Selective modulation of the major histocompatibility complex class II antigen presentation pathway following B cell receptor ligation and protein kinase C activation.

We noticed that B cell receptor ligation or phorbol 12-myristate 13-acetate treatment induced intracellular vesicles containing major histocompatibility complex (MHC) class II and invariant chain (Ii), and increased the amount of transmembrane p12 Ii fragments coimmunoprecipitated with class II molecules. To determine the influence of protein kinase C activation on the MHC class II presentation pathway, we analyzed the subcellular distribution of Ii, the induction of SDS-stable forms of class II molecules, and their ability to present different antigens. Ii chains visualized with luminal and cytoplasmic directed antibodies appeared in early endosomal compartments accessible to transferrin in response to phorbol 12-myristate 13-acetate treatment, whereas transmembrane Ii degradation products equivalent to the p12 Ii fragments were colocalized with the B cell receptors internalized after cross-linking. Protein kinase C activation delayed in parallel the formation of SDS-stable forms of class II molecules and reduced the presentation of antigenic determinants requiring newly synthesized class II αβ-Ii complexes. These data indicate that B cell activation affects Ii processing and MHC class II peptide loading in endosomal compartments intersecting the biosynthetic pathway.

MHC 1 class II molecules bind in their groove antigenic peptides generally derived from endocytosed proteins for their presentation to specific CD4 ϩ T lymphocytes (reviewed in Refs. [1][2][3]. Shortly after their biosynthesis, the MHC class II ␣ and ␤ chains assemble with Ii through a sequence from amino acid 81 to 104 of Ii called CLIP for class II associated Ii peptide (4 -6), and form (␣␤) 3 Ii 3 nonameric structures (7,8). Ii prevents the association of antigenic peptides with class II ␣␤ heterodimers (9 -11) through CLIP peptide (5), which occupies the peptidebinding groove in crystallized MHC class II molecules (12). The trimerization of Ii cytoplasmic domains, containing two dileucine motives each, drives the targeting of newly synthesized class II molecules to specialized endosomal compartments (13)(14)(15), which were further characterized as compartments of antigen processing and of peptide loading (16 -19). The amount of class II molecules located in these intracellular compartments can differ to a considerable extent, depending on the cell type (20 -23), and proteolytic cleavage of Ii occurs at this stage as indicated by ultrastructural observations (22). Upon removal of CLIP from class II molecules catalyzed by HLA-DM or its murine equivalent H2-M class II molecules (24 -26), peptide-loaded MHC class II complexes are exported to the cell surface by a poorly defined pathway. Recycling of class II molecules, from the plasma membrane through endosomes, has also been observed in B cells (27)(28)(29), allowing binding and presentation of different antigens to T cells (27,30). MHC class II molecules can therefore gain access to the antigen-processing compartments by at least two different routes, a direct targeting of newly synthesized ␣␤-Ii complexes and an indirect targeting of resident ␣␤ heterodimers. These two pathways of antigen presentation coexist in B cells. One is sensitive to protein synthesis and membrane transport inhibitors (31)(32)(33), and requires the expression of Ii (34). The other one requires preexisting ␣␤ class II heterodimers with intact cytoplasmic domains (30).
In B lymphocytes, the most efficient pathway of antigen uptake is mediated by the BCR (reviewed in Ref. 35). Upon BCR ligation, antigens are internalized independently of a phosphorylation of the immunoreceptor tyrosine-based activation motives present on the cytoplasmic tail of the coreceptors Ig␣ and Ig␤ molecules (36). Ligation of the BCR transduces activation signals, through the Ig␣ and Ig␤ coreceptors, leading to a cascade of protein tyrosine kinase activation (reviewed in Refs. 37 and 38) and to the production of second messengers, such as inositol triphosphate, and PKC activators, such as diacylglycerol (39). Several routes dependent on protein tyrosine kinase and PKC are then converging at the level of the activation of the mitogen-activated protein kinase pathway (40).
