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The Journal of Immunology, 2003, 171: 6478-6487.
Copyright © 2003 by The American Association of Immunologists

CD40-Induced Aggregation of MHC Class II and CD80 on the Cell Surface Leads to an Early Enhancement in Antigen Presentation 1

Abigail Clatza*, Laura C. Bonifaz*, Dario A. A. Vignali{dagger} and José Moreno2,*

* Research Unit on Autoimmune Diseases, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico City, Mexico; and {dagger} Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105-2794


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ligation of CD40 on B cells increases their ability to present Ag and to activate MHC class II (MHC-II)-restricted T cells. How this occurs is not entirely clear. In this study we demonstrate that CD40 ligation on Ag-presenting B cells (APC) for a short period between 30 min and 3 h has a rapid, augmenting effect on the ability of a B cell line and normal B cells to activate T cells. This is not due to alterations in Ag processing or to an increase in surface expression of CD80, CD86, ICAM-1, or MHC-II. This effect is particularly evident with naive, resting T lymphocytes and appears to be more pronounced under limiting Ag concentrations. Shortly after CD40 ligation on a B cell line, MHC-II and CD80 progressively accumulated in cholesterol-enriched microdomains on the cell surface, which correlated with an initial enhancement in their Ag presentation ability. Moreover, CD40 ligation induced a second, late, more sustained enhancement of Ag presentation, which correlates with a significant increase in CD80 expression by APC. Thus, CD40 signaling enhances the efficiency with which APC activate T cells by at least two related, but distinct, mechanisms: an early stage characterized by aggregation of MHC-II and CD80 clusters, and a late stage in which a significant increase in CD80 expression is observed. These results raise the possibility that one important role of CD40 is to contribute to the formation of the immunological synapse on the APC side.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4+ T cells recognize protein Ags as peptides bound to MHC class II molecules (MHC-II)3 (1, 2). Peptide-loaded MHC-II (peptide-MHC-II) is the ligand for the TCR that, upon recognition by CD4+ T cells, marks the beginning of an adaptive immune response. Although recognition of peptide-MHC-II is essential, it is insufficient for Ag-specific CD4+ T cell activation, as a number of additional cell surface interactions are required (3). Among these are costimulatory signals, of which the interaction of CD80 or CD86 surface molecules on the APC with CD28 on the T cell is the best characterized (4, 5). Costimulation is a reciprocal process, as activated T cells also provide signals leading to APC maturation. The best-characterized cognate signal provided by T cells to the APC is the interaction of CD154 (CD40 ligand), a member of the TNF family, with its receptor CD40 on APC, a TNF receptor family molecule.

Signaling through CD40 induces a number of functions that differ depending on the type of APC (6, 7). CD40 ligation on dendritic cells (DC) induces the release of IL-12 and plays a role in their final maturation. On macrophages, CD40 ligation induces their activation and hence plays a role in the defense against intracellular pathogens (8, 9). The effects of CD40 signaling have been studied in detail in B cells, where CD40 ligation modulates the expression of many genes (10), leading to B cell proliferation and survival (11, 12), differentiation into Ab-secreting cells (13, 14, 15), isotype switching (13, 16), and an enhancement of their ability to activate T cells during Ag presentation. The latter is due at least in part to up-regulation of CD80 expression (17, 18, 19, 20). Others have proposed that CD40-induced enhancement of Ag presentation could be related to changes in the ability of B cells to process Ags (21). These findings are not mutually exclusive.

In many cell systems it has been demonstrated that receptor signaling is greatly enhanced by incorporation into cholesterol-enriched cell membrane microdomains (22, 23, 24), also referred to as lipid rafts. During T cell Ag recognition, TCR signaling induces the aggregation of TCR and costimulatory receptor-containing rafts, eventually leading to the formation of the immunological synapse, which polarizes the reciprocal activation processes of the T cell and the APC (25, 26, 27, 28). Lipid rafts containing MHC-II also exist on the APC surface before T cell Ag recognition (29). These appear to be essential to increase the number of TCR/CD3 complexes engaged during Ag presentation. Clustering of these lipid rafts on the APC side during early T cell Ag recognition could lead to a local increase in MHC-II and costimulatory molecule density. Therefore, the increased overall T cell-APC avidity would result in more efficient T cell activation. Besides an increase in the efficiency of Ag presentation, this could lead to enhanced signaling through MHC-II on the APC itself (30).

During DC maturation, MHC-II molecules are transported from intracellular processing compartments to the cell surface (31, 32). Moreover, engagement by specific TCR triggers the export of MHC-II molecules from intracellular compartments in a unidirectional manner to the immunological synapse on DC (31, 32). Although it is not known whether a similar phenomenon occurs during Ag presentation by B cells, candidate signals that may induce the recruitment of MHC-II to the cell surface and the immunological synapse are surface Ig, CD40, and the ligands for the different Toll-like receptors. Indeed, MHC-II and CD40 can be associated on the cell surface, and reciprocal stimulation through either molecule increases their presence in detergent-insoluble fractions (33, 34).

