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The Journal of Immunology, 2001, 167: 6849-6858.
Copyright © 2001 by The American Association of Immunologists

In Vivo and In Vitro Modulation of HLA-DM and HLA-DO Is Induced by B Lymphocyte Activation1

Corinne Roucard2,*, Claire Thomas{dagger}, Marie-Anne Pasquier*, John Trowsdale{ddagger}, Jean-Jacques Sotto*, Jacques Neefjes§ and Marieke van Ham§,¶

* Groupe de Recherche sur les Lymphomes, Institut Albert Bonniot, Domaine de la Merci, La Tronche, France; {dagger} Molecular Neuropathobiology Laboratory, Imperial Cancer Research Fund, London, United Kingdom; {ddagger} Division of Immunology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom; § Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; and Department of Pathology, Unit Experimental Oncopathology, Free University Hospital, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ag presentation via HLA class II molecules in B lymphocytes depends on the coordinated action of HLA-DM, the catalyst of class II-peptide loading, and HLA-DO, a pH-dependent modulator of DM, the expression of which is almost completely restricted to B lymphocytes. The relative expression levels of both class II modulators are critical for the composition of the HLA class II peptide repertoire. The data in this work demonstrate that DO and DM expression are both dependent on the cellular activation status in primary human B lymphocytes. In vivo low-density activated primary human B lymphocytes show a prominent reduction in DO and DM expression when compared with high-density resting primary B lymphocytes. In vitro, reduction of DO and DM expression can be induced by B lymphocyte activation via the B cell receptor or by use of the phorbol ester, PMA. Specific inhibition of protein kinase C resulted in a significant reduction of HLA-DO and is potentially due to protein degradation in lysosomal compartments as the phenomenon is reversed by chloroquine. Thus, the expression of the dedicated HLA class II chaperone DM and its pH-dependent modulator DO is regulated and tightly controlled by the activation status of the B lymphocyte.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class II molecules present Ags derived from exogenous sources to the TCR on CD4+ T lymphocytes. In the human, MHC class II molecules are composed of the HLA-DR, -DQ, and -DP. Shortly after synthesis, the DR{alpha} and DR{beta} chains form a complex with the invariant chain (Ii)3 (1), which targets the class II/Ii complex into the endocytic pathway (2). During transport to the Ag loading compartments (dubbed MIIC for MHC class II-containing compartment), Ii is degraded until only a class II-associated Ii peptide (CLIP) remains bound to the class II peptide binding groove. In the MIICs, binding of incoming Ag to class II requires the action of HLA-DM that resides in endosomal/lysosomal compartments, as it catalyzes the release of CLIP from the class II backbone (3, 4). Finally, the class II-peptide complex is transported to the cell surface (5) where interaction with an appropriate T cell can occur.

In B lymphocytes, another MHC class II-like heterodimer, termed HLA-DO, associates with DM (6). The expression of the DO complex (7, 8) and the murine equivalent H2-O, is unusual in that it seems to be restricted to B lymphocytes and thymic epithelium (9, 10). B lymphocytes have unique features as APCs, in that they can take up Ags via a specific receptor, the B cell receptor (BCR). Recent studies demonstrated that DO is a negative modulator of the catalytic activity of DM (11, 12, 13). The action of DO depends on the pH of the compartment in which the DM/DO complex resides. DO effectively blocks DM function at the endosomal pH, while allowing DM action at the more acidic pH of the MIICs (11, 14, 15). In this way, DO action may skew the class II-peptide loading process toward acidic compartments. This may favor presentation of specific peptides internalized via the BCR, as these preferentially form a complex with class II in the MIICs (16, 17), while counteracting presentation of peptides that are taken up via fluid phase endocytosis. Indeed, DO modulates the antigenic peptide repertoire associated with class II molecules both qualitatively and quantitatively (13). Moreover, mice knockout for H2-O have enhanced class II presentation of fluid phase Ags as opposed to Ags internalized via BCR (11).

In this work, the expression and regulation of both modulators of class II-mediated Ag presentation as well as HLA class II molecules were studied in primary human B lymphocytes isolated from peripheral blood. The expression of DO in primary B lymphocytes is much higher than anticipated on the basis of previous studies with B cell lines. Strikingly, in in vivo low-density activated B lymphocytes the expression of both DM and DO is strongly reduced compared with high-density resting B lymphocytes. In vitro B cell stimulation via the BCR or with the phorbol ester PMA induced a reduction in DM and DO expression. Inactivation of protein kinase C (PKC) with the bisindolylmaleimide Ro 31-8220 resulted in a significant loss of HLA-DO expression and, in some cases, of HLA-DM. Lysosomal degradation could be involved as chloroquine (CQ) reverses the effect of Ro 31-8220. Thus, activation of primary B lymphocytes enable the cells to control the efficacy of MHC class II-mediated Ag presentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and peptides

The mouse mAbs 5C1 (anti-DM{alpha}) (18), DA6-147 (anti-DR{alpha} cytoplasmic tail) (19, 20), D1.12 (anti-DR{alpha}) (21), and CerCLIP (22) were previously described. The anti-PKC{alpha} and anti-PKC{beta} mAbs were obtained from BD Transduction Laboratories (Lexington, KY) and the anti-actin mAb was obtained from Oncogene Research Products (Cambridge, MA).