Since the BCR delivers signals leading to PKC activation (37,41), it was of interest to analyze the effects of BCR cross-linking and of direct PKC activation, through phorbol ester, on the biosynthetic transport and the function of MHC class II molecules. Our results show that different mechanisms of PKC activation decreased the endosomal degradation of MHC class II associated Ii chains and induced an intracellular accumulation of p12 Ii protein fragments previously identified in MHC class II-Ii transfectants (20) and equivalent to the p10 Ii protein fragment SLIP (23,42). This is correlated with a selective modulation of antigen presentation, and a regulation of vesicular traffic as shown for the uptake of transferrin (43). * This work was supported by institutional grants from INSERM, CNRS, and by grants from the Association pour la Recherche sur le Cancer (ARC) and the Ligue Nationale de lutte contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18

EXPERIMENTAL PROCEDURES
Antibodies and Reagents-Anti-Ii rabbit polyclonal Abs were raised against synthetic peptides of the cytoplasmic and CLIP domain of the mouse Ii (␣Cyt.Ii and ␣CLIP). Anti-MHC class II rabbit polyclonal Ab was raised against cytoplasmic domains of the mouse I-A␤ molecule (␣Cyt.IA␤). The corresponding amino acid sequences of the peptides used for immunization were: M 1 DDQRDLISNHEQLPILGNRPREPES-RCSRY 31 , YR 77 MKLPKSAKPVSQMRMATPLLMRPMSMDNMLLG 109 , and YH 222 RSQKGPRGPPPAGLLQ 238 for the ␣Cyt.Ii, ␣CLIP, and ␣Cyt.IA␤ respectively. Peptides were coupled to keyhole limpet hemocyanin as a protein carrier with the cross-linker bisdiazobenzidine. The biochemical characterization of ␣CLIP and ␣Cyt.IA␤ Abs was previously performed (44). The rabbit antiserum ␣Cyt.Ii was conjugated to the NHS-LC-biotin (Pierce) for double staining with other rabbit antiserum. The mouse hybridoma 10.2.16 producing an I-A k -specific mAb was obtained from the American Type Culture Collection (Rockville, MD). The rabbit antiserum against luminal domain of human Ii (␣Lum.Ii), the mouse anti-lgp110-B mAb GL2A7 (45), and the mouse anti-Golgi apparatus mAb CTR433 (46) were kindly provided by Drs. Salamero, Amigorena, and Bornens (Curie Institute, Paris) respectively. The secondary reagents (donkey anti-mouse Igs, anti-rabbit Igs, and streptavidin) coupled to FITC, Texas Red, or Cyanine 5 suitable for multiple labeling experiments and unlabeled donkey anti-mouse Igs to cross-link the BCR were purchased from Jackson Immunoresearch (West Grove, PA). Human transferrin (Sigma) was conjugated to FITC and purified on G25 column. PMA and leupeptin were from Sigma and ICN respectively.
Cells-The F6 B cell lymphoma was derived from the M12C3 B cell line, I-A negative, transfected for expression of I-A k molecules (47) and the B lymphoma, the CH27 cell line used in antigen presentation experiments expressed H-2 k molecules (48). The 3A9 T cell hybridoma (49) and the TS12 T cell hybridoma (50) are both I-A k -restricted and specific for HEL 46 -61 and RNase A 43-56 peptides, respectively. Cell lines were cultured in Dulbecco' modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 20 M ␤-mercaptoethanol, 1 mM sodium pyruvate, and 1 mM glutamine.
