which were migrated on 10% SDS-PAGE gels with (+) or without (-) boiling to detect free HLA-DR {alpha} or SDS-stable {alpha}ß complexes. The contaminating band, as a result of the maturation stimulus (CD40 antibody), is indicated (IgH). (C) Detection of empty MHC class II molecules at the cell surface. Empty MHC class II molecules are detected at the cell surface of DCi. DCi and DCm were incubated with 50 µg/ml Iisol-FITC (solid lines) or with a control protein TI-FITC (dotted lines) for 1 h at 4°C before analysis on a FACScan cytometer.

 
Detection of peptide-bound HLA-DR in DIG fractions
As SDS-stable HLA-DR complexes were more abundant in TCL from mature DCs compared with immature DCs (Fig. 1C) , we next analyzed the SDS stability of the DIG-localized HLA-DR in Fraction 8 from the fluorometric analysis. The 55-kDa heterodimers were only weakly detected in DIGs isolated from immature DCs (Fig. 3B) . The 30-kDa band corresponding to the free DR{alpha} chain was detected after denaturation. In contrast, in mature DCs, a 55-kDa band corresponding to the heterodimer was clearly detected, and the band corresponding to free DR{alpha} chains was increased after boiling. The HLA-DR{alpha} free chains are always more readily detected than the heterodimers as a result of the differential exposure of epitopes before and after boiling. Together, these data indicate that peptide-stabilized HLA-DR molecules are more enriched in DIGs isolated from mature DCs than in DIGs from immature DCs.

Cell-surface expression of empty MHC class II molecules on immature and mature DCs
Empty MHC class II was detected at the cell surface with a FITC-labeled soluble invariant chain (Ii) probe (Iisol-FITC) [31 ]. Binding of the probe to immature and mature DCs was quantified by FACS analysis (Fig. 3C , solid line). The FITC-labeled TI [31 ] (Fig. 3C , dotted line) provided a nonspecific binding control. The histogram plots reveal that immature DCs express empty MHC class II molecules at the cell surface, which are absent from mature DCs despite the higher overall level of expression of surface HLA-DR (Fig. 3C) .

Characterization of HLA-DR-enriched DIGs
The data presented in Figure 3 indicate that a fraction of cell-surface HLA-DR localizes in DIGs. Triton X-100 lysates were prepared from immature and mature DCs, stimulated or not via HLA-DR to characterize total cellular DIG constituents. The specific DIG marker ganglioside GM1 was present in Fractions 7 and 8 in immature and mature DCs and was unchanged by HLA-DR-engagement (Fig. 4 ). CD45, a transmembrane protein, which is constitutively excluded from DIGs [36 ], was exclusively found in the soluble fractions (Fractions 1 and 2) of the gradients in immature and mature DCs. HLA-DR was readily detected in the fractions corresponding to DIGs (Fractions 7 and 8) in immature and mature DCs (Fig. 4) , although the majority was confined to the high-density fractions (Fractions 1–3).



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  		Figure 4. Western blot analysis of sucrose gradients from DCi and DCm: Sucrose density gradients of TCL were analyzed by immunoblotting. Detection of HLA-DR and HLA-DM in DIGs from DCi and DCm: The localization of HLA-DR and HLA-DM in high-density (soluble) and low-density (DIG) fractions was analyzed by immunoblotting, and HLA-DR and HLA-DM are constitutively present in DIGs (Fractions 7 and 8) in DCi and DCm. Fractions were separated on PAGE gels, transferred on a PVDF membrane, and immunoblotted. Localization of PKC-{delta} to DIGs was observed only in mature cells, and further recruitment of PKC-{delta} was induced in mature DCs by HLA-DR-mediated signaling. Lyn was expressed constitutively in lipid-rich microdomains from immature or mature DCs. Maturation increased the Lyn expression in DIGs. Actin and tubulin detection in DIGs in DCi and DCm: Distribution of actin and tubulin in sucrose gradient was analyzed with specific mAb by Western blotting. Actin was detected in DIGs from DCi and DCm. Maturation and MHC class II engagement increased the actin expression in DIGs. Tubulin was specifically localized in DIGs from DCi, whereas maturation led to a diffuse expression throughout the gradient. Localization of DIG-marker ganglioside GM1 and of non-DIG-marker CD45 throughout the sucrose gradients: Membranes were incubated with a CD45 antibody or with a HRP-labeled cholera toxin ß subunit. Fractions 7 and 8 contained the DIG-specific marker GM1, whereas CD45 was solely detected in the soluble fractions. Intermediate fractions (4–6) contained neither GM1 nor CD45.