The mAbs anti-CD80 labeled with FITC and anti-CD86 labeled with PE were obtained from Immunotech (Marseille, France), and the rabbit F(ab')2 Abs against human IgD labeled with FITC, IgG labeled with FITC, and IgM labeled with PE were from DAKO (High Wycombe, U.K.).

The rabbit anti-DO{beta} serum was generated by immunizing a rabbit with the C-terminal peptide (SGNEVSRAVLLPQSC) of DO{beta} (8) conjugated via glutaraldehyde (Sigma-Aldrich, Poole, U.K.) to keyhole limpet hemocyanin (Calbiochem, La Jolla, CA). The DO{beta} peptide was synthesized using F-moc/tBu strategy, and structure and purity were confirmed by HPLC analysis.

Analysis of Endoglycosidase H-resistant molecules was conducted and showed that the 5C1 mAb and the rabbit anti-DO{beta} serum recognized mature populations, as DO{beta} molecules were all resistant and DM{alpha} presented one level of sensitivity to Endoglycosidase H as reported for mature MHC class II {alpha} molecules (data not shown).

Cell lines and culture conditions

The Burkitt’s lymphoma B cell lines Raji (African origin, EBV-positive) and Ramos (American origin, EBV-negative) were obtained from the American Type Culture Collection (Manassas, VA). The cell lines were routinely grown in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (Life Technologies), penicillin/streptomycin, and glutamin in the presence of 5% CO2 at 37°C.

Preparation of B lymphocytes

Buffy-coats were obtained from healthy donors from the Isère and Savoie Blood Transfusion Center (n = 30; Grenoble, France), the London North Blood Bank (n = 19; London, U.K.) or the Central Laboratory for the Blood Transfusion Service (n = 13; Amsterdam, The Netherlands). Mononuclear cells were isolated by centrifugation on Ficoll-Hypaque gradient (Pharmacia Biotech, Uppsala, Sweden). B lymphocytes were purified with anti-CD19 Dynabeads and detached with DETACHaBEAD (Dynal Biotech, Oslo, Norway), according to the manufacturer’s instructions. B lymphocytes isolated by this method were extensively shown to remain quiescent (23). The cell purity and viability was >99% as determined by cytofluorometric analysis (data not shown).

Purification of high- and low-density B lymphocytes

Freshly purified CD19+ B lymphocytes were separated on a discontinuous Percoll (Pharmacia Biotech) density gradient of 60, 50, 40, and 30% Percoll layers as previously described (24). Lymphocytes obtained from the bottom of the 60% fraction are referred to as high-density B lymphocytes and correspond to cells in a resting state, and lymphocytes collected from the 50–60% interface are referred to as low-density B lymphocytes and correspond to cells with a more activated phenotype. Routinely, the amount of cells obtained from the 50–60% interface was ~10 times less than the amount obtained from the bottom of the 60% fraction. For analysis, 5 x 106 B lymphocytes were immediately lysed in 1% Nonidet P-40 lysis buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, 1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride HCl, 100 µM iodoacetamide, 10 µM leupeptin) for 20 min at 4°C and centrifuged for 30 min at 10,000 x g. The supernatants were stored at -20°C until further use.

In vitro activation of high-density B lymphocytes

B lymphocytes obtained from the bottom of the 60% fraction, high-density B lymphocytes, were cultured at 1 x 106 cells/ml for 24 h at 37°C in 5% CO2 in RPMI 1640 supplemented with 10% FCS, and activated with 20 ng/ml PMA (Sigma-Aldrich). Alternatively, cells were stimulated using the anti-DR mAb D1.12 (10 µg/ml) followed by cross-linking by F(ab')2 of goat anti-mouse IgG (20 µg/ml) (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were activated by BCR stimulation by addition of a F(ab')2 of goat anti-human IgM, Fc5µ fragment specific (5 µg/ml, azide free; Jackson ImmunoResearch Laboratories). Finally, cells were activated using a combination of IL-4 (10 ng/ml; Eurocetus, Amsterdam, The Netherlands) and anti-CD40 mAb (0.5 µg/ml, clone mAb89, azide free; Immunotech). After activation, cells were lysed in a 1% Nonidet P-40 lysis buffer and centrifuged, and supernatant was stored at -20°C until further use.

PKC inhibition

Freshly purified high-density B lymphocytes were incubated for 4 h with the PKC inhibitor bisindolylmaleimide Ro 31-8220 (final concentration 10 µM; stock solution in DMSO; Calbiochem). Control cells were incubated with equivalent amounts of DMSO, representing less than 0.1% of the total volume. For inhibition of de novo protein synthesis, cells were preincubated for 1 h with cycloheximide (CHX) (final concentration 20 µM; stock solution in ethanol; Sigma-Aldrich) followed by a 4-h incubation in the presence or absence of Ro 31-8220 (10 µM). To neutralize the acidic pH of endosomal/lysosomal compartments, cells were preincubated with 200 µM CQ for 15 min, followed by 4 h with both CQ and Ro 31-8220 (10 µM) or CQ alone.