Immunofluorescence Staining and Confocal Microscopy-F6 cells cultured to 75% confluence on glass coverslips were treated or untreated with 50 ng/ml PMA or with 100 M leupeptin. Cells were fixed for 15 min at room temperature with 4% paraformaldehyde in PBS. After washing, cells were permeabilized with PBS containing 0.05% saponin, then incubated for 30 min at room temperature with primary Abs diluted in PBS containing 0.05% saponin and 1% bovine serum albumin. Unbound Abs were removed by washing in the same medium, then cells were incubated for 30 min with secondary labeled Abs. After washing in PBS and distilled water, the coverslips were mounted onto glass slides with Mowiol plus 1,4-diazabicyclo[2.2.2]octane (Sigma). For double immunofluorescence using two rabbit antisera, the first staining was performed with the first primary Ab followed by the secondary labeled Ab as described above. The cells were then incubated with rabbit preimmune serum, washed, and post-fixed with 2% paraformaldehyde before being incubated for 30 min with the second primary antibody coupled to biotin. The cells were then rinsed and labeled with FITC or Texas Red-conjugated streptavidin. To label the endosomal compartments, FITC-coupled transferrin was endocytosed for 20 min, or surface Igs were cross-linked with anti-Ig Ab for 30 min at 37°C, just before fixation and multiple labeling of other internal molecules. Confocal microscopy was performed using the Confocal Laser Scanning Microscopy TCS 4D (Leica Lasertechnik GmbH, Heidelberg Germany) interfaced with an argon/krypton ion laser and with fluorescence filters and detectors allowing to record simultaneously FITC, Texas Red, and Cyanine 5 markers. For the visualization of triple labeling, we have avoided the combination of red, green, and blue that produces too many hues leading to color interpretation problems. Instead, we defined a fluorescence threshold of 10% with an image processing program that automatically draws a black line around FITC-positive structures; the black contours are then superimposed to the conventional red-green pseudocolor look-up table representing the Texas Red and Cyanine 5 fluorescent markers (from D. Demandolx, CIML, Marseille).
Western Blotting-F6 cells were either untreated or treated with 100 M leupeptin, with 50 ng/ml PMA, or with 10 g/ml donkey anti-Ig Ab to cross-link the BCR for 1 h at 37°C. Cells were washed in ice-cold PBS and were solubilized in 1 ml of lysis buffer (1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 7.5) containing a mixture of protease inhibitors (0.25 mM phenylmethylsulfonyl fluoride, 0.5 mM iodoacetamide, and 1 g/ml leupeptin, pepstatin, and aprotinin). Half of the lysate was directly charged on 15% SDS-polyacrylamide gel electrophoresis (PAGE). Separated proteins were transferred onto Immobilon-P membrane (Millipore). Ii and Ii fragments containing intact cytoplasmic tail were detected with the ␣Cyt.Ii Ab. The second half of the lysate was incubated for 2 h with 10.2.16 mAb bound to the protein A-Sepharose beads (Pharmacia Biotech Inc.). Then immunoprecipitates were washed, and pellets were suspended in reducing sample buffer (10 mM Tris-HCl, 2 mM EDTA, 33% glycerol, 2% SDS, 5% ␤-mercaptoethanol). Immunoprecipitates were boiled in SDS and run on 15% SDS-PAGE. After transferring onto membrane, proteins were blotted with ␣Lum.Ii, ␣Cyt.Ii, or ␣CLIP Abs. After washing, membranes were incubated with anti-rabbit Ab conjugated to horseradish peroxydase (Jackson Immunoresearch). Labeled proteins were detected using the ECL immunodetection kit (Amersham Corp.). 35 S Metabolic Labeling-F6 cells were washed twice and incubated for 45 min at 37°C in cysteine/methionine-free RPMI medium (Life Technologies, Inc.). In 3 ml of this medium containing 5% dialyzed FCS, cells were incubated for 30 min at 37°C with 0.6 mCi of [ 35 S]cysteine/ methionine in the presence or in the absence of 50 ng/ml PMA. When indicated, pulse-labeled cells were chased for different periods at 37°C in RPMI medium containing cold cysteine and methionine with or without PMA. Cells were washed with ice-cold PBS and solubilized in lysis buffer previously described. Lysates were precleared with protein A-Sepharose beads, and the supernatants were mixed with ␣Cyt.Ii Ab or 10.2.16 mAb previously bound to protein A-Sepharose beads. Immunoprecipitates were extensively washed and pellets were resuspended in reducing sample buffer. To detect SDS-stable ␣␤ dimers, immunoprecipitates were left at room temperature 1 h before being resolved on 15% acrylamide SDS-PAGE. Radiolabeled proteins were intensified and revealed by autoradiography.