 
In immature DCs, ligand binding to HLA-DR did not modify the distribution of HLA-DR. In contrast, signaling via HLA-DR in mature DCs led to MHC class II (HLA-DR and -DM) enrichment in fractions of intermediate density (Fractions 4–6) as well as in GM1-containing fractions (see Fractions 7). HLA-DR engagement in mature DCs initiated a coordinated redistribution of HLA-DR and HLA-DM. Presence of the nonclassical MHC class II molecule HLA-DO was examined by screening TCL, and expression was not detected in immature or mature DCs (data not shown).

PKC-{delta} is recruited to DIGs via HLA-DR-mediated signaling in mature DCs
Lipid rafts have been described to provide a highly favorable microenvironment for signal transduction as a result of the local concentration of specific kinases and phosphatases (reviewed in ref. [37 ]), and previous studies have revealed the intimate association between MHC class II molecule signaling and the PKC family [15 , 38 ]. The PKC-{delta} isoenzyme has been attributed a role as a proapoptotic isoenzyme in different cell types including mature DCs [39 40 41 ]. In immature DCs, PKC-{delta} expression was restricted to detergent-soluble fractions, and stimulation via HLA-DR did not modify this distribution (Fig. 4) . In contrast, in mature DCs, PKC-{delta} was also present in the intermediate fractions (Fractions 4–6) and in the GM1-containing DIG fraction (Fractions 7 and 8). HLA-DR ligand binding increased the amount of PKC-{delta} in intermediate and DIG fractions.

DC maturation recruits Lyn to DIGs
Src tyrosine kinases readily localize to lipid microdomains through their myristoylated N-terminal domains. The recruitment and activation of the src-family kinase Lyn to DIGs have been reported upon HLA-DR signaling in THP-1 monocytes [27 ]. Lyn expression was concentrated in DIGs from immature DCs, and the level of expression was increased by maturation. In contrast to the THP-1 cell line, Lyn is therefore constitutively present in DIGs isolated from primary human DCs.

Differential expression of actin and tubulin in DIGs isolated from mature versus immature DCs
The actin cytoskeleton is important for optimal antigen presentation via MHC class II molecules [42 ] and plays an active role in the formation of the immunological synapse at the interaction site of the TCR with the APC [43 44 45 ]. Actin-dependent cytoskeletal modifications have been identified as key events in TCR-mediated signaling [46 ].

Actin was absent from the DIG fractions of immature DCs, whereas it was constitutively expressed in DIGs isolated from mature cells (Fig. 4) . In immature DCs, HLA-DR signaling did not significantly alter the localization of actin. In contrast, in mature DCs, actin was recruited to intermediate density and DIG fractions by HLA-DR engagement.

Tubulin was examined in parallel, as it is a cytoskeletal component that is excluded from DIGs in lymphocytes [36 ], although it has been described as a DIG constituent in neurons [47 ]. In immature DCs, tubulin is a specific DIG constituent, and maturation of DCs led to diffusion of tubulin throughout the gradient (Fig. 4) . HLA-DR engagement did not influence the distribution of tubulin in immature or mature DCs. The microdomain association of the tubulin and actin cytoskeletons is therefore remodeled by DC maturation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
DCs play a key role in the immune system because of their unique capacity to activate naïve helper T lymphocytes by peptide-specific antigen recognition. Such activation involves the formation of an immunological synapse enriched in costimulatory molecules and signaling proteins (reviewed in ref. [18 ]). Recent studies have revealed that there is not a single type but rather a variety of immunological synapses and that their formation can even occur in the absence of exogenous antigen [48 ]. Lipid-enriched microdomains accumulate at the site of the immunological synapse [49 ], and the integrity of the lipid rafts is essential for optimal signal transduction via the TCR [50 ].