SDS-PAGE and Western blot analysis

The protein content of cell lysates was quantified using the bicinchoninic acid protein assay (Pierce, Rockford, IL). SDS-PAGE of cell lysates was performed on 10% polyacrylamide gels after boiling of the samples for 10 min in reducing Laemmli sample buffer. For Western blot analysis, equal amounts of proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon P; Millipore, Bedford, MA) in 25 mM Tris, 192 mM glycine, 5% methanol, pH 8.3. In the case of transfer of high amounts of protein (i.e., up to 150 µg of protein per lane), a modified transfer buffer was used consisting of 10 mM 3(cyclohexylamino)-1-propane sulfonic acid, 5% methanol, pH 11. Membranes were blocked in 3% skimmed milk in PBS. Ab binding (in 1.5% skimmed milk in PBS) was detected by incubation with secondary HRP-conjugated rabbit anti-mouse or swine anti-rabbit Ig Abs (DAKO), followed by ECL detection (Amersham, Arlington Heights, IL). Washes were performed using 0.1% Triton X-100 in PBS.

For semiquantitative analysis of the Western blots, subsaturated autoradiograms were scanned and the signals were analyzed by densitometry (TINA 2.09 software; Raytest, Straubenhardt, Germany). The different samples of one experiment were loaded on the same gel and were therefore transferred and treated in the same conditions. The autoradiograms for actin were used as the reference for protein loading. The lane corresponding to the control (high-density cells or control of the experiment) was used as the reference and accorded an arbitrary value of 1. The ratio for the other lanes compared with the control was determined. The corresponding densitometric values for DR, DM, and DO were corrected by the obtained ratios. The corrected value for the control was accorded an arbitrary value of 100 and the corresponding percentage was calculated for the other conditions. After this standardization was done for every donor, the values were pooled together and the mean and SD were calculated.

FACS analysis

Cells (5 x 105 cells per experiment) were washed in PBS containing 0.02% NaN3 and incubated for 15 min on ice in PBS containing 0.02% NaN3, 5% FCS, 100 µg/ml human gamma-globulins (Calbiochem). Subsequently cells were washed, incubated with the unlabeled Abs for 30 min in PBS, 0.02% NaN3, and 1% BSA on ice, washed, and incubated with a secondary FITC-labeled F(ab')2 of goat anti-mouse Ig (DAKO) or with Abs directly labeled with FITC or PE, for 30 min on ice before washes and analysis on a FACScan cytometer (BD Biosciences, Moutain View, CA). Ten thousand events were acquired on living cells gated on forward scatter.

Statistical analysis

Two nonparametric tests (Kruskal-Wallis and Mann-Whitney) were used for statistical analysis of DR, DM, and DO expression in different conditions in Figs. 2Go, 4Go, and 6Go.



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  		FIGURE 2. A, Analysis of IgM and IgD cell surface expression on primary high- and low-density B lymphocytes. Freshly purified CD19+ B lymphocytes from human peripheral blood were separated into high- and low-density B lymphocytes by Percoll density gradient centrifugation and analyzed by flow cytometry. B, Western blot analysis of DR, DM, DO, and actin expression in high- and low-density B lymphocytes. High- and low-density freshly purified B lymphocytes from the same donor were analyzed by Western blotting as in Fig. 1GoB. Molecular weights are indicated on the left. A representative experiment of four is shown. C, Semiquantitative analysis of DR, DM, and DO expression in high-density vs low-density B lymphocytes from four donors, and correlation with the expression of a control protein (actin). Autoradiograph films were scanned and signals were quantified by densitometric analysis. The values for DR, DM, and DO expression levels were standardized by comparison with the respective value obtained for actin as detailed in Materials and Methods. Statistical analysis revealed p = 0.343 (nonsignificant) for DR and p = 0.029 (significant) for both DM and DO when comparing low- to high-density B lymphocytes.

 


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  		FIGURE 4. Modulation of DR, DM, and DO expression after in vitro activation. Freshly purified high-density B lymphocytes were cultivated for 24 h in vitro in 1) culture medium only (control), or upon addition of various stimuli: 2) PMA, 3) D1.12 anti-HLA-DR mAb, 4) soluble anti-IgM Ab, and 5) IL-4 plus anti-CD40 mAb. Subsequently, cells were lysed and analyzed by Western blot as before. Semiquantitative analysis from three different samples is shown, correlated to the level of the control protein actin (as described in Fig. 2GoC). A representative Western blot experiment is shown. Statistical analysis revealed p = 0.407 (non significant) for DR and p = 0.03 (significant) for both DM and DO when comparing the conditions PMA and anti-IgM to the other conditions.

 


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  		FIGURE 6. Semiquantitative Western blot analysis of DR, DM, and DO expression upon PKC inhibition. High-density B lymphocytes were incubated for 4 h with DMSO (control) or with equal amounts of DMSO containing Ro 31-8220 (final concentration 10 µM). Cell lysates were treated and analyzed as before. The presented results are means of five different samples, and a representative Western blot experiment is shown. Statistical analysis revealed p = 0.69 (nonsignificant) for DR, p = 0.151 (nonsignificant) for DM, and p = 0.008 (significant) for DO when comparing the condition Ro 31-8220 to the control.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primary mature B lymphocytes express high amounts of HLA-DO

So far, the function of the modulators of human MHC class II-mediated Ag presentation, HLA-DM and HLA-DO, has been studied in immortalized cells (6, 12, 13, 14). However, an important question remains; i.e., how do these modulators regulate the Ag presentation process and consequently the composition of the MHC class II antigenic peptide repertoire in the in vivo situation? Therefore, the physiological, in vivo expression levels of the various components of MHC class II-dependent Ag presentation were studied in human primary B lymphocytes isolated from the peripheral blood of healthy donors.

