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The Journal of Immunology, 2003, 171: 5812-5819.
Copyright © 2003 by The American Association of Immunologists

MHC Class II-Peptide Complexes in Dendritic Cell Lipid Microdomains Initiate the CD4 Th1 Phenotype 1

Vanessa Buatois2,*, Marjorie Baillet2,*, Stéphane Bécart{dagger}, Nuala Mooney{dagger}, Lee Leserman*,{ddagger} and Patrick Machy3,*,{ddagger}

* Centre d’Immunologie Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique-Université de la Méditerranée de Marseille-Luminy, Marseille, France; {dagger} Institut National de la Santé et de la Recherche Médicale Unité 396, Immunogénétique Humaine, Centre de Recherches Biomédicales des Cordeliers, Paris, France; and {ddagger} Centre National de la Recherche Scientifique Groupement de Recherche 2352, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated differentiation of CD4 T cells responding to Ag presented by bone marrow-derived dendritic cells (DC) in association with MHC class II (MHC II) molecules. Peptides encapsulated in liposomes opsonized by IgG were taken up by endocytosis. MHC II-peptide-specific T cells responding to this Ag were polarized to a Th1 cytokine profile in a CD40-, CD28-, MyD88-, and IL-12-dependent manner. Th2 responses were obtained from the same transgenic T cell population exposed to the same DC on which MHC-peptide complexes had dispersed for 48 h following uptake of FcR-targeted liposomes. DC that took up the same FcR-targeted liposomes and then were exposed to methyl-{beta}-cyclodextrin, which chelates cholesterol and dissociates lipid microdomains, also stimulated Th2 differentiation. Incubation of T cells with DC incubated with peptides directly binding to MHC II resulted in Th2 responses, whether or not the DC were coincubated with opsonized liposomes as a maturation stimulus. CD4 Th1 polarization thus appears to depend on MHC II-peptide complex clustering in DC lipid microdomains and the time between peptide loading and T cell encounter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inflammatory and cell-mediated responses are typically Th1 (producing IFN-{gamma}), while Th2 cells (producing IL-4) dominate in humoral immunity and allergy (1, 2). Mechanisms of Ag presentation cited as influencing Th1/Th2 orientation include Ag density; heterogeneity of the APC populations; the form of the Ag; duration of signaling through the TCR; and the cytokine environment (3, 4). Many studies of T cell polarization have used peptide or protein Ag to stimulate T cell proliferation or cytokine production. Nevertheless, Ag responsible for the evolution of the immune system were microbes: particulate Ag expressing determinants that can promote APC activation via Toll-like receptors (5), or bind Ab and activate APC via IgG FcR (6, 7, 8). In this study, we compared Th1/Th2 polarization of cells either responding to Ag in the form of free peptide, or capable of binding directly to MHC II molecules at the cell surface or to Ag released from liposomes in intracellular compartments. Liposomes can highly concentrate soluble Ag. They can also be opsonized by Ab for uptake by the FcR (7), activating the dendritic cells (DC) 4 and delivering Ag into compartments physiologically selected for its association with MHC II molecules (9, 10, 11). We evaluated the differentiation of naive CD4 T cells from TCR transgenic (Tg) mice specific for well-studied hen egg lysozyme (HEL) (12, 13) and OVA (14) peptides, which bind to MHC II I-Ak and I-Ab molecules, respectively. APC were DC derived from bone marrow with GM-CSF (15).

Although of increasing interest for their role in T cell activation (16), lipid microdomains have been little studied in DC. Increased Ag density in B cell microdomains reduced the amount of Ag necessary to stimulate T hybridoma cells (17). Ag in opsonized liposomes taken up by DC and re-expressed at the cell surface in lipid-rich microdomains efficiently stimulated naive Th cells (18). FcR targeting of exogenous Ag was necessary for its presentation to CD8 T cells (8, 19) in a CD4-dependent manner (18). We concluded that signals from Th cells necessary for class I presentation by DC pass through molecules concentrated in lipid-rich microdomains of DC (18). The present study shows that the spatial orientation of MHC II-peptide complexes regulates also the polarization of Th cells. T cells incubated with DC-expressing MHC II-peptide complexes expressed as a consequence of uptake of Ag in FcR-targeted liposomes differentiated into Th1 cells. T cell differentiation into the Th2 pathway followed dispersion of these structures, or occurred when T cells were stimulated with DC MHC II molecules directly loaded with free peptide. In addition, this study reveals that Th1 differentiation depends on DC production of IL-12, which in turn requires TCR engagement by MHC II-peptide complexes in lipid-rich microdomains.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