In the present study we found that CD40 ligation on B cells had a dual positive effect on Ag presentation. An early effect was characterized by a rapid clustering of MHC-II and CD80 in cholesterol-enriched domains on the cell surface that correlates with an increased ability to stimulate both T cell hybridomas and naive T cells, which is followed by a more pronounced late effect, which correlates with increased expression of CD80.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Ags

Hen egg-white lysozyme (HEL) was obtained from Sigma-Aldrich (St. Louis, MO). Culture medium was RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 25 mM HEPES, 2 mM glutamine, sodium pyruvate, penicillin, streptomycin, and 10% FBS (HyClone Laboratories, Logan, UT). Synthetic HEL or bovine RNase A peptides were either synthesized at the Hartwell Center, St. Jude Children’s Research Hospital, or were purchased from Research Genetics (Huntsville, AL). Methyl-{beta}-cyclodextrin was from Sigma-Aldrich.

T cell hybridomas and other cell lines

HEL-specific, IAk-restricted T cell hybridomas C10 (48-62) and A6.B3 (34-45) were gifts from Dr. L. Glimcher (Harvard University, Boston, MA) (35), E907.D (33-47) was generated by one of us (J.M.) and has been described previously (36). The bovine RNase A43–56-specific T cell hybridoma TS12 was a gift from Dr. P. Allen (Washington University, St. Louis, MO) (37). The mouse B cell hybridoma LK-35.2 (H-2k,d; American Type Culture Collection, Manassas, VA) (38) was used as APC. The IL-2-dependent cell line CTLL-2 (39) was obtained from American Type Culture Collection. All cells were maintained in culture in complete medium at 37°C in 5% CO2.

Mice and T cell purification

Mice bearing the rearranged {alpha}{beta}-chain genes from the anti-HEL46–61 T cell hybridoma 3A9 in C3H (H2k) background (3A9 mice) have been described previously (40). 3A9 CD4+ T cells were purified by B220, CD8, IAk, NK1.1, CD14, and CD69 negative selection (BD PharMingen, San Diego, CA) FACS using a Mo-Flo flow cytometer (Cytomation, Fort Collins, CO).

Monoclonal Abs

The hybridoma H116.32 (secreting IgG2b anti-Ia{alpha}k) (41) was a gift from Dr. G. Hämmerling (German Cancer Research Center, Heidelberg, Germany). Hybridoma secreting the rat-derived anti-CD40 mAb 1C10 (42) was a gift from Dr. A. Heath (University of Sheffield, Sheffield, U.K.), the rat hybridomas secreting anti-CD11b mAb 70.15.11.5.HL (IgG2b), GL-1 (IgG2a anti-CD86), and the hybridoma 1G10 (IgG2a anti-CD80) were acquired from American Type Culture Collection. All mAbs were used either as culture supernatants or purified from ascites in Sepharose 4B-protein A or protein G columns (Amersham Pharmacia Biotech, Piscataway, NJ). For the confocal microscopy experiments, purified 1G10 Ab was labeled with Texas Red (Pierce, Rockford, IL) as described in the product brochure. Anti-MHC-II (H116-32) was either biotinylated (Pierce) or labeled with Texas Red. Finally, anti-CD54-biotin (clone 3E2), and anti-CD80-FITC (clone 1G10) were purchased from BD PharMingen.

Ag presentation assays

For these studies a variable number of APC (LK-35.2) were cultured with 5 x 104 T cell hybridoma cells for 24 h in the absence or the presence of exogenous HEL or the relevant peptide in triplicate wells in 96-well plates in a final volume of 200 µl. At 24 h, 100 µl of supernatant was recovered and cultured for 36 additional h with 104 CTLL-2 cells. During the last 16 h of culture 1 µCi [3H]TdR was added to each well. Cultures were harvested with a semiautomatic cell harvester (Tomtec, Hamden, CT), and DNA synthesis was determined in a scintillation counter (Wallac, Gaithersburg, MD).

Cytokine quantitation

IL-2 and IFN-{gamma} concentrations were determined using a multiplexed, particle-based, flow cytometric assay as previously described (43).

Flow cytometry

This was conducted after reacting cells with the appropriate mAbs and fluorescent dye-labeled second reagents. All Ab reactions were conducted at 4°C in PBS containing 0.1% sodium azide and 1% rabbit serum to prevent binding of mAb to Fc receptors. Some Abs were biotinylated (as indicated in the figure legends), followed by fluorescent dye-labeled streptavidin (BD PharMingen or Jackson ImmunoResearch Laboratories (West Grove, PA)). One- to four-color flow cytometry was conducted in a dual laser FACSort flow cytometer (BD Biosciences, San Jose, CA).

Confocal microscopy

LK35.2 cells (104) were cultured at 37°C on sterile glass slides in a volume of 200 µl for 24 h, after which 10 µg/ml anti-CD40 or anti-CD11b (control) was added and incubated at 37°C for varying lengths of time. Slides were washed, fixed in 4% paraformaldehyde, stained for the expression of MHC-II (Texas Red or Alexa 488), ganglioside M1 (GM1; Alexa 488-cholera toxin B (Alexa 488-CTB)) and CD80-Texas Red. Cover glasses were mounted on Vectashield (Vector Laboratories, Burlingame, CA), sealed with acrylic resin, and observed under a confocal microscope (model LSM510; Carl Zeiss, New York, NY) equipped with a dual laser. Fluorescence detection was conducted simultaneously with excitation/emission at 488/520 nm for FITC or Alexa 488, and at 596/633 nm for Texas Red. Images (x40 and x100) were acquired with a 2024 x 2024 pixel resolution and stored in the computer, after which they were processed and analyzed by means of the LSM5 Image Examiner software (Carl Zeiss).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD40 ligation induces enhanced Ag presentation in a manner that is not related to Ag processing

The aim of our initial experiments was to confirm that CD40 ligation on APC contribute to their Ag-presenting functions and to examine whether this phenomenon occurred with more than a single epitope. Treatment of the B cell hybridoma LK-35.2 with agonist anti-CD40 mAb in the presence of varying concentrations of HEL enhanced the ability of the T cell hybridomas C10 (48-62), A6.B3 (34-45), and E.907D (33-47) to see Ag and to release IL-2. For A6.B3 and E.907D, this was more evident at low Ag dose (Fig. 1A). Similar results were obtained with a T cell hybridoma specific for another Ag, bovine RNase A (data not shown and Fig. 2). Thus, CD40 ligation induces an enhancement of Ag presentation of at least four different epitopes from two proteins.