First, the phenotypic characteristics of the isolated primary B lymphocytes were compared with those of two Burkitt’s lymphoma B cell lines, Raji and Ramos, which are considered to represent different maturation stages of B cell differentiation (25). Human B lymphocytes purified from peripheral blood were >80% IgM+ and IgD+ and <10% IgG+ (Figs. 2GoA and 1A, respectively), demonstrating that they constitute a population of mature B lymphocytes. Ramos had an IgM+, IgG-, and IgD- phenotype and Raji did not express any surface Ig (Fig. 1GoA), as described before (25).



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  		FIGURE 1. A, Cell surface phenotype of primary B lymphocytes and two B cell lines. Purified B lymphocytes and Raji and Ramos B cells were analyzed by flow cytometry after cell surface labeling with rabbit anti-IgM (directly labeled with PE) and anti-IgG (directly labeled with FITC) Abs, anti-CD80 (directly labeled with FITC) and anti-CD86 (directly labeled with PE) mAbs, or anti-HLA-DR mAb (D1.12) followed by a goat anti-mouse IgG F(ab')2-FITC. The mean fluorescence intensity (MFI) is presented in parenthesis, after subtraction of background values. The IgG+ population is gated in a circle, and the percentage is shown above. B, Expression of HLA-DR, -DM, and -DO in total cell lysates of CD19+ freshly purified B lymphocytes and Raji and Ramos B cell lines. Equal amounts of cellular proteins were analyzed by Western blot (15 µg for DO, and 5 µg for DR, DM, and actin) as demonstrated by the comparable amounts of actin, as well as the comparable staining of the cross-reactive band obtained with the rabbit anti-DO{beta} serum. The slightly higher m.w. position of DM{alpha} observed with Ramos was due to additional glycosylation, as demonstrated by N-glycosidase F (Roche, Meylan, France) digestion (data not shown). It was previously reported not to interfere with the catalytic activity of DM (22 ). The results shown are derived from a representative experiment performed on five different primary isolates and five independent cell lysates of the B cell lines. Molecular weights are indicated on the left. C, Western blot analysis of DO expression in primary B lymphocytes and Raji and Ramos cell lines using an anti-DO{beta} rabbit polyclonal serum on varying total amounts of cellular proteins. Complete protein transfer was checked by Coomassie brilliant blue staining of the polyacrylamide gel after transfer in 3(cyclohexylamino)-1-propane sulfonic acid buffer (data not shown).

 
The costimulatory molecules CD80 and CD86, ligands of CD28 and CTLA-4 on T lymphocytes, are crucial for optimal Ag presentation, and their expression levels are increased upon activation. CD80 and CD86 were observed at a low level in both primary B lymphocytes and Ramos (Fig. 1GoA), suggesting that they were relatively unactivated. In contrast, Raji expressed a high level of CD80 and CD86 (Fig. 1GoA), implying a higher activation status for Raji, which is in line with the presence of Fc, C3, and EBV receptors on the cells (25). Increased expression of MHC class II molecules has been correlated with B lymphocyte activation as well (26, 27). Indeed, the cell surface expression of HLA-DR was much higher in Raji cells than on Ramos or primary cells (Fig. 1GoA).

The expression levels of the various proteins that contribute to the efficiency of MHC class II Ag presentation were examined by Western blot analysis of the B cell lines and different isolates of primary B lymphocytes (Fig. 1GoB). In total, Raji has a far more abundant HLA-DR expression than Ramos, in agreement with the higher cell surface levels of HLA-DR (Fig. 1GoA). Both DM and DO expression were higher in Raji than in Ramos as well (Fig. 1GoB). In primary B lymphocytes the total HLA-DR expression was of an intermediate level and DM expression was consistently slightly higher (Fig. 1GoB). However, in marked contrast, DO expression was greatly enhanced in all primary isolates tested (Fig. 1GoB). Western blot analysis of increasing protein levels between primary B lymphocytes and the B cell lines demonstrated that for Raji a total of 100–150 µg of protein was necessary to obtain a DO signal equivalent to that observed in primary B lymphocyte lysates containing 5–10 µg protein (Fig. 1GoC). Thus, the in vivo DO expression level in primary B lymphocytes seems to be much higher than anticipated so far from studies using immortalized B cell lines, which is strongly indicative of an important physiological role for DO in primary B lymphocytes.

Attenuation of DM and DO expression upon B cell activation

Next, we investigated whether the components of the MHC class II Ag presentation pathway are subject to modulation in vivo via B cell activation. For this, primary B lymphocytes were subjected to Percoll gradient centrifugation, which separates the lymphocytes in fractions according to their specific cellular density, because activated lymphocytes have a lower density than resting lymphocytes (24). Typically, ~10% of the B lymphocytes migrated to a low-density fraction and 90% to a higher-density fraction (data not shown). As anticipated, FACS analysis of primary cells isolated from the lower-density fractions shows a different scatter pattern than for the cells isolated from the higher-density fractions (Table IGo). Each fraction contained B lymphocytes that were >80% of IgM+ and IgD+ (Fig. 2GoA) and <10% IgG+ (data not shown), demonstrating that the majority of both types of B lymphocytes were of a mature phenotype. FACS analysis showed that the B lymphocytes obtained from the lower-density fractions expressed both more CD80, CD86, and HLA-DR (Table IGo). Therefore, the B lymphocytes from the lower-density fraction have a more activated phenotype than those migrating in the higher-density fraction.