The 3A9 TCR Tg mice are specific for the HEL peptide 46-61 in the context of I-Ak. The T cell hybridoma 3A9, the source of the TCR expressed by these mice, has been reported to respond equally well to this peptide and to peptides processed from intact HEL (20). The 3A9 Tg mice were backcrossed to CBA/J mice (IFFA-CREDO, L’Arbresle, France). OT-II mice are specific for the OVA peptide 323–339 in the context of I-Ab (14). They were maintained as homozygotes on a B6 background. Mice were used at 6–10 wk of age. CD4 T cells from spleens of these mice and DC from CBA/J, (CBA x B6) F1, or MyD88-/- mice on a B6 129F2 background (21) (kindly provided by S. Akira, courtesy of J.-P. Gorvel, Centre d’Immunologie, Marseille, France) femur bone marrow were obtained, as described (18). Briefly, bone marrow cells were cultured for 3 days in DMEM supplemented with 10% FCS, antibiotics, 2 mM glutamine, 50 µM 2-ME, and 30% conditioned medium from NIH3T3 cells containing GM-CSF. They were then diluted 1/1 in the same medium, and after an additional period of 3–4 days of culture, plastic-nonadherent cells were washed, resuspended in supplemented RPMI 1640, and used as APC. CD11c+ DC (80–90%) expressed low levels of MHC II and costimulatory molecules. The phenotype of these cells has been reported previously (19).

Antibodies

U7.27.7, an IgG2a anti-DNP mAb, was provided by Z. Eshhar (Weizmann Institute, Rehovot, Israel). The 10.2.16 (IgG2a) binds to I-Ak molecules in a peptide-independent manner (22). Other mAb include the hamster anti-CD3 (145.2C.11) (23) and anti-CD28 (37.51) (24). P. Lane (University of Birmingham, Birmingham, U.K.) kindly provided soluble CD40, CD152, and CD154. M5-114, an I-Ab-specific mAb; PE-conjugated anti-CD154, CD3, CD69, CD25, CD44, CD28, and MHC class I H-2Kk and FITC-conjugated CD4 mAbs; fluorescent IL-4- and IFN-{gamma}-specific mAbs; and neutralizing IL-4- and IL-12-specific mAbs were purchased from BD PharMingen (San Diego, CA).

Liposomes and other reagents

Liposomes were made from 65% (mol/mol) dimyristoyl phosphatidylcholine, 34.5% cholesterol (Sigma-Aldrich, St. Louis, MO), and 0.5% DNP-caproyl-phosphatidylethanolamine (Molecular Probes, Eugene, OR). They were formed by exposing lipids evaporated from organic solvents to 2 mg/ml (1.4 mM) 46-61, 20 mg/ml HEL protein, or 60 mg/ml OVA (both 1.4 mM) (Sigma-Aldrich) in PBS; to PBS only (empty liposomes); or to 10 mM carboxyfluorescein (Molecular Probes) (for determination of encapsulation efficiency), followed by extrusion, as reported (18).

Methyl-{beta}-cyclodextrin (MCD) and HRP-labeled cholera toxin {beta}-subunit were from Sigma-Aldrich. Recombinant IL-4 and IL-12 were from PeproTech (Rocky Hill, NJ). CFSE was from Molecular Probes.