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  		FIGURE 1. A, CD40 ligation induces enhanced Ag presentation, which is not related to Ag processing. LK-35.2 cells (2.5 x 104) were cultured a with 5 x 104 T cell hybridomas in the presence of 10 µg/ml of the mAbs 1C10 (anti-CD40) or 70.15.11.5.HL (anti-CD11b, control) and varying concentrations of HEL (A) or the indicated peptide (B), which was added either before (extreme right) or after (left and center) CD40 and cell fixation with paraformaldehyde. After a 24-h culture, 100 µl of supernatant was transferred to a new plate with 104 CTLL-2 cells in a total volume of 200 µl. Twenty hours later 1 µCi [3H]TdR was added, and cells were cultured for an additional 18 h, then harvested in a semiautomated harvester (Tomtec). DNA synthesis ([3H]TdR incorporation) was measured in a scintillation counter. Results are expressed as counts per minute. Data shown are representative of at least five experiments conducted with these and other HEL-specific T cell hybridomas.

 


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  		FIGURE 2. CD40 ligation induces a dual enhancement on the ability of APC to activate T cells during Ag presentation in B cell lymphomas. A, LK-35.2 cells were cultured for different lengths of time in six-well plates (2 x 105 cells/well in 2 ml) in the presence or the absence of 10 µg/ml anti-CD40 or anti-CD11b mAb (control). Alter the culture period, cells were reacted with the following mAb (with or without streptavidin-PE): anti-CD80-FITC, anti-ICAM-biotin, anti-CD86-Alexa 488, or anti-MHC-II-biotin. Results are expressed as stimulation index minus 1, which represents the number of times over baseline surface expression of each surface marker in unstimulated LK-35.2 cells. B, Left, cells were precultured with anti-CD40 or control Abs as in A, after which were stained with anti-CD80-FITC and examined in a flow cytometer. Results are expressed as the mean fluorescence intensity. The two lower right panels show the same LK-35.2 cells depicted in the left panel, used for the Ag presentation assay after fixation with 1% paraformaldehyde and used in Ag presentation assays (2.5 x 104 cells) with 5 x 104 T cell hybridomas A6.B3 (HEL34–45) or TS12 (RNase A43–56) in the presence of varying concentrations of the relevant synthetic peptides. The results shown depict the data obtained with optimal peptide concentrations (HEL33–47 and bovine RNase A43–56; 0.3 and 3 µM, respectively). After a 24-h culture, 100 µl of supernatant were transferred to a new plate with 104 CTLL-2 cells and processed as in Fig. 1. Results are expressed as counts per minute x 10-3. The data are representative of three experiments with similar results. Fixed LK-35.2 cells failed to induce IL-2 release by T cell hybridomas in response to native HEL (10 µg/ml).

 
The enhancing effect of CD40 stimulation on Ag presentation also occurs without the need for Ag processing, as CD40 ligation on LK-35.2 cells also enhances their capacity to induce IL-2 release by the same T cell hybridomas in response to their corresponding synthetic peptides (Fig. 1B). The enhancement occurred regardless of the time of peptide addition, as this occurred when the peptides were added to live LK-35.2 cells and T cell hybrids during the incubation with anti-CD40 (Fig. 1B, extreme right) or if the peptide were added to CD40-activated APC that were fixed with paraformaldehyde just before the addition of peptide (Fig. 1B, left and center). Therefore, to avoid possible confusion caused by the effect of anti-CD40 on intracellular Ag handling, all additional experiments were performed with APC stimulated with anti-CD40 for varying lengths of time and fixed with paraformaldehyde before the addition of synthetic peptides.

CD40 ligation-induced enhancement of the ability of APC to activate T cells during Ag presentation occurs in two sequential phases

We next examined the kinetics of anti-CD40-induced enhancement of Ag presentation and its relation to the expression of the costimulatory molecules CD80 and/or CD86. Increased CD80 expression was seen, but only after a 12-h incubation in the presence of anti-CD40 (Fig. 2A). The expression of CD86, ICAM-1, and MHC-II did not change after 72 h in the presence of anti-CD40.

It was important to define whether the enhancement of Ag presentation correlated with the kinetics of CD80 expression. We, therefore, conducted experiments with LK-35.2 cells treated with anti-CD40 for varying lengths of time and fixed with paraformaldehyde before the addition of synthetic peptides. CD40 ligation enhanced the ability of APC to stimulate IL-2 release by T cell hybridomas specific for HEL34–45 or bovine RNase A43–56 as early as 30 min after the addition of anti-CD40 (Fig. 2B, center and right). This enhancement had a first peak at 3 h, before any detectable increase in CD80 expression (Fig. 2B, left). At 12 h, there was a further enhancement of the ability of LK-35.2 cells to induce IL-2 release, which was coincident with a significant increase in CD80 expression. These results allow us to conclude that the increase in CD80 expression is not involved in the early enhancement of Ag presentation induced by CD40 ligation. Moreover, as the preprocessed Ag (synthetic peptide) was added to metabolically inactive APC previously activated through CD40, the enhancing effect is unrelated to Ag uptake and/or intracellular handling.