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  		Table I. Cell surface expression of CD80, CD86, and HLA-DR on high- and low-density (in vivo activated) primary human B lymphocytes

 
DR, DM, and DO total expression levels were compared between high- and low-density B lymphocytes derived from the same donor (Fig. 2GoB). In total, 11 of 15 donors presented highly reduced levels of both DM and DO in the low-density B lymphocytes. Semiquantitative analysis of the protein expression levels on four representative samples confirmed that the reduction of both DM and DO expression upon B cell activation was significant (p = 0.029 for both), whereas the total amount of DR was not significantly reduced by B cell activation (p = 0.343) (Fig. 2GoC). Therefore, the expression of DM and DO is highly dependent on the activation status because they are down-modulated upon B cell activation.

DM and DO expression and the cell surface deposition of DR/CLIP complexes

As variations in the relative expression levels of DM and DO may affect exchange of CLIP for antigenic peptide onto class II molecules, we compared the relative amount of DR/CLIP complexes vs total DR complexes that reach the cell surface on Ramos, Raji, and high- and low-density primary B lymphocytes from three donors. Ramos did not significantly express any DR/CLIP at its cell surface, while considerable amounts of cell surface exposed DR/CLIP were observed on Raji (Fig. 3Go). This correlates with the observation that Ramos expressed relative low amounts of DR, hardly any DO, and only slightly less DM than Raji (Fig. 1GoB), a situation that will ensure optimal CLIP removal from the relatively low amount of newly synthesized DR/CLIP through an active DM pool that is only marginally restrained by a minor pool of DO.



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  		FIGURE 3. Cell surface ratio of DR/CLIP vs total DR. Ramos and Raji cell lines, high- and low-density B lymphocytes from three different donors, were analyzed for cell surface expression of DR/CLIP complexes (with CerCLIP mAb) and compared with the total level of DR complexes (with D1.12 mAb). MFI value for CerCLIP labeling obtained after subtraction of control value was divided by the MFI value of D1.12 labeling (after subtraction of control) and expressed as percentage. MFI for Raji and Ramos are a mean of three independent experiments.

 
All primary B lymphocytes expressed clearly detectable levels of DR/CLIP at the cell surface (Fig. 3Go), indicating that in vivo in B lymphocytes a fraction of newly synthesized DR/CLIP routinely escapes the editing action of DM and DO. The relative amount of DR/CLIP varied highly between donors (Fig. 3Go), possibly due to allelic variation between donors, as the strength of HLA-DR association with CLIP differs with the DR allele. The ratio of DR/CLIP vs total DR was neither strongly nor consistently affected by in vivo activation of the B lymphocytes (Fig. 3Go). This correlates with the finding that B lymphocyte activation down-modulates expression of both DM (positively affecting DR/CLIP dissociation) and DO (negatively affecting DR/CLIP dissociation) (Fig. 2GoC) in a coordinated fashion.

Down-modulation of DM and DO upon in vitro activation of primary B lymphocytes

Because in vivo the down-modulation of DO and DM expression correlates with activation of the B lymphocytes, freshly isolated high-density B lymphocytes were cultured for 24 h in the presence of various in vitro stimuli. Soluble anti-IgM Ab (inducing B cell proliferation) (28) was used to stimulate B lymphocytes via BCR cross-linking. The mAb D1.12, recognizing an epitope near the peptide binding groove on the DR{alpha} chain (29), was used to stimulate the B lymphocytes via HLA-DR (30, 31). IL-4 and anti-CD40 mAb, a well-known combination for B lymphocyte activation and proliferation, were applied as well (32, 33). Finally, cells were incubated with PMA, a potent B lymphocyte activator via modulation of PKC (34, 35). All stimuli induced various levels of B cell homotypic adhesion, except in the control, indicative of effective stimulation/activation (36) (data not shown). The consequences of in vitro activation on expression of DR, DM, and DO were examined. Semiquantitative analysis of three independent isolates showed a trend of increased class II levels compared with control-treated cells with all stimuli (Fig. 4Go). The levels of both DM{alpha} and DO{beta} were significantly (p = 0.03 for both) and consistently decreased upon activation with PMA and soluble anti-IgM Ab, while other in vitro stimuli did not affect their expression (Fig. 4Go). Similar results were obtained with a rabbit serum against DO{alpha} (data not shown). BCR stimulation with soluble anti-IgM Ab induced a reduction in DO and DM expression in 12 subsequently tested samples. PMA-induced down-modulation of DO and DM expression was observed in all samples tested subsequently (n = 14). Thus, the observed reduction of both DM and DO in in vivo low-density B lymphocytes can be mimicked in vitro by prolonged activation by PMA and, more importantly, after application of a physiological stimulus like triggering of the BCR.