Ag presentation assays

A total of 1–2 x 106 DC was incubated in 1 ml of RPMI 1640 medium containing 5% FCS, 2 x 10-5 M 2-ME, 2 mM glutamine, and antibiotics (supplemented RPMI 1640) in six-well tissue culture plates. Free 46-61, or liposome-encapsulated peptides were added at the indicated concentrations for 6 h (or for other times, as indicated) in the presence (FcR-targeted liposomes) or absence (untargeted liposomes) of 5 µg/ml anti-DNP mAb. In some experiments, cells were incubated with free 46-61 peptide and either FcR-targeted empty liposomes to induce activation (equivalent to the quantity of peptide-containing liposomes for a concentration of 0.1 nM 46-61), or targeted liposomes containing 46-61. DC were then washed before the addition of 1–2 x 106 CD4 T cells in supplemented RPMI 1640 for different periods. Supernatant fluids obtained at 24-h intervals were tested for IL-2 secretion using the IL-2-dependent cell line CTLL (18). T cell division was assessed by counting cells or by FACS analysis of T cells labeled with 10 µM CFSE at 37°C for 10 min in PBS. In this case, cells were washed in PBS, then fixed with 2% formaldehyde, and fluorescence intensity was analyzed. For cholesterol depletion, DC were treated with 10 mM MCD for 1–2 h at 37°C and washed. T cells were then added directly or at daily intervals, as specified. DC viability was unchanged by this treatment, and residual MCD had no effect on the T cells. In some experiments, we added soluble ILs; Abs to ILs or cell surface molecules; or soluble CD molecules to DC cultures before addition of CD4 T cells.

FACS analysis of intracellular cytokines

After 7 days of coculture of T cells with DC, T cells were restimulated overnight with anti-CD3 (0.3 µg/ml) and anti-CD28 (5 µg/ml) Ab. Cells were then treated for 2 h at 37°C with brefeldin A (Sigma Aldrich; 8 µg/ml), after which cells were treated with anti-FcR (2.4G2). Cells were either washed and incubated in PBS with PE-conjugated anti-CD4 for 30 min at 4°C, then washed again, or directly fixed with 2% formaldehyde for 20 min at 4°C. Cells were permeabilized with saponin (Sigma Aldrich; 0.5%) in PBS before incubation for 45 min at 4°C with allophycocyanin-conjugated anti-IL-4 or anti-IFN-{gamma} (double labeling), or FITC-conjugated anti-IFN-{gamma}.

Sucrose gradient fractionation of DC lysates

A total of 107 DC was incubated with either 0.1 nM 46-61 in FcR-targeted liposomes, or 10 nM free 46-61 with or without FcR-targeted empty liposomes. Incubation was from 3 h (day 1) to 3 days at 37°C. In some experiments, DC that had been incubated with 46-61 in FcR-targeted liposomes were treated with 10 mM MCD for 1 h at 37°C. Cells were washed in PBS and shifted to 4°C for 30 min before lysis with 0.5% Triton X-100 in 350 µl of PBS buffer containing protease inhibitors (25). Cell lysates were mixed v/v with an 85% sucrose solution containing protease inhibitors at 4°C. The lysates were overlaid with 2 ml of a 35% sucrose solution and followed by 1 ml of a 5% sucrose solution, both containing protease inhibitors. The samples were ultracentrifuged for 18 h at 4°C at 200,000 x g in a SW55Ti rotor (Beckman Instruments, Fullerton, CA). Eight fractions of 500 µl were collected. Fractions 2 and 3 correspond to the lipid-enriched fraction migrating at the 5/35% interface. The fractions containing MHC II molecules or GM1 were examined by Western blotting with 10.2.16 or M5-114 anti-MHC II mAb, and HRP-labeled {beta} unit of cholera toxin, respectively.

IL-12p40 secretion

The p40 subunit of IL-12 was measured by ELISA (BD PharMingen). A total of 2 x 104 DC was incubated in 100 µl of supplemented RPMI 1640, in wells of 96-well flat-bottom tissue culture plates. Cells were incubated with 46-61 in FcR liposomes (0.1 nM) in the presence or absence of 10 nM free 46-61, or alternatively, with LPS (Escherichia coli 055:B5), from Sigma-Aldrich. After an overnight incubation, DC were washed and, in some experiments, incubated with soluble CD40, CD152, CD154, or anti-CD28, or treated with 10 mM MCD before addition or not of 104 T cells, or left for 3 days before addition of T cells. Supernatant fluids were tested at the intervals indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD4 T cell activation by Ag presented by DC

DC were incubated with the HEL 46-61 peptide encapsulated in FcR-targeted or nontargeted liposomes, or with free 46-61, then I-Ak/46-61-specific CD4 T cells were added. FACS profiles of CFSE-labeled T cells are presented in Fig. 1. Optimal T cell proliferation required 10 nM peptide, either in free form or in untargeted liposomes, while the same proliferation was induced by as little as 0.1 nM peptide encapsulated in FcR-targeted liposomes. DC were also incubated with various concentrations of peptide in the presence of empty FcR-targeted liposomes from aliquots of the same lipids using the same extrusion device, before addition of T cells. This is because the standard Limulus assay for endotoxin contamination does not work well with liposome preparations and its sensitivity may vary according to liposome lipid composition after destruction of the liposomes by detergent (26). As seen in Fig. 1, the presence of these liposomes and the targeting Ab did not alter the sensitivity of I-Ak/46-61-specific CD4 T cells to the Ag in free form.