Short-term anti-CD40-activated B cells have an increased ability to activate naive CD4+ T cells

The former Ag presentation experiments were conducted with T cell hybridomas, which have a lower threshold for activation. Moreover, if enhanced costimulation via CD80-CD28 is the major benefit of short-term CD40 ligation, T cell hybridomas are representative of secondary T cells, which are less dependent of CD80-CD28 interactions. As it has been shown that CD40 signaling enhances the ability of resting B cells to activate naive T cells (44), it was important to examine whether the early, costimulator-independent activation of LK-35.2 cells was sufficient to enhance their ability to activate naive T cells. Thus, we tested the capability of anti-CD40-treated LK-35.2 cells to activate resting naive T cells from 3A9 transgenic mice, which bear the rearranged {alpha}- and {beta}-chain TCR genes from a HEL48–62-specific T cell hybridoma (40). To ensure that other accessory cells and activated cells were not present during the assay, spleen CD4+ T cells from 3A9 mice were purified by FACS-negative selection after staining with a mixture of PE-labeled mAbs specific for MHC-II (IAk), B220, NK1.1, CD14, CD8, and CD69. The remaining cells were typically small lymphocytes and were >90% CD4+ with <1% cells positive for any of the mixture mAbs. For these experiments, LK-35.2 cells were incubated with 10 µg/ml anti-CD40 for 3 h in the absence of Ag, washed, and fixed in 1% paraformaldehyde. The peptide HEL48–63 was added at increasing concentrations to the fixed APC and 3A9 cells, directly to the tissue culture wells.

Anti-CD40-treated LK-35.2 cells were significantly better at inducing naive 3A9 CD4+ T cell proliferation after a 48-h incubation period compared with control LK-35.2 cells precultured for the same length of time in the presence of an irrelevant isotype control Ab (Fig. 3, top panel). When examined at later times, the differences in the T cell activation capacity of anti-CD40-treated and control LK-35.2 cells were less apparent, but still present (data not shown). These results clearly indicate that short-term CD40 stimulation prepares APC for Ag presentation to and activation of resting T cells.



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  		FIGURE 3. Short-term anti-CD40-activated B cells have an increased ability to activate naive CD4+ T cells. Naive resting CD4+ spleen T cells (5 x 104) from anti-HEL48–62 transgenic mice (3A9) were cultured in the presence of 2.5 x 104 anti-CD40 or control-treated (3 h), paraformaldehyde-fixed LK-35.2 cells for 48 h in duplicate plates (triplicate wells), after which 1 µCi of [3H]TdR was added per well (top), or 100 µl of supernatant was removed (middle and bottom). For T cell proliferation, plate 1 was cultured for an additional 18 h, after which it was harvested in a semiautomated cell harvester and counted in a scintillation counter. Results are expressed as counts per minute x 10-3. Supernatants from plate 2 were tested for the presence of the cytokines IL-2 (middle), IFN-{gamma} (bottom), and IL-4 (not shown) by a flow cytometric assay after incubation with fluorescent beads coupled to anti-cytokine mAb, followed by a PE-labeled second anti-cytokine mAb and read in a FACSCalibur flow cytometer with Luminex equipment and software. Cytokines were plotted against a pattern curve and run in the presence of known concentrations of cytokine for each cytokine. Results are representative of two experiments.

 
To examine whether activation of naive CD4+ T cells by anti-CD40-activated APC induced the release of cytokines, supernatants obtained from the previous experiment were tested for the presence of IL-2, IL-4, and IFN-{gamma}. CD40-activated APC induced IL-2 and IFN-{gamma} (Fig. 3, middle and bottom panels), but no IL-4 (data not shown), release by freshly isolated 3A9 T cells. The results indicate that CD40 ligation primes APC to have an enhanced ability to activate T cells and to induce an early release of effector cytokines.

Short-term CD40 ligation on normal B cells is sufficient to enhance their Ag-presenting capacity

Among the mechanisms needed to turn resting B cells into professional APC are IL-4, which induces MHC-II expression, Ig cross-linking by Ag, and T cell-derived signals, particularly CD40 ligation (5, 45). These events act in concert to allow B cells to enter into an activated state in which they can activate Ag-specific T cells. Although the precise role of these events has not been established, the results shown to date suggest that CD40 could be the major stimulus leading to the enhancement of the Ag-presenting function of B cells. Therefore, we examined the effect of anti-CD40 on the expression of CD80, CD86, MHC-II, and ICAM-1 by freshly isolated spleen B cells as well as on their ability to induce IL-2 release by T cell hybridomas. To ensure that the effect was not due to the possible presence of LPS in the medium, these experiments were conducted in the presence of polymyxin B. Anti-CD40 induced an increase in MHC-II, CD80, and ICAM-1 expression to near-optimal levels at 72 h (Fig. 4A). CD86 was induced at 3 h to decline at 12 h. This differs from LK-35.2 cells, which are constitutively positive for all these markers, except for CD80, which is weak in unstimulated LK-35.2 cells and was induced by CD40 ligation (Fig. 2A). CD40 ligation for a short period of 3 h was again sufficient to render normal B cells into fully competent APCs for T cell hybridomas (Fig. 4B).