Although the DC is the most potent APC, little attention has been devoted to the characterization of DIGs in DCs or to the outcome of HLA-DR-mediated signaling. Specific ligand binding to HLA-DR in the myelomonocytic cell line THP-1 led to a recruitment of HLA-DR to lipid rafts, which was necessary for the HLA-DR-mediated activation of the src-kinase Lyn [27 ]. In a B cell line, ligand binding to HLA-DR led to enrichment of the HLA-DR content in DIGs in parallel with the recruitment of the F-actin cytoskeleton to the site of ligand binding [51 ]. A role for lipid-enriched microdomains in antigen presentation via MHC class II molecules has been proposed, as lipid raft localization of the MHC class II proteins enhanced peptide presentation under conditions of limited peptide availability [28 ]. The present study examined DCs obtained by a protocol developed for the generation of immature and mature DCs for clinical use [30 ]. As differentiation and maturation steps differ from the more traditional protocols, the phenotypical analysis was carefully performed, and the results convincingly demonstrated that bona fide DCs were obtained by this protocol.

The soluble invariant chain (Iisol) has been used to detect empty MHC class II molecules, and its binding was abrogated by peptide loading [31 ]. In the present study, fixation of Iisol was restricted to immature DCs despite the higher cell-surface expression of MHC class II molecules on mature DCs, reinforcing the notion that MHC class II molecules on immature DCs can efficiently be loaded with a given peptide before maturation to provide optimal antigen presentation for therapeutic applications.

The increased expression level of MHC class II proteins at the surface of mature DCs was not reflected by an increase in DIG-localized cell-surface HLA-DR. This could be the consequence of a cell-dependent limitation in the amount of HLA-DR molecules, which can be accommodated within lipid-rich microdomains at a given time. However, we demonstrated that peptide-stabilized HLA-DR molecules are more enriched in DIGs isolated from mature DCs than in DIGs from immature DCs. This indicates that maturation leads to the exchange of nonpeptide-loaded MHC class II molecules by peptide-loaded MHC class II molecules within existing DIGs or that existing MHC class II-containing DIGs are progressively replaced by de novo synthesized DIGs.

Engagement of MHC class II proteins with a specific ligand induces signaling and provides a model for some of the events following APC peptide-specific T cell interaction. HLA-DR engagement in mature DCs redistributed HLA-DR and -DM to intermediate density fractions without any decrease of DIG-clustered HLA-DR and -DM molecules. Fractions of intermediate density are characteristic of microdomains undergoing lipid enrichment and have been referred to as "barges" [52 ]. As HLA-DM is not expressed at the cell surface of mature DCs [12 ] and our observation, the HLA-DM localization to barges is likely to be of intracellular origin, and the coordinated relocalization of HLA-DR and -DM suggests a redistribution of endosomal compartments. As neither CD45 nor GM1 was observed in intermediate fractions, the recruitment of proteins to such fractions is not the consequence of incomplete detergent solubilization or of cross-contamination of sucrose gradient fractions.