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  		FIGURE 1. Proliferation of T cells in response to presentation by DC of Ag in different forms. DC were incubated for 6 h with the indicated concentrations of free or liposome-encapsulated HEL peptide 46-61. After washing the DC, CFSE-labeled CD4 46-61-specific T cells were added for 4 days, at which time T cell division was analyzed by flow cytometry. Numbers refer to the percentage of cells that had divided. Graphs are representative of the results obtained in five different experiments.

 
Characteristics of CD4 T cell activation by different forms of the same Ag

We examined T cell activation after loading DC with either free 46-61 or 46-61 in FcR-targeted liposomes. T cell proliferation and cell number were determined at intervals of 24 h after T cell addition. The extent of proliferation (Fig. 2a) and the T cell number (Fig. 2b) were similar when DC were loaded with 10 nM free peptide with or without empty FcR-targeted liposomes as a maturation stimulus, or with 0.1 nM peptide in FcR-targeted liposomes, when T cells were added to DC immediately after incubation with Ag. In addition, expression of cell surface CD3, the coreceptor CD4, and CD44 and MHC class I molecules was similar after encounter with DC loaded with either free peptide or peptide in FcR-targeted liposomes (Fig. 2c). However, up-regulation of the activation marker CD69, the IL-2R CD25, and the costimulatory molecules CD154 and CD28 on T cells was much more pronounced following stimulation by DC presenting MHC II-peptide complexes formed after endocytosis of FcR-targeted liposomes containing peptide, than after stimulation with DC incubated with free peptides (Fig. 2c).



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  		FIGURE 2. Quantitative and qualitative aspects of T cell responses to different forms of Ag. DC were incubated for 6 h with peptide in FcR-targeted liposomes diluted to a concentration of 0.1 nM, or with 10 nM free 46-61 with or without empty FcR-targeted liposomes. These stimulated equivalent proliferation of T cells (see Fig. 1). Cells were washed, and CFSE-labeled (a) or unlabeled (b and c) HEL-specific T cells were added. Cell division (a) and cell number (b) were evaluated daily over 6 days. c, Expression of the indicated T cell surface molecules is expressed as relative fluorescence intensity from FACS profiles after labeling of T cells with FITC-conjugated anti-CD4, alone or in combination with PE-conjugated Abs. Right panel, Representative FACS profiles of T cells are shown 3 days after their addition to cultures. Molecules whose expression differed strongly when incubated with free (dashed line) or liposome-encapsulated (thick line) Ag are presented. Curves are representative of the results obtained in three to five experiments.

 
Duration of cytokine production and of T cell-stimulatory capacity as a function of Ag form

The stimulatory activity of these different Ag forms evolved differently (Fig. 3). T cells were added to DC incubated with free or liposome-encapsulated Ag, and IL-2 production was determined daily. DC pulsed with 46-61 peptide in FcR-targeted liposomes stimulated IL-2 production from T cells for 3 days. DC pulsed with free peptides, either in the presence or absence of empty FcR-targeted liposomes, lost half their capacity to stimulate IL-2 production after 2 days and were inactive at 3 days (Fig. 3a). In the variant of this experiment presented in Fig. 3b, DC were incubated with free peptides or with FcR-targeted Ag-containing liposomes, followed or not by brief treatment with the cholesterol chelator MCD. This agent leads to disruption of lipid microdomains (27). T cells were added immediately, or after intervals of 1–4 days, and their proliferation was determined 4 days after their addition. DC incubated with FcR-targeted Ag were capable of stimulating T cells for at least 3 days following Ag exposure, whereas DC that had been treated with MCD following exposure to FcR-targeted Ag lost most of their stimulatory capacity in 2 days, in a manner similar to DC incubated with free 46-61 (Fig. 3b). Thus, the functional t1/2 of peptide-MHC II complexes formed after endocytosis of FcR targeted peptide-containing liposomes appeared lipid dependent and was more durable than that of complexes formed directly at the cell surface with free peptides.