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  		FIGURE 4. CD40 ligation enhances the Ag-presenting ability of normal B cells. A, Resting spleen B cells from C3H/HeJ mice were purified by negative selection panning (CD3-/CD11b-) and were cultured in six-well plates (1.5 x 106 cells/well in 2 ml) in the presence of 10 µg/ml anti-CD40 or anti-CD11b mAb for the indicated lengths of time. At the end of the culture, cells were fixed with 1% paraformaldehyde, reacted with the indicated mAb, and examined in a flow cytometer. B, The same B cells (5 x 104) shown in A were cultured with the T cell hybridoma A6.B3 in the presence of varying concentrations of HEL33–47 peptide for 24 h, after which 100 µl of supernatant was removed and tested for the presence of IL-2 by their ability to induce CTLL-2 cell proliferation. The results shown correspond to 0.3 µM peptide and are representative of two experiments.

 
CD40 ligation induces early redistribution and aggregation of MHC-II on the APC surface

It was recently shown that MHC-II on the APC surface concentrate in lipid rafts, even before any contact with T cells, and that raft disruption impairs Ag presentation (29). The stimulus (or stimuli) leading to MHC-II clustering in lipid rafts is as yet unknown. As CD40 is a major costimulator for APC, and it has been shown to associate with class II on the surface of B cells (33, 34), it was possible that some of the enhancing effects of CD40 ligation on Ag presentation could be through the induction of MHC-II cluster formation. LK-35.2 cells cultured in the presence of anti-CD40 for different lengths of time, stained with Texas Red-labeled anti-MHC-II, and viewed under a confocal microscope (Fig. 5A). In unstimulated cells, MHC-II are evenly distributed in small clusters over the cell surface, and anti-CD40 induces a progressive aggregation into larger clusters, reaching a peak at 3 h. Before any stimulus, the percentage of large (>1.5 µm) clusters was ~4.1 at 1 h; CD40-induced clusters occurred in 69 ± 14% of cells rising to 83 ± 2.5% at 3 h and 85 ± 2% at 24 h (Fig. 5B). These clusters presented in two main patterns; nearly half the cells had a patchy distribution of large clusters, whereas the remaining cells showed a complete polarization of MHC-II molecules into a very large high density structure (Fig. 5 and data not shown). Cluster aggregation was not just a passive effect of anti-CD40 cross-linking, as it did not occur in LK-35.2 cells cultured with anti-CD40 in the presence of 0.1% NaN3 (Fig. 6).



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  		FIGURE 5. CD40 ligation induces an early redistribution and coaggregation of MHC-II on the APC surface. A, LK-35.2 cells (104/slide) were cultured for varying lengths of time in culture slides in 200 µl of culture medium in the presence of 10 µg/ml anti-CD40 or anti-CD11b (control) mAb, after which cells were fixed with 4% paraformaldehyde and reacted with biotinylated anti-MHC-II Ab, followed by streptavidin-Texas Red. Slides were observed under a confocal microscope with an excitation filter of 540 nm and an emission filter at 590 nm. Cells are shown above as Nomarsky and red fluorescence. Below, we show the three-dimensional graphic representation of the fluorescence for the particular, representative cell shown above (height equals fluorescence intensity). Each cell shown is representative of >50 cells counted. These experiments were conducted at least four times with similar results. B, Percentage of cells with small (<1.5 µm; ) or large (>1.5 µm; {square}) clusters (including polarized). Data are shown as the mean of 50–200 cells counted in independent experiments (two or three, except for *, where one field with >100 cells was counted). SDs are shown in squares for each set counted.

 


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  		FIGURE 6. MHC-II clustering induced by CD40 ligation is energy dependent. LK-35.2 cells (104/slide) were cultured for 3 h in culture slides as described in Fig. 5 in the presence of 10 µg/ml anti-CD40 or anti-CD11b (control) with or without the addition of 0.1% sodium azide, after which cells were observed under a confocal microscope as described in Fig. 5. Experiments in C were performed twice with similar results.

 
Anti-CD40-induced aggregation of surface MHC-II includes CD80

In dendritic cells, MHC-II colocalize with the costimulatory molecules CD80/86, even before they reach the cell surface (46). Therefore, we examined whether, in addition to MHC-II cluster aggregation, CD40 ligation could induce colocalization of MHC-II with the costimulatory molecule CD80 on the APC surface. For these experiments, LK-35.2 cells were cultured on sterile glass slides in the presence of anti-CD40 for varying lengths of time, after which cells were fixed and stained for the expression of MHC-II (Alexa 488) plus CD80 (Texas Red). LK-35.2 cells have a very faint basal expression of CD80, which is patchy and colocalizes with MHC-II in all cells that were positive for CD80, which were nearly 100% (Fig. 7). As shown by flow cytometry, incubation with anti-CD40 induces an increased expression of CD80 after 24 h, but not after 3 h. However, the distribution of CD80 follows that of MHC-II in the aggregated clusters induced by anti-CD40. These results indicate that MHC-II and CD80 are present in the same clusters before CD40 ligation, providing ligands for the TCR and the costimulatory receptor CD28, respectively. These clusters coaggregate into larger clusters to become a higher avidity target for T cells, which may result in more efficient T cell activation.