Intracellular Distribution of MHC Class II and Ii
Molecules-To determine whether B cell activation influenced the accumulation of Ii and of class II molecules into specialized intracellular compartments, we used confocal microscopy and immunofluorescence labeling of I-A k and Ii molecules in F6 B lymphoma cells. In untreated cells, MHC class II molecules labeled with ␣Cyt.IA␤ polyclonal Ab were localized mainly at the cell surface (Fig. 1A). Ii chain visualized with ␣Cyt.Ii polyclonal Ab gave mostly a reticulated intracellular staining consistent with the endoplasmic reticulum (Fig. 1B). After crosslinking of the surface Igs, MHC class II molecules appeared in intracellular vesicles (Fig. 1C, arrows) colocalized with Ii (Fig.  1D, arrows). A similar colocalization of MHC class II and Ii was found in PMA-treated cells (Fig. 1, E and F, arrows).
The presence in class II-rich compartments of Ii proteins and Ii degradation fragments was reported in human B cell lines (22) and in murine B lymphoma cells treated with the leupeptin protease inhibitor (23). To visualize the subcellular distribution and the processing of Ii molecules in PMA (Fig. 2B)-and in leupeptin ( Fig. 2A)-treated cells, we double labeled the cells with rabbit ␣Cyt.Ii and ␣Lum.Ii Abs. A set of vesicles was found to contain colocalized luminal and cytoplasmic Ii epitopes, likely from full length Ii proteins (Fig. 2, A and B, yellow vesicles), while many peripheral vesicles were labeled with the ␣Cyt.Ii Ab only (Fig. 2, A and B, red vesicles). These vesicles contain only short Ii fragments lacking their luminal domains. Since distinct vesicular compartments contained different forms of Ii, it was of importance to localize Ii fragments with respect to Golgi, lysosomal, and endosomal markers in PMA-treated cells. The mouse Golgi CTR433 mAb which recognizes a marker present in the Golgi complex (46), was partially colocalized with the ␣Lum.Ii (Fig. 2C, yellow vesicles) and weakly colocalized with ␣Cyt.Ii labeling (Fig. 2D). Some vesicles positive for the ␣Lum.Ii Ab and many peripheral vesicles, containing Ii degradation products recognized by the ␣Cyt.Ii Ab, were not coincident with this Golgi marker (Fig. 2, C and D,  red vesicles). Next, to identify the lysosomal compartments, we used a mAb directed against the lgp110-B marker (45) and found no superimposition of the ␣Cyt.Ii Ab after PMA treatment (Fig. 2E), suggesting that Ii fragments do not reach or do not persist in the lysosomes (51). Since antigens bound to the BCR are transported to peptide-loading compartments for processing and presentation by the MHC class II molecules (35), it was of importance to determine whether the BCR could reach vesicles containing the different forms of Ii. Internalization of the BCR was triggered by cross-linking with anti-Ig Ab in PMA-treated cells. The anti-Ig Ab internalized for 0.5 h were highly colocalized with peripheral vesicles containing cytoplasmic Ii fragments (Fig. 2F).
To further define the intracellular localization of Ii degradation products with respect to the early endosomes in activated cells, FITC-coupled transferrin was internalized through its receptor during PMA treatment. The cells were then fixed and processed for double immunofluorescence staining with the ␣Lum.Ii and ␣Cyt.Ii Abs. We found a limited level of colocalization between the early endosomes loaded with FITC-transferrin and the Ii positive vesicles labeled with the ␣Cyt.Ii Ab (Fig. 2G, yellow vesicles). To determine whether the Ii-positive early endosomes contained also the luminal Ii epitope, we used an image processing program to surround FITC-transferrinpositive structures above a fluorescence value of 10% with black contours. These contours were then superimposed on the double fluorescence image obtained with ␣Lum.Ii and ␣Cyt.Ii Abs (Fig. 2H). This representation shows triple positive vesicles as double labeled objects surrounded with black contours. Some early endosomes contain the two Ii epitopes (Fig. 2H, yellow vesicles with black contours), while only a few contain short Ii cytoplasmic fragments (Fig. 2H, red vesicles with black contours). We think that newly synthesized MHC class II-Ii complexes bearing unprocessed Ii chains gain access to early endosomal compartments. PKC activation also generates Ii fragments that are colocalized with internalized surface Igs in later elements of the endosomal pathway (Fig. 2F).