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  		FIGURE 3. Duration of cytokine production and of T cell-stimulatory capacity as a function of Ag form. a, DC were incubated for 6 h with 10 nM free peptide or 0.1 nM peptide in FcR-targeted liposomes, as above. Cells were washed, and unlabeled HEL-specific T cells were added. IL-2 secretion was monitored daily for 4 days. Results are presented as quantity of IL-2 secreted as a function of the day or assay. b, DC were incubated with free or liposome-encapsulated peptide for 6 h and washed as in a. Some wells were subsequently incubated with MCD and washed again. CFSE-labeled CD4 T cells were added immediately or after periods of from 1 to 4 days, as indicated, and division was monitored by flow cytometry 4 days after addition of the T cells. Curves present the percentage of T cells in division as a function of the day of addition of T cells and are representative of the results obtained in three to five experiments.

 
Localization of MHC II molecules in lipid-rich fractions

Ultracentrifugation of lysates of these cells over sucrose gradients showed that fractions 2 and 3, corresponding to the lipid-enriched fraction on the basis of their localization at the 5/35% interface and by the localization of GM1, were devoid of MHC II molecules for cells incubated without Ag. (Fig. 4a). {alpha}{beta} SDS-stable heterodimers of compact MHC II molecules were seen in the lipid-enriched fractions as early as 3 h for cells incubated with FcR-targeted liposomes (Fig. 4b). After a 72-h incubation post-Ag pulsing, MHC II complexes were no longer observed in low-density fractions, and a similar absence of MHC II dimers was seen for cells treated with FcR-targeted liposomes for 3 h and then MCD (Fig. 4b). GM1 was also relocalized to higher density fractions by MCD treatment. MHC II dimers were absent for cells incubated with free 46-61 (Fig. 4c), but were detected in lipid-enriched fractions of cells incubated with free peptide plus empty FcR-targeted liposomes, due to DC activation via the FcR (Fig. 4d).



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  		FIGURE 4. Localization of MHC II molecules in lipid-rich fractions. a, DC were incubated for 3 h without Ag. b, DC were incubated with FcR-targeted liposomes at a concentration of 0.1 nM HEL peptide for 3 or 72 h. For the cells treated with FcR-targeted liposomes for 3 h, after washing, one aliquot was treated for additional 1 h at 37°C with 10 mM MCD, and the other was treated with PBS. c, DC were treated for 3 h with 10 nM free 46-61, to which were added empty FcR-targeted liposomes in d. In each case, cells were lysed with 0.5% Triton X-100 following the indicated incubation period. Lysates were ultracentrifuged over a sucrose gradient (5–85%). Unboiled aliquots were run in SDS-PAGE, transferred to nitrocellulose filters, and probed for I-Ak or for GM1. Western blots are representative of the results of three different experiments.

 
Polarization of CD4 T cells by DC expressing class II molecules after uptake of different forms of Ag

We next evaluated the influence of the form of the Ag taken up by DC on the cytokine expression in responding T cells. T cells initially incubated with DC pulsed with Ag (HEL protein or 46-61 peptide) in FcR-targeted liposomes were polarized to Th1, as 20–30% of these cells produced IFN-{gamma} (Fig. 5a). A 3-day period of preincubation of these DC before T cell addition, which induced equivalent proliferation and IL-2 production in primary culture (see Fig. 3), resulted in a change in cytokine profiles. The dominant pattern became Th2, as 15–20% of these cells produced IL-4 (Fig. 5a). A similar pattern of Th2 priming (25% of the cells produced IL-4) was seen for DC incubated with FcR-targeted liposomes and then briefly treated with the cholesterol chelator MCD (Fig. 5a). These data indicate that Th1 priming depends on Ag presentation in cholesterol-rich domains of DC, and that the capacity of these clusters to stimulate Th1 responses may be modified over time or by pharmacological manipulation.