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  		FIGURE 7. CD40 ligation induces coaggregation of MHC-II and CD80 on the APC surface LK-35.2 cells were cultured for varying lengths of time in culture slides as described in Fig. 3. Cells were fixed with 4% paraformaldehyde and reacted with biotinylated anti-MHC-II Ab, followed by streptavidin-Alexa 488 and anti-CD80-Texas Red. Slides were observed under a confocal microscope with long pass excitation filters from 488–540 nm and emission filters at 530 and 590. Results represent three experiments with comparable results.

 
CD40 ligation-induced MHC-II clusters are in cholesterol and sphingolipid enriched domains on the APC surface

Lipid rafts appear to contain molecules that are the focal points for the initiation of signal transduction. Recent observations indicate that MHC-II molecules can be found in lipid rafts (29). Thus, we examined whether the MHC-II clusters and higher order aggregates induced by CD40 ligation were contained within sphingolipid (hence, cholesterol)-enriched microdomains as defined by CTB binding. For these studies, LK-35.2 cells were cultured on sterile glass slides in the presence or the absence of anti-CD40 for different lengths of time, after which they were stained for GM1 (Alexa 488-CTB) and MHC-II (Texas Red) and observed under a confocal microscope. Before CD40 ligation, LK-35.2 cells express MHC-II with a basal colocalization with GM1 in small patches along the cell surface (Fig. 8). Incubation with anti-CD40 at 37°C for as little as 1 h (data not shown) was sufficient to increase the patch size, which reached a maximum at 3 h and was always colocalizing with GM1. All MHC-II clusters on the cell surface were contained within GM1-positive spots. Moreover, MHC-II clusters on the cell surface were disrupted by methyl-{beta}-cyclodextrin, which depletes cholesterol. Thus, CD40 ligation induces aggregation of small MHC-II-containing cholesterol/sphingolipid-enriched rafts into larger clusters. Moreover, although we did not carry out simultaneous staining for CD80 expression, the almost complete colocalization of MHC-II with either GM1 or CD80 also suggests CD80-MHC-II association in lipid rafts. In contrast, methyl-{beta}-cyclodextrin failed to disrupt MHC-II clusters in LK-35.2 stimulated through CD40 for 24 h. This suggests that although the initial CD40-induced aggregation of MHC-II essentially takes place in lipid rafts, these organize later into more complex structures.



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  		FIGURE 8. Short-term CD40 ligation-induced MHC-II clusters are contained in cholesterol- and sphyngolipid-enriched domains on the APC. LK-35.2 cells were cultured for varying lengths of time in culture slides as described in Fig. 5, except that in the indicated wells, methyl-{beta}-cyclodextrin (10 mM) was added for the last 10 min of culture. Cells were fixed with 4% paraformaldehyde and reacted with biotinylated anti-MHC-II Ab, followed by streptavidin-Texas Red and cholera toxin B-streptavidin-Alexa 488. Slides were observed under a confocal microscope with long pass excitation filters from 488–540 nm and emission filters at 530 and 590. Data are representative of two experiments.

 
As it has been reported that in some human B cell lines CD40 and MHC-II are associated on the cell surface (33, 34), we examined whether such an association also occurs in LK-35.2 cells both before and after CD40 ligation. However, CD40 expression by LK-35.2 cells under the confocal microscopy was faint, not allowing us to ascertain its possible colocalization with MHC-II (data not shown).

MHC-II and CD80 accumulation in lipid rafts induced by CD40 ligation correlates with an early enhancement in Ag presentation

As CD40 ligation was followed by an aggregation of MHC-II and CD80 lipid rafts into larger clusters together with an early enhancement of Ag presentation, it was of interest to examine whether disruption of such lipid rafts would abrogate the enhancing effects of CD40 ligation on Ag presentation. LK-35.2 cells were treated as described above, in the presence or the absence of methyl-{beta}-cyclodextrin. Fig. 9 shows that methyl-{beta}-cyclodextrin, in addition to disrupting MHC-II clusters, reverses the early positive effects of CD40 ligation on Ag presentation. Thus, the presence of larger MHC-II/CD80 lipid rafts directly correlates with the early enhanced Ag presentation induced by short-term CD40 ligation. This was not the case for the enhancing effect of anti-CD40 on cells stimulated for 24 h, which was not reversed by methyl-{beta}-cyclodextrin and correlated with an increase in CD80 on the APC surface and a relative resistance to raft disruption by methyl-{beta}-cyclodextrin, suggesting, again, that these higher order clusters are held together by a different mechanism.



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  		FIGURE 9. MHC-II clustering in lipid rafts correlates with early enhancement of Ag presentation. LK-35.2 cells were treated as described in Fig. 8 and were cultured (2 x 105/well) in the presence of 10 µg/ml of the indicated mAb, after which 10 mM methyl-{beta}-cyclodextrin was added to the indicated wells for the last 10 min of culture. Cells were fixed in 0.5% paraformaldehyde, washed, and cultured (2.5 x 104/well) with the T cell hybridoma E907.D or TS12 (5 x 104 cells/well) in the presence of 0.3 µM peptides HEL33–47 and RNase A43–56, respectively for 24 h, after which 100 µl of supernatant was transferred to a new plate with 104 CTLL-2 cells and processed as described in Fig. 1. Results are expressed as counts per minute x 10-3 and are representative of three experiments. Mean values of control wells: fixed LK-35.2 and E907.D cells plus HEL (10 µg/ml): control, 1079 cpm; anti-CD40, 987 cpm; M{beta}C-treated LK-35.2 and E907.D cells plus HEL (10 µg/ml): control, 589 cpm; anti-CD40, 1067 cpm, which do not differ from the counts obtained in the absence of Ag.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it has been known for a while that CD40 ligation exerts a positive effect on B cells as APC, it is not entirely clear how it is achieved. One mechanism that has been conclusively demonstrated is enhanced costimulation via induction of CD80 expression on B cells (17, 18, 19, 20). It has also been proposed that CD40 ligation improves Ag processing, through an as yet unknown mechanism (21). In the current studies we examined the nature of this phenomenon and found a positive effect of CD40 ligation on B cells as APC due to at least two mechanisms, both unrelated to Ag processing: 1) an early effect due to induced aggregation of MHC-II and CD80 lipid rafts into larger clusters, and 2) a late effect that correlates with an increase in costimulation via CD80. The early effect is sufficient to confer on B cells the ability to activate naive T cells, which in response secrete both IL-2 and IFN-{gamma}.