Generation of Ii Fragments and Induction of Class II Compact Forms-
The cytoplasmic Ii fragments identified in endosomal compartments after PKC activation are probably equivalent to cytoplasmic derived SLIP Ii protein fragment associated with intracellular class II molecules (23,42) and to the p12 Ii fragments identified in ␣␤-Ii-transfected fibroblasts (20). To test this hypothesis, we performed Western blotting with ␣Cyt.Ii, ␣Lum.Ii, and ␣CLIP antisera in total cell lysates from leupeptin (100 M)and PMA (50 ng/ml)-treated cells (Fig.  3A, lanes L and P, respectively). The ␣Cyt.Ii polyclonal Ab recognized the p10, p12, p31, and to a lesser extent the p41 forms of Ii in the total cell lysate. After leupeptin or PMA treatment, there was no change in the amount of p10 and p31 Ii forms; however, a slight increase in the p12 Ii form was detected in PMA treated cells (Fig. 3A). Since Ii is produced in excess compared to class II molecules, we identified also I-A kassociated Ii proteins on Western blots after immunoprecipitation with the nonconformational 10.2.16 anti-I-A k mAb (Fig.  3B). Compared to the controls, no change occurred for p31 and p41 Ii forms associated with class II molecules; however, PMA and leupeptin treatments increased the class II-associated p12 Ii fragments, revealed with rabbit ␣Cyt.Ii and ␣CLIP Abs (Fig.  3B, lanes L and P). The cytoplasmic derived p10 Ii fragments identified in the total cell lysates do not bind to class II molecules (Fig. 3, A and B). In another set of experiments, we compared the effect of PMA treatment and BCR ligation (Fig. 4,  lanes P and B) on the pattern of MHC class II-associated Ii fragments revealed with the ␣Cyt.Ii Ab (Fig. 4A) and the ␣CLIP Ab (Fig. 4B). Both PKC activation pathways increased the level of association of p12 Ii fragments with class II molecules (Fig. 4, A and B).
To test whether PKC activation modified Ii turnover, we performed a 35 S pulse-chase labeling followed by SDS-PAGE analysis of Ii products immunoprecipitated with the ␣Cyt.Ii Ab. In untreated cells, Ii was rapidly degraded and a p12 Ii fragment appeared after 30 min of 35 S pulse (Fig. 5A) analog to SLIP. Faint p10 Ii fragments were detected after 2 h of chase, and most of the p31 and p41 Ii forms were degraded within 3 h. In cells treated with PMA, degradation of Ii was consistently reduced with more p12 fragment at 2 h and more p31 form at 3 h of chase compared with the untreated cells (Fig. 5A). Rather than increasing the biosynthesis of Ii, PMA reduced the rate of Ii turnover. This could affect peptide loading on newly synthesized ␣␤ class II dimers as in leupeptin-treated cells (52). To analyze the kinetics of antigen binding we monitored the resistance of class II complexes to denaturation by SDS detergent at 20°C reflecting the presence of peptide loaded class II ␣␤ heterodimers (53). In untreated cells, newly synthesized class II molecules acquired resistance to SDS 1 h after their biosynthesis; the mature form of class II ␣ chains (␣m) appeared after 30 min of chase, while immature ␣ chains (␣i) disappeared after 1 h (Fig. 5B). Immature ␤ chains (␤i) disappeared after 30 min of chase, but since mature ␤ chains (␤m) migrate at the same position as the p31 Ii chains, we could not really detect the initial rate of ␤ chain maturation on these SDS-PAGE. In PMA-treated cells, SDS-resistant ␣␤ dimers were delayed for 2.5 h following biosynthesis (Fig. 5B), but PMA did not affect class II ␣ and ␤ chain maturation patterns. We think therefore that PKC activation affects the peptide loading process thought to occur in specialized endosomal compartments (16 -19) in which the persistence of Ii fragments can reduce the rate of formation of SDS-resistant class II ␣␤ dimers.