The cell surface level of HLA-DR/CLIP complexes was investigated on PMA and BCR-stimulated B lymphocytes (data not shown). Both the total level of cell surface expressed DR and the total level of DR/CLIP were enhanced upon stimulation, without significantly modifying the ratio of CLIP occupied class II vs total class II, in line with the concomitant diminution of both DM and DO expression.

PKC modulates DM and DO expression in primary human B lymphocytes

Addition of PMA initially induces a rapid activation of classical ({alpha}, {beta}, and {gamma}) and novel ({delta}, {epsilon}, {epsilon}', {eta}, and {theta}) isoforms of the PKC family and subsequently depletes the cellular PKC pool upon prolonged incubation times (34, 35). The most abundantly expressed PKC isoforms in B lymphocytes that can be modulated by phorbol esters are PKC{alpha} and PKC{beta} (37) and were therefore explored in our study.

The effect of BCR triggering on DM and DO expression was examined in more detail using the soluble anti-IgM Ab in a time course experiment on high-density B lymphocytes. The decrease in DM and DO was observed after 5 h and is marked at 15 h of BCR stimulation (Fig. 5GoA). Interestingly, a slight reduction of PKC{beta} but not of PKC{alpha} was observed at 15 h of stimulation (Fig. 5GoA).



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  		FIGURE 5. Time course experiments of anti-IgM soluble Ab and PMA addition to freshly purified high-density B lymphocytes and its effect on DR, DM, DO, and PKC{alpha} and -{beta} expression. The cells were analyzed as before. The figure shown is a representative experiment performed on one isolate for PMA and one for IgM stimulation (n = 5 for PMA and n = 3 for anti-BCR).

 
As PMA induced a reduction in DM and DO as well, a similar time course experiment was conducted with PMA. DM and DO started to diminish after 15 h of stimulation (Fig. 5GoB). Five hours after PMA addition a drop in the level of PKC{beta} became apparent and from 15 h onwards PKC{alpha} decreased as well, until after 24 h both PKC{alpha} and PKC{beta} could hardly be detected (Fig. 5GoB). Thus, depletion of both PKC{alpha} and PKC{beta}, the major phorbol ester-sensitive PKC pools in the B lymphocytes, preceded the decline in DM/DO expression, suggesting involvement of PKC in DM and DO modulation. As previously noted, the total expression level of DR was not negatively affected during the course of PMA incubation (Fig. 5GoB), and might be explained by the different behavior of DR molecules as, once charged with a peptide, they acquired a more stable structure and were targeted to the cell surface (5).

It is interesting to note that, in contrast to PMA stimulation, no total loss of PKC was observed after BCR stimulation. We cannot exclude that, to regulate the activation pathway via the BCR (38), a desensitization or inactivation of PKC without degradation could occur once the intracellular signal is delivered (39, 40, 41). The modulation of DM and DO expression seems to be selectively activated by some stimuli, because neither HLA-DR nor CD40 and IL-4 stimulation can modulate DM and DO, and it also appeared to be independent of cell proliferation as obtained with CD40 and IL-4 stimulation (data not shown and Ref. 33).

If PKC depletion is directly controlling the expression of DM and DO, specific inhibition of PKC should yield a similar but more timely effect. To examine this, primary high-density B lymphocytes from five different donors were incubated for 4 h with Ro 31-8220, a competitive and selective inhibitor of classic and novel PKCs. Semiquantitative analysis on these samples showed a marked and significant reduction in expression of DO (p = 0.008) and a trend toward reduction in DM (nonsignificant, p = 0.151) (Fig. 6Go). Subsequent testing of a large panel of donors showed this result in 75% percent of the donors (11 of 15). The discrepancy concerning the modulation of HLA-DM when comparing BCR and PMA stimulation with PKC inhibition by Ro 31-8220 could be due to 1) a weaker effect of Ro 31-8220, 2) a larger duration of inhibition required for DM modulation, or 3) the influence of other factors besides PKC on DM expression. Thus, in primary B lymphocytes PKC activity may, either directly or indirectly, regulate the expression levels of the modulators of HLA class II Ag presentation.

PKC regulates DM and DO expression via modulation of their degradation rate

Modulation of the steady state expression levels of DM and DO occurs either via manipulation of their synthesis rates or via regulation of the DM and DO life span. A coordinated transcriptional DM and DO regulation, without affecting DR expression as well, is unlikely because DMA, DMB, DOA, DRA, and DRB genes are under the control of the same transcriptional regulator, CIITA (42, 43). The coordinated modulation of DM and DO expression at the protein level is more likely, because it has been shown that DO depends on its association with DM for its steady state expression level and endoplasmic reticulum exit (6). Subcellular fractionation studies of DM-mutant B cell lines confirmed that also in B lymphocytes, DO expression (and endoplasmic reticulum egress) depends on DM expression (data not shown). The mode of DM and DO regulation was investigated by PKC inhibition studies in the presence of CHX, an inhibitor of initiation of protein translation (44). In CHX-treated B lymphocytes, inhibition of PKC activity by Ro 31-8220 led to the same reduction of DO as with Ro 31-8220 alone, and again less for DM, in most isolates tested (Fig. 7Go). Thus, PKC-mediated regulation of DO expression and, to a lesser extent, of DM, is independent from de novo protein synthesis and may involve a post-translational control mechanism.