Among the TNF receptor family members, CD40 has one of the widest spectra of functions in the immune system. Its capacity to enhance the ability of APC to activate T cells in response to Ag has been demonstrated for DC (6, 47, 48) and B cells (44). The Ag-presenting function of B cells requires a change from a resting, tolerogenic, nonprofessional Ag-presenting B cell into an activated, professional APC fully capable of activating T cells. B cells, but not other APC from CD40-null mice, have a defect in Ag presentation (49). These studies together with our current findings place CD40 ligation as the major stimulus driving B cell Ag-presenting functions.

Previous findings that CD40 ligation induces an enhancement of the ability of B cells to activate T cells in response to an intact protein (pigeon cytochrome c) but not in response to the peptide have suggested that CD40 improves Ag processing (21). However, unlike other studies in which CD40 ligation augments processing in the class I MHC pathway via increased expression of peptide transporters (50), the mechanisms involved in the increased processing for MHC-II presentation are not clear. We found an enhancement of presentation to T cells specific for two different Ags, which differ in Ag processing requirements (51). Although anti-CD40 ligation could have an enhancing effect on Ag processing, the presentation of synthetic peptides was also enhanced by anti-CD40, indicating that additional mechanisms are involved.

In recent years increasing evidence indicates that many receptors signal while associated with lipid rafts. They provide a mechanism for increasing receptor density, which allow the association with intracellular signal transduction modules that include, among others, G proteins, phospholipases, and their substrates. It has been shown that MHC-II can accumulate in cholesterol-enriched domains (29), which may be necessary for signaling into the APC via activation of tyrosine kinases (52). Moreover, T cell recognition of MHC-II in lipid rafts on DC induces an enhanced response by CD8+ T cells (53). Thus, MHC-II accumulation in lipid rafts is important for these molecules to function as signaling receptors.

The former scenario has only recently been considered for MHC-II molecules as ligands. Indeed, Anderson et al. (29) found that MHC-II accumulation in lipid rafts is necessary for efficient Ag presentation to CD4+ T cells under limiting Ag conditions (29). However, it is not known whether MHC-II molecules need a stimulus to integrate into lipid rafts and whether lipid rafts in nonactivated and activated APC are different. We found that CD40 ligation induces small MHC-II and CD80 containing cholesterol/sphingolipid-enriched rafts to aggregate into larger clusters. Moreover, although we did not do simultaneous staining for CD80 expression, the almost complete colocalization of MHC-II with either GM1 or CD80 suggests that the early association of CD80-MHC-II takes place in lipid rafts. Whether this is due to intracellular preassembly into secretory modules or occurs at the cell surface is not clear. At later times, MHC-II clusters were not disrupted by cholesterol depletion, which correlated with a relative resistance of APC to methyl-{beta}-cyclodextrin in Ag presentation assays. This suggests that these early cholesterol-mediated aggregates further organize into higher order structures, which could be held together by the cytoskeleton. We have not yet tested this formally. In any case, the initial association of MHC-II with CD80 by itself in LK-35.2 cells is not dependent on CD40 ligation.

Costimulator induction has largely been considered the mechanism by which a resting B cell becomes a professional APC (44, 54, 55, 56). In addition to the classical CD80/CD86-CD28-mediated costimulation, other forms of costimulation have been described in CD40-activated B cells (55). We found that CD40-induced aggregation of MHC-II and CD80 on the B cell surface was sufficient to enhance Ag presentation to naive CD4+ T cells. Although LK-35.2 cells have a baseline expression of MHC-II and costimulatory molecules, there was no need for an increase in the baseline levels of costimulation, at least through CD80-CD28, to enhance their ability to activate naive T cells, although more conclusive data await studies with CD86 as well. Thus, it appears that MHC-II plus the low level CD80 clustering in lipid rafts are sufficient for B cells to function as professional APC. Before CD40 ligation, all CD80 was already found in association with MHC-II clusters, with the remaining MHC-II appearing diffuse along the LK-35.2 cell surface.

The mechanism leading to cluster formation after CD40 ligation is not readily clear. Most studies of the functional effects of CD40 ligation have been conducted for longer than the 3-h incubation period used in this study. CD40 ligation is followed by the recruiting of adaptor proteins of the TNF receptor-associated factor family (57). Although the immediate events following TRAF recruitment have not been completely defined, CD40 ligation on B cells induces or inhibits the expression of >100 genes through three main signal transduction pathways that include members the mitogen-activated protein kinase p38 and phosphatidylinositol 3-kinase and translocation into the nucleus of members the NF-{kappa}B/Rel transcription factors (58). The early appearance of MHC-II/CD80 cluster aggregation after CD40 ligation makes it unlikely to be transcriptionally regulated. On the cell surface, CD40 resides in lipid rafts that also contain other molecules involved in CD40 signaling (59). It will be of relevance to examine whether MHC-II and CD80 reside in the same lipid rafts and if local changes in the organization of signaling modules in these rafts are related to the effects described herein. We are currently undertaking studies to define the signal transduction pathways involved in CD40 ligation-induced MHC-II/CD80 cluster aggregation on B cells.