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  		FIGURE 5. Polarization of CD4 T cells in response to Ag in different forms. a, DC were incubated with 0.1 nM HEL 46-61 peptide or HEL protein in FcR-targeted liposomes for 3 h (or 72 h) at 37°C and washed. Some DC were subsequently treated with 10 mM MCD for 1 h at 37°C. b, DC were incubated for 3–6 h with 10 nM 46-61 peptide, to which empty FcR-targeted liposomes were added in c. HEL-specific CD4 T cells were then added to all wells for 7 days. The T cells were then restimulated with CD3- and CD28-specific Ab for 16 h. Intracellular lymphokines were detected in a, with allophycocyanin-conjugated IL-4- and FITC-conjugated IFN-{gamma}-specific mAbs. b and c, Intracellular expression of lymphokines was monitored using allophycocyanin-conjugated IL-4- or IFN-{gamma}- and FITC-conjugated CD4-specific mAbs. Dot plots of FACS analyses are representative of the results of five different experiments.

 
In contrast to DC incubated with FcR-targeted liposomes containing 46-61 of HEL, cells incubated with free HEL peptide 46-61 stimulated a typical Th2 response (Fig. 5b). This response was unchanged by the addition of empty FcR-targeted liposomes (Fig. 5c).

Dependence of Th1 polarization on IL-12 secretion by DC

Induction of a Th1 profile in CD4 T cells is IL-12 dependent (28). IL-12 was not produced when DC were loaded with free peptide (Fig. 6a). At low Ag concentrations, IL-12 was produced only in cultures of DC loaded with peptide encapsulated in FcR-targeted liposomes in the presence of specific T cells. We confirmed that the cells producing the IL-12 were the CD11c-expressing DC population (data not shown). IL-12 was not produced by DC from MyD88-/- mice exposed to liposomes containing OVA, in the presence of OT-II T cells cognate for the relevant I-Ab OVA-derived peptide 323–339 (14). DC from MyD88-sufficient mice exposed to FcR-targeted liposomes produced IL-12 in response to the same liposomes in the presence of the same T cells, whereas 100- to 1000-fold higher Ag concentrations were required for IL-12 production when nontargeted liposomes were used as an Ag source (Fig. 6b). DC produce IL-12 during a short period following activation with LPS (29). DC incubated without T cells produced IL-12 in the presence of LPS, but not in the presence of opsonized liposomes (Fig. 6c). Nevertheless, we considered the possibility that contamination of the liposome preparation by an agent such as endotoxin might be insufficient by itself to induce IL-12, but could do so in the presence of stimulation by T cells. Consequently, in Fig. 6a, we used DC incubated in parallel either with FcR-targeted liposomes containing the Ag or with FcR-targeted empty liposomes, together with an amount of the same Ag in free form sufficient to induce equivalent T cell proliferation. Only FcR-targeted liposomes containing the Ag stimulated IL-12 production. We conclude that the difference in the results with respect to IL-12 production and the Th1 or Th2 orientation of the responding cells can be explained neither by some component of the Ag, the liposomes, the opsonizing Ab, nor the engagement of the FcR. Thus, a critical factor in Th1 polarization must be the topology of MHC II-peptide complexes and relevant associated molecules. Nevertheless, equivalent expression of MHC II molecules in lipid microdomains can be observed as a consequence of LPS or FcR engagement, in I-Ab-specific Western blots from DC of both MyD88-/- and MyD88-sufficient mice (Fig. 6d). Thus, expression of these molecules is necessary, but not sufficient for IL-12 production.



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  		FIGURE 6. Lymphokine production in response to FcR-targeted liposomes and CD4 T cells. a, DC were incubated without Ag or for 6 h with 10 nM free 46-61 and empty FcR-targeted liposomes or 0.1 nM peptide in FcR-targeted liposomes. CD4 T cells were then added or not, and IL-12p40 secretion was determined at daily intervals. Curves are representative of the results of five different experiments. b, DC from (CBA x B6)F1 or MyD88-/- mice were incubated with various dilutions of FcR-targeted liposomes containing OVA. CD4 T cells from OT-II Tg mice were added for 4 days, and IL-12 was measured. c, DC from (CBA x B6)F1 (filled bars) or MyD88-/- (open bars) mice were incubated overnight in medium, with 5 µg/ml LPS, or with liposomes in the absence or presence of 5 µg/ml opsonizing anti-DNP Ab. Cells were then washed and IL-12p40 was determined at day 3. d, Sucrose gradients of (CBA x B6)F1 or MyD88-/- DC were prepared, as in Fig. 4. After incubation of cells with 5 µg/ml LPS or FcR-targeted liposomes and Western blots were probed for I-Ab. Results are representative of two experiments.