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  		FIGURE 7. Western blot analysis of DR, DM, and DO expression during inhibition of de novo protein synthesis and inhibition of PKC. High-density B lymphocytes were incubated for 1 h with CHX (20 µM), followed by incubation for 4 h with or without Ro 31-8220 (10 µM). Control cells were incubated with equivalent amounts of DMSO. The presented results are means of three different samples and a representative analysis of one donor is shown.

 
Regulation of DM and DO expression via manipulation of their life span could involve manipulation of their lysosomal degradation, as DM and DO mainly reside in the acidic environment of the MIICs (6, 13). This was investigated by examining PKC-mediated regulation of DO and DM expression in cells treated with the weak base CQ. CQ raises the lysosomal pH by acting on the H+ ATPases (45, 46). Indeed, CQ treatment consistently abrogated the various levels of reduction in DO and DM expression that were induced by inhibition of PKC activity by Ro 31-8220 in the three different donors that were tested (Fig. 8Go). An interesting result was obtained with CQ alone: by itself it modulates negatively the levels of both DM and DO but not of DR (Fig. 8Go). This further supports the idea that DM and DO may be modulated in lysosomal compartments.



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  		FIGURE 8. Western blot analysis of DR, DM, and DO expression after neutralization of lysosomal compartments by CQ and PKC inhibition. High-density B lymphocytes were either preincubated for 15 min with CQ (200 µM) followed by a 4-h incubation with CQ alone or with Ro 31-8220 (10 µM), or incubated for 4 h with Ro 31-8220 alone. Control cells were incubated for 4 h with DMSO alone. The presented results are means of three different samples, and a representative experiment is shown.

 
Together, our data reveal the existence of a mechanism regulating the expression levels of both modulators of class II-mediated Ag presentation via the cellular activation status. In vitro studies demonstrated that DO expression and, to a lesser extent, DM expression can be regulated by PKC via a previously unknown mechanism possibly by the lysosomal degradation rate of the various components of the HLA class II Ag presentation pathway.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In B lymphocytes, binding and subsequent presentation of most antigenic peptides to MHC class II molecules requires the action of DM, which in turn is modulated by DO in a pH-sensitive manner. Thus, the delicate balance of the relative levels of DM and DO expression could influence the peptide repertoire presented by class II molecules in B lymphocytes. We have studied the expression of both modulators of class II-mediated Ag presentation and class II expression itself in human, primary B lymphocytes. Because the initial state of B cell activation of every individual tested was unclear and the class II haplotype not known, some heterogeneity in the results was expected and obtained. Still, comparison between immortalized B cell lines and primary B lymphocytes invariably demonstrated markedly higher DO expression in primary cells. This strongly indicates that the in vivo role of DO on human class II-mediated peptide presentation is more prominent than previously anticipated.

Separation of primary B lymphocytes into low- and high-density populations revealed reduced expression of both DM and DO in in vivo activated B lymphocytes. Further in vitro activation of high-density B lymphocytes showed that down-modulation of DM and DO could be induced via the BCR stimulation or with the tumor promoter PMA. PMA thus provides a pharmacological and the BCR a physiological modulator of DM and DO expression. This effect on DM and DO expression was further explored on the basis that a long exposure to PMA results in degradation of PKC. The latter was previously shown to be able to control expression at the RNA or protein level (for review see Refs. 47 and 48). In this study, modulation of DM and DO expression is proposed to occur via a novel mechanism involving the PKC activity and the manipulation of lysosomal degradation of both DM and DO. Lysosomes are usually referred to as "dead-end organelles" but have also been reported to be prone to exocytosis (reviewed in Ref. 49) in certain conditions such as elevated intracellular calcium concentration (50, 51). Sundler and colleagues (52, 53) reported exocytosis of preformed lysosomal contents in mouse macrophages induced by zymosan particles. The same effect was obtained by increasing the lysosomal pH and was further enhanced by PKC activation. However, down-regulation of PKC inhibited the lysosomal exocytosis induced by zymosan or increase of lysosomal pH. These data could provide a parallel to our results where CQ alone induced down-regulation of DM and DO. Is this effect mediated by exocytosis of lysosomal contents? Another hypothesis could be that CQ, by modifying the environment and behavior of the proteins present in endo/lysosomal compartments, suppresses the identity of these compartments. The latter, not recognized by the cellular machinery, becomes prone to destruction and therefore presents a reduced half-life. Again, in concordance with Sundler et al. (52, 53), PKC inhibitor and CQ applied together resulted in an inhibition of the observed diminution of DM and DO. Although it seems improbable, because Ro 31-8220 and CQ act in different compartments (cytosol and lysosomes respectively), we cannot exclude the possibility that the two components act together in an unexpected way on DM and DO expression. In conclusion, our results reveal two phenomena: 1) a basal activity of PKC is needed to maintain the physiological expression level of DO and DM, and 2) DM/DO can be down-regulated by acting on PKC activity or on lysosomal pH by a mechanism that remains to be elucidated.