Why does aggregation of MHC-II/CD80 clusters have such a dramatic impact on the ability of B cells to activate T cells? We suggest that large MHC-II/CD80 clusters provide a higher avidity target that engages greater numbers of TCRs from the start of T cell-APC interaction. This eventually leads to formation of the immunological synapse (60) and indeed provides a better means for reciprocal signaling between T cells and APC, leading to full activation of both cells. It has been recently shown that MHC-II-peptide-containing lipid rafts accumulate at the immunological synapse (61). Our results place CD40 ligation as an important inducer for such accumulation and indicate that in addition to MHC-II, the costimulatory molecule CD80 is included in such raft accumulation. This provides CD4+ T cells with the complete set of signals for proper activation.

Although CD40 ligation is not the initial stimulus for the APC, it is an early event, as CD154 expression by T cells occurs within a few hours after their initial activation by Ag. If our observations are relevant in vivo, we could consider at least two possible scenarios. We favor the view that CD40 ligation could play its main role in Ag presentation shortly after the initial stimulus via TCR, which is responsible for CD154 induction, by enhancing transport of MHC-II to the TCR-APC interphase, leading to synapse formation. This is not opposed to the observation that MHC ligation by the TCR down-modulates peptide-MHC complexes from the cell surface (62), because the increased clustering would occur mainly at the T cell-APC interphase. Thus, cluster induction by CD40 ligation is not needed for the initial interaction. However, for T cell Ag recognition, foreign peptides need to compete against a myriad of self peptides contained within the cell, first to bind to MHC-II and subsequently to be engaged by the appropriate number of TCRs to initiate T cell activation. CD40 ligation would increase the overall flow and nonspecific grouping of MHC-II molecules on the cell surface. The relevant MHC-II-peptide complexes would then be selected among them by the TCR to form the immunological synapse, as it has recently been shown (61). Preliminary findings with LK-35.2 cells that neither TLR-9 activation nor B cell Ag receptor cross-linking provides a stimulus comparable to that of CD40 ligation, suggest that even though CD40 is not the initial stimulus to increase B cell Ag presentation, it still is an early event, and its relevance is supported by the inability of CD40-/- B cells to become professional APC (49).

An alternative explanation could be that the CD40 ligand could be expressed by a bystander cell. This seems unlikely, however, because although CD154 has been reported to be expressed by many cell types (63, 64), the only ones that have been convincingly and reproducibly shown to express CD154 are activated T cells and platelets (65, 66). Platelets are unlikely to be present in the lymphoid interstitium in sufficient numbers to ligate CD40 and activate B cells. Moreover, it is not desirable to trigger the professional Ag-presenting activity of B cells in the absence of the immunizing Ag.

In conclusion, the present studies provide evidence that CD40 ligation enhances the Ag-presenting function of B cells, which is related to an association of TCR and costimulatory receptor ligands on the APC surface. Thus, according to our working model, 1) the initial APC for CD4+ T cells, DC, induce CD154 expression on T cells, mainly via TCR signaling; 2) CD154 on activated CD4+ T cells interacts with CD40 on the resting B cell, which turns into a professional APC in two steps: an initial increase in local MHC costimulator density (rafts), providing a higher avidity ligand, followed by increased expression of CD80 that results in more efficient costimulation, eventually conducing to the formation of the immunological synapse at the T cell-B cell interphase; and 3) reciprocally, MHC-II rafts should allow better signal transduction of TCR-delivered signals into Ag-presenting B cells.


   Acknowledgments
 
We thank all the scientists who kindly made available to us cell lines and reagents: Drs. Günter Hämmerling, Laurie Glimcher, Paul Allen, and Andy Heath. The comments of Drs. Elizabeth Langley, Fernando Esquivel, and Carlos Rosales and the assistance of Paty Rojo, Kate Vignali, and Augusto Aguilar are especially appreciated. We thank Dr. Leobardo Mendoza for assistance with the confocal microscope. Some of the experiments were conducted by J. Moreno as a visiting scientist to St. Jude Children’s Research Hospital (DV laboratory). We also thank Creg Workman for advice, Janet Gatewood for the cytokine analysis, and Richard Cross for cell sorting.


   Footnotes
 
1 This work was supported by Grant 30899-M from Consejo Nacional de Ciencia y Tecnologial México (to J.M. and L.C.B.), and by Cancer Center Support Center of Research Excellence Grant CA21765 and the American Lebanese Syrian Associated Charities (to D.A.A.V.). Back

2 Address correspondence and reprint requests to Dr. José Moreno, Research Unit on Autoimmune Diseases, Centro Médico Nacional Siglo XXI, Apartado Postal A-047, 06703, Coahuila No. 5, Col. Roma, México. E-mail address: jmoreno49{at}prodigy.net.mx Back

3 Abbreviations used in this paper: MHC-II, class II MHC molecule; CTB, cholera toxin B; DC, dendritic cell; GM1, ganglioside M1; HEL, hen egg-white lysozyme. Back

Received for publication March 3, 2003. Accepted for publication October 1, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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