Effect of PMA on Antigen Presentation-In order to determine the effects of PKC activation on the MHC class II antigen presentation pathway, we studied the response of two T cell hybridoma requiring different pathways of antigen processing. The I-A k -restricted presentation of HEL to the HEL 46 -61 specific 3A9 T cell hybridoma (49) is sensitive to protein synthesis and membrane transport inhibitors (31)(32)(33), and requires Ii expression (34). The presentation of RNase A to the RNase A 43-56-specific TS12 T cell hybridoma requires neither protein synthesis nor Ii chain expression (34,50). These two antigenic determinants are thought to use different pathways of processing for their presentation to helper T cells. Using cycloheximide-treated F6 and CH27 B lymphoma cells, we confirmed that, in B cells, protein synthesis inhibitor blocked the presentation of HEL to the 3A9 T cell hybridoma, but not the presentation of RNase A to the TS12 T cell hybridoma. 2 The RNase A 43-56 peptide is therefore presented here by resident MHC class II molecules, whereas the HEL 46 -61 peptide is presented by newly synthesized class II molecules.
In order to assess the function of newly synthesized and of resident class II molecules depending on PKC activation, we delivered the antigen by uptake from the fluid phase for 2 h in F6 B lymphoma cells (Fig. 6, A and B), and in the nonadherent CH27 B lymphoma cells for the sake of comparison (Fig. 6, C  and D). At low doses of antigen, the presentation of HEL to the I-A k -restricted 3A9 T cell hybridoma was reduced by about 10-fold when the APCs were pretreated for 1 h with 50 ng/ml PMA (Fig. 6, A and C). The presentation of HEL became insensitive to PMA at high concentration of antigen. In contrast to HEL, the efficiency of the presentation of RNase A to the I-A k -restricted TS12 specific T cell hybridoma was not affected by pretreatment with PMA for 1 h in F6 and in CH27 B cell lines (Fig. 6, B and D). PKC activation altered selectively the presentation pathway of the HEL-derived 46 -61 peptides, which are thought to meet newly synthesized MHC class II molecules after processing of the protein.

DISCUSSION
The ligation of activation receptors such as surface Igs and the direct activation of PKC generate a cascade of phosphorylation events in B cells (37)(38)(39)(40) which influences protein-protein interactions in the cytoplasm, activates gene transcription and modifies the cell morphology. Our goal was to investigate whether B cell activation influences the MHC class II presentation pathway. Initial experiments showed that surface Ig cross-linking and phorbol ester treatment can induce the appearance of MHC class II/Ii-containing intracellular vesicles in murine B lymphoma cells. These observations led us to evaluate the effects of PKC activation on Ii processing and transport, MHC class II peptide loading, and antigen presentation.  D). Interleukin-2 production was determined using the growth of the interleukin-2-dependent cell line CTLL with an 3-(4,5dimethyl thiazol-2-xy)-2,5-diphenyl tetrazolium bromide assay. they appeared after 30 min and were susceptible to protein synthesis inhibitors. 2 The Ii-positive vesicles were further divided into two subsets according to the stage of Ii processing. One subset contained colocalized luminal and cytoplasmic Ii epitopes and the other contained only the cytoplasmic Ii epitope. Short term treatment with the leupeptin serine protease inhibitor induced a similar redistribution of Ii proteins and Ii fragments (Fig. 2, A and B).