 
Dependence of cytokine production by CD4 T cells on other molecular interactions

T cell cytokine expression was Th1 when they were incubated with either anti-IL-4 or IL-12 and Th2 when incubated with anti-IL-12 or with IL-4 (Table I). Blocking interactions between CD40/CD154 inhibited IL-12 production. Under such conditions, priming of T cells to the Th2 phenotype was induced. Abs to CD40 and to CD154 molecules and with Abs to CD80 or CD86 also inhibited IL-12 production, and T cells were oriented toward the Th2 phenotype (data not shown). Nevertheless, agonist Ab to CD28 that induced blockade of IL-12 production induced a Th1 phenotype, as long as the MHC II-peptide complexes were presented in lipid-rich domains (Table I).


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  		Table I. Th1 polarization of T cells is dependent on CD40/CD154 and CD80:86/CD28 interactionsa

 
MCD treatment of DC or delay of addition of T cells prevented IL-12 production, and T cells were polarized toward the Th2 phenotype. Strikingly, stimulation of DC with FcR-encapsulated 46-61, in the presence of free 46-61 (10 nM), did not induce IL-12 production, and Th2 cells were generated (Table I). Thus, MHC II-peptide complexes at the surface of DC in lipid-rich microdomains are capable of transmitting signals that induce Th1 differentiation in a CD40- and CD28-dependent manner. Competition between lipid microdomain-associated and free MHC-peptide complexes can interfere with signaling, block IL-12 production, and induce Th2 differentiation.

Finally, treatment of DC with LPS resulted in IL-12 production, and oriented T cells toward the Th1 phenotype, whether FcR targeted or free 46-61, were used.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
T cell activation has been reported to depend on the expression of both MHC-peptide complexes of sufficient density (30) and costimulatory molecules (31, 32) by APC. We show in this study that the spatial organization of MHC II-peptide complexes on DC has an important role in T lymphocyte polarization. We determined consequences of priming CD4 T cells with Ag presented by DC on cytokine production by both the DC and the T cells. The data indicate that DC provide signals necessary and sufficient for priming either Th1 or Th2 responses from Ag-specific T cells, depending on the manner in which the Ag is presented by the DC. MHC II-peptide complexes formed in endosomes after FcR-mediated internalization of Ag appear to be concentrated in lipid-rich microdomains on DC, in that they are susceptible to dissociation by chelation of cholesterol by MCD (Fig. 4). Concentration of these molecules appears required for signals from the TCR and other T cell molecules inducing IL-12 synthesis by the DC, orienting T cells toward the Th1 phenotype. In contrast, dispersed MHC II-peptide complexes stimulated equivalent proliferation and IL-2 production by T cells, but not production of IL-12 by the DC. Consequently, the T cells were oriented toward the Th2 phenotype. These results are consistent with those showing that LPS activation of DC that had taken up Ag resulted in its transport into compartments containing MHC II molecules and lipid microdomains before MHC II-peptide expression at the cell surface (9). These data confirm those obtained by Langenkamp et al. (29) for LPS-activated DC, and extend them to DC activated by Ag acquired by a physiological Ag receptor, the FcR.

Upon incubation of DC for 3 days before T cell addition, T cells of the Th2 phenotype were induced, presumably as a consequence of the diffusion of MHC-peptide complexes in the plasma membrane of DC. In addition, treatment of DC that took up peptide in FcR-targeted liposomes and normally stimulated Th1 responses induced Th2 responses instead if the DC were incubated with MCD (Fig. 3b). Interestingly, Th2 cytokine priming was dominant, being induced by concomitant incubation of DC with free peptides and peptides in FcR-targeted liposomes (Table I). The functional dominance of Th2 priming by free peptides, despite uptake of liposome-encapsulated peptides sufficient to induce Th1 responses, presumably resulted from competition for TCR binding between concentrated and dispersed peptide-MHC II complexes. Under these conditions, DC did not produce IL-12 (Table I). Th2 differentiation may occur if IL-4 production becomes autonomous in cells in which the IL-12R is not up-regulated by IL-12 from DC and IFN-{gamma} from Th1 cells (33, 34). Collectively, these data suggest that lipid microdomain-localized MHC II molecules inducing Th1 differentiation are enriched for the expression of 46-61 peptides released after endocytosis of the FcR-targeted liposomes. These molecules are part of a signaling complex necessary for the activation of IL-12 production by DC after TCR engagement. DC from MyD88-/- mice are capable of up-regulating costimulatory molecules and have intact NF-{kappa}B and mitogen-activated protein kinase cascades (35). MHC-peptide complexes were expressed in lipid-enriched fractions in DC from MyD88-/- mice (Fig. 6d), in response to LPS and to FcR-targeted liposomes. These were not sufficient for IL-12 production by these cells in the presence of cognate T cell help, thus showing that expression of class II molecules in lipid-enriched structures is MyD88 independent, but that signaling through these complexes is MyD88 dependent.