Evidence for DIGs providing an environment beneficial to signal transduction has been provided by studies of the TCR and the BCR [36 , 53 ]. The only DC study addressing the role of DIGs in signaling concerns CD40 and revealed that the DIG localization of CD40 was essential for TNF receptor-associated factor recruitment, ultimately leading to cytokine production [54 ]. The DC response to MHC class II-mediated signals depends on their state of maturation. Mature DCs undergo apoptosis, whereas immature DCs are relatively resistant [26 ]. MHC class II engagement has been reported to lead to differential regulation of maturation as well as apoptosis in human monocyte-derived DCs [35 ]. Mature B lymphocytes also undergo apoptosis via HLA-DR [55 ], and humanized HLA-DR mAb have been successfully used to induce apoptosis of malignant APCs in vivo in a primate model [56 ]. We have recently described a PKC-{delta}-dependent HLA-DR-mediated pathway of mature DC apoptosis [41 ]. The present study characterizes the PKC-{delta} isoenzyme as a constituent of HLA-DR and Lyn kinase-containing lipid rafts in mature DCs. In T lymphocytes, PKC-{theta} is recruited to lipid rafts before their polarization to the T cell–APC contact site [49 ]. PKC-{delta}, which shares high pseudosubstrate homology with PKC-{theta}, could be implicated in the cytoskeletal modifications of the APC in a similar way to that described for PKC-{theta} [57 ].

Tubulin is excluded from lipid microdomains in the lymphoid lineage [36 ], whereas neuron-derived lipid rafts contain tubulin [47 ]. In oligodendrocytes, activation of the Fyn kinase in lipid rafts is critical for tubulin recruitment and rearrangement at the site of process outgrowth [58 ]. The data presented in this study raise the possibility that tubulin colocalization with Lyn in lipid raft domains plays a role in the dendrite outgrowth induced by DC maturation. In addition to actin accumulation in T lymphocytes at the site of the immunological synapse [59 ], microscopic examination of DC–T lymphocyte conjugates revealed F-actin accumulation in DCs at the cell–cell interface [60 ], indicating an active participation of the DC–actin cytoskeleton in the formation of the immunological synapse.

Accumulation of MHC class II peptide complexes has been described at the site of an immune synapse initiated by agonist peptide MHC class II [61 ]. The accumulation not only concerned high-affinity interactions, as nonagonist MHC class II peptide complexes also concentrated at the site. Such nonagonist complexes were entitled "accessory ligands", as they strongly enhanced T lymphocyte activation. HLA-DR molecules recruited to DIGs following HLA-DR engagement could provide such accessory ligands. The present study reveals qualitative differences in terms of signaling proteins and cytoskeletal components between HLA-DR-enriched DIGs in immature versus mature human primary DCs prepared for DC-mediated immunotherapy.

Moreover, the HLA-DR molecules found in the DIGs differ between mature and immature DCs, as seen by the differences in their association with peptide. This is in agreement with recent observations reporting the effect of the antigen concentration during peptide loading in the formation of the immune synapse [62 ]. In light of our data, DC maturation leads to the generation of mature HLA-DR-containing DIGs. In addition, recruitment of MHC class II molecules to DIGs by CD4+ T cells has been proposed to play a role in the MHC class I presentation of exogenous antigens by DCs [63 ], which indicates that DIG-localized MHC class II molecules participate in complex signaling pathways. Finally, DIG-clustered MHC class II signaling induced by lymphocyte-activated gene-3 could induce migration of immature DCs to secondary lymphoid tissues given the profile of chemokine production observed [64 ], as well as initiating maturation and activation of these DCs [34 ]. Taken together, we propose that certain signaling pathways are dependent on MHC class II being localized to DIGs, and the characterization of the signaling molecules in HLA-DR containing DIGs reported here supports this notion. Taken together, these differences contribute to explain the distinct abilities of immature versus mature DCs to generate an efficient immune response, as reported in previous immunotherapy studies [24 ].


   ACKNOWLEDGEMENTS
 
The Fondation de France and the European Community (Project QLK3-CT-2002-02026) supported this research. N. S. was a recipient of an INSERM fellowship, and C. R. was supported by the Fondation St. Louis. N. S. and C. R. made equal contributions. We thank Professor John Trowsdale (Cambridge, UK) for kindly providing the 5C1 anti-HLA-DM{alpha} and the anti-HLA-DOß antibodies, Dr. Y. Richard (INSERM U131) for kindly providing the anti-CD40 mAb, and C. Klein (IFR58) for help with confocal microscopy.

Received January 27, 2003; revised March 11, 2003; accepted March 14, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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