B lymphocytes become activated after Ag uptake and appropriate costimulatory signals (reviewed in Refs. 54, 55, 56). Their most efficient pathway of Ag uptake is mediated via the BCR and targeting of the BCR-Ag complex to the MIICs (57, 58, 59). Cross-linking of the BCR leads to activation of signaling cascades initiated by ITAM phosphorylation of Ig{alpha}{beta} and recruitment of tyrosine kinases such as Syk and Btk. They subsequently activate extracellular signal-related kinase, phospholipase C, Ras, and phosphatidylinositol 3 kinase. The resulting effects on B lymphocyte activation and differentiation are therefore due to multiple pathways, some of them being mediated by PKC (56, 60, 61, 62, 63). Results from several studies indicate a role for PKC isoforms in the B cell immune response. PKC{beta}-deficient mice develop impaired humoral immune responses and a reduced sensitivity to BCR stimulation, pointing to a crucial function of PKC{beta} in this pathway, as its absence cannot be compensated for by other members of the PKC family (64). Interestingly, the differentiation of B lymphocytes into Ab-secreting plasma cells in mice is accompanied by an increase in PKC{alpha} and loss of PKC{beta} expression (65). Taken together these observations indicate that PKC is an important factor for B lymphocyte activation and differentiation. Previous studies have already revealed an impact of signal transduction on the class II processing and presentation pathway (reviewed in Ref. 66), notably via the BCR (17) and more recently via the Fc{alpha}R (67, 68). Signalization via the BCR or via Fc{alpha}R results in fusion and acidification of the subcellular MIICs in which Ag processing and class II loading occur. PKC is involved in the modification of the MIICs as PMA/ionomicine stimulation reproduces the effect of BCR stimulation (17). The Ag associated to its receptor is specifically targeted to these modified compartments, and Lang et al. (68) propose a role for phosphatidylinositol-dependent protein kinase 1 and protein kinase B{alpha} in this process. Although we cannot exclude that other factors are acting in the observed down-regulation of DM and DO, our results support the notion that PKC has an important and crucial role, either directly or indirectly, in the maintenance of these modulators of class II-mediated Ag processing.

What is the physiological rationale and need for regulation of DM and DO expression during B lymphocyte activation? The answer is likely to lie in the need for tight control of class II-mediated Ag presentation in B lymphocytes to avoid presentation of undesirable Ags. The data presented in this work show that high-density B lymphocytes seem to be optimally set for class II loading with specific Ag; the observed high expression of DM will ensure effective class II peptide loading, while the high levels of DO will skew peptide loading to the proper, acidic Ag loading compartments. This presetting of the B lymphocyte before Ag encounter may be required as the timeframe observed for uptake, and class II loading of specific Ag is too short (< 20 min) (58, 59) for efficient up-regulation of DM and DO synthesis at this stage. Moreover, in lymphoid organs, after Ag encounter by the B lymphocyte there are strict temporal requirements for the recruitment of T cell help necessary for B lymphocyte survival and development of an immune response. In this study, B lymphocyte activation results in enhanced down-regulation of particularly DM and DO. The observation that B lymphocyte activation via the BCR leads to fusion and acidification of the MIICs is of interest (17, 69), because this process is likely to enhance the protease activity in the MIICs. The latter would not only result in the rapid generation of the desired antigenic peptides but may also lead to an elevated turnover rate of MIIC resident proteins, as observed in this study for DM and DO. This process would affect the turnover rate of class II molecules far less, as they only reside in the MIICs for a limited time period before being exported to the plasma membrane. Indeed, this is what we observed. Physiologically, a reduction of DM and DO expression at this stage of B lymphocyte activation would not affect presentation of the specific Ag, because this rapid process would precede DM and DO degradation. However, it would skew the activated B lymphocyte to a state where subsequent class II loading with unrelated, and therefore less desired, peptides would be minimized. The observation that, like dendritic cells (70), low-density B lymphocytes express relatively more DR on their cell surface and less intracellularly than high-density cells (Table IGo vs Fig. 2Go) is in keeping with this scheme.

In conclusion, we report selective modulation of DM and DO during B lymphocyte activation. Moreover, a novel and tightly controlled picture of B lymphocyte-specific regulation of class II-mediated Ag presentation begins to emerge. The regulation of both modulators of class II-mediated Ag presentation may ensure that they are optimally expressed at a stage where B lymphocytes need to be ready for an efficient and fast processing of specific Ag and subsequent class II loading.


   Acknowledgments
 
We thank Peter Cresswell for providing the CerCLIP mAb; Nuala Mooney, Frédéric Garban, Dominique Charron, and Christian Villiers for helpful discussions and support; Christophe Chiquet for statistical analysis; and colleagues from our respective laboratories for useful hints and discussions.


   Footnotes
 
1 C.R. was supported by grants from Fondation de France and the Association Espoir-Isère Contre le Cancer. M.v.H. was supported by a Pioneer Grant from the Netherlands Organization for Scientific Research. J.T. was supported by a program grant from the Wellcome Foundation. J.N. and J.T. were supported by Training and Mobility of Researchers Network Grant CT960069 from the European Community. Back

2 Address correspondence and reprint requests to Dr. Corinne Roucard, Groupe de Recherche sur les Lymphomes, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, France. E-mail address: Corinne.Roucard{at}ujf-grenoble.fr Back

3 Abbreviations used in this paper: Ii, invariant chain; BCR, B cell receptor; CHX, cycloheximide; CLIP, class II-associated Ii peptide; CQ, chloroquine; MIIC, MHC class II compartment; PKC, protein kinase C; MFI, mean fluorescence intensity. Back

Received for publication March 23, 2001. Accepted for publication October 15, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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