CD40 localizes in lipid microdomains after cross-linking (36), and may signal DC for IL-12 production (37). Blocking CD40/CD154 interaction inhibited IL-12 production and priming for Th1 differentiation and oriented CD4 T cells toward the Th2 phenotype (Table I). Agents blocking CD40/CD154 interactions failed to block Th2 priming when DC were pulsed with free peptides (data not shown). Similarly, blocking CD80:86/CD28 interaction blocked IL-12 production and Th1 differentiation in response to targeted liposomes (Table I), but not Th2 differentiation in response to soluble peptide (data not shown). Nevertheless, agonist Ab for CD28 that blocked IL-12 production by DC induced the Th1 phenotype when DC were incubated with FcR-targeted liposomes (Table I). Blocking CD80:86/CD28 interactions reduced clustering of MHC II molecules at the interface between T cells and B cells used as APC (31). It has recently been reported that CD152 localizes in lipid-rich structures containing TCR, contributing to the regulation of T cell function (38). T cells driven into the Th1 pathway by cytokine exposure have been reported to cluster their TCR in lipid-rich structures when exposed to peptide-pulsed splenic APC. Under the same conditions, the TCR of Th2 cells was dispersed (39). These clustering patterns correlate with those seen for MHC II-peptide complexes at the surface of DC that induce Th1 or Th2 differentiation, respectively, in the present study. T cells primed toward the Th1 phenotype up-regulated expression of markers (CD69, CD25, CD154, and CD28) more than CD4 T cells oriented toward the Th2 phenotype (Fig. 2). This is in agreement with results obtained by cross-linking the TCR by MHC-peptide spacers of different length, which showed that the increasing intermolecular distance between TCR molecules reduced expression of CD69 and CD25 (30). The data indicate that the conditions necessary to activate CD4 T cells for Th2 differentiation are less stringent than those necessary for induction of Th1 differentiation.

MHC II molecules have recently been described in tetraspan as well as in lipid microdomains (40). The identity and organization of molecules in microdomains in proximity to MHC II molecules are of obvious interest for their role in stimulation of T cells and signal transduction in DC. The role of the APC in the formation of the immunological synapse has been considered to be passive, as shown for T cells interacting with B cells (41, 42) or with model phospholipid bilayers (42). Nevertheless, a study in which DC were used as APC has shown that these cells polarize their actin cytoskeleton in a cytochalasin D-inhibitable manner during the formation of synapses with naive T cells (43). The data presented in this work are consistent with those results, in that they suggest that MHC II localization in lipid-rich microdomains of DC precedes and may induce movement of the TCR on interacting CD4 T cells. This localization is not necessary for T lymphocyte activation, but appears essential for Th1 differentiation. If clustering of MHC II molecules occurs after the addition of T cells to peptide-pulsed DC, it does not prime for the Th1 phenotype.


   Acknowledgments
 
We thank Laurent Giraudo, Hervé Sanchez, and Anne Gillet for assistance, and Anne-Marie Schmitt-Verhulst, Sylvie Guerder, and Nathalie Auphan-Anezin for helpful discussions.


   Footnotes
 
1 This work was supported by Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique, and by grants from the Association pour la Recherche sur le Cancer and the European Community No. GLG1-1999-00622. V.B., M.B., and S.B. were supported by fellowships from the Ministère de l’Education Nationale. Back

2 V.B. and M.B. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Patrick Machy, Centre d’Immunologie de Marseille-Luminy, Case 906, 13288 Marseille cedex 9, France. E-mail address: machy{at}ciml.univ-mrs.fr Back

4 Abbreviations used in this paper: DC, dendritic cell; HEL, hen egg lysozyme; MCD, methyl-{beta}-cyclodextrin; Tg, transgenic. Back

Received for publication February 20, 2003. Accepted for publication September 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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