Most of the vesicular compartments containing the luminal Ii epitope were also labeled with the CTR433 mAb, which is characterized at the ultrastructural level as a marker of the mid-Golgi compartment (46). However, many vesicles containing only transmembrane Ii fragments were not labeled with this Golgi marker and all of them were distinct from the lysosomes identified with the lgp110-B marker (45). Ii-positive vesicles were also distinct from the H2-M molecules present in the lysosomes (54), and from the late endosomes 2 defined by the anti-Rab7 mAb and by the presence of the cation-independent mannose 6 phosphate receptor (55). The absence of colocalization with lysosomal and late endosomal markers indicates that Ii cytoplasmic fragments are present in vesicles equivalent to endosomal compartments defined by subcellular fractionation in murine B cells (16,17). Using multiple immunofluorescence and confocal microscopy, we found that PKC activation induces the redistribution of intact Ii from the endoplasmic reticulum to the Golgi complex and to other elements of the endosomal pathway. After cross-linking and internalization of the BCR, numerous endocytic vesicles defined by their Ig content were labeled with the ␣Cyt.Ii Ab.
To determine whether the vesicles in which MHC class II-Ii complexes are delivered correspond to early endosomes, we performed a double immunofluorescence staining with ␣Lum.Ii and ␣Cyt.Ii Abs following internalization of FITC-transferrin (Fig. 2, G and H). Some early endosomes labeled with transferrin contain no Ii molecules, whereas other ones are labeled with both luminal and cytoplasmic Ii-directed Abs. Intact Ii chains are probably reaching the early endosomal compartments first, whereas later elements of endosomal pathway are loaded with Ii fragments partially colocalized with internalized Igs. This scheme is compatible with the steady state distribution of Ii degradation products found in distinct compartments in human B cell lines at the ultrastructural level (22). Our results are also in agreement with recent analyses of subcellular fractions derived from leupeptin-treated cells (23) and with subcellular fractionation experiments performed in murine B lymphoma cells (56), since class II molecules were colocalized with Ii proteins and Ii fragments after BCR engagement or after PMA treatment. In conclusion, PKC activation triggers the accumulation of intact Ii presumably associated with newly synthesized MHC class II molecules in vesicular structures accessible to transferrin uptake. Later elements of the endosomal pathway, in which surface Igs are internalized, contain apparently many Ii fragments, suggesting an initial targeting of ␣␤-Ii complexes to transferrin positive compartments as reported in leupeptin treated cells (23,56).
Peptide Loading of MHC Class II Molecules-Looking at the biosynthetic pathway of MHC class II transport, we report here that PKC activation reduces Ii turnover and delays the induction of class II SDS stable forms. PMA treatment also triggers the accumulation of CLIP containing p12 Ii fragments previously identified in fibroblast transfectants (20) and analogous to p10 cytoplasmic derived fragments obtained in leupeptintreated B cells (23,42). The p12 Ii fragments contain in their cytoplasmic portion two dileucine based targeting motives able to direct Ii chain and Ii chimeric constructs to endosomal compartments (13,14). The fact that Ii proteins and p12 Ii frag-ments remain associated with ␣␤ heterodimers for a longer time in PMA treated cells provides an explanation for the intracellular retention of ␣␤-Ii complexes and for the delay in the induction of SDS stable forms. Moreover, intracellular Ii and MHC class II molecules remained highly colocalized in PKC activated cells, indicating that the CLIP portion of p12 Ii fragments presumably lies in the groove of newly synthesized MHC class II molecules and impairs peptide loading in the endosomes.
To identify whether PKC activation influences the antigen presentation capacity of B cells in relation to the biochemical perturbation of Ii processing, we have analyzed the presentation efficiency of Ii-dependent and Ii-independent epitopes. In the B cell populations selected here, newly synthesized class II molecules are required to present HEL to the HEL 46 -61 specific I-A k restricted, 3A9 T cell hybridoma (31,32), and this antigen presentation pathway is critically dependent on Ii expression in B cells (34). We showed here that this presentation event is partially impaired by PMA treatment. However, we found no correlation with a reduction in class II surface expression analyzed by flow cytometry and no correlation with a reduction in the presentation of the HEL 46 -61 peptide. 2 The MHC class II recycling pathway of antigen presentation was resistant to protein synthesis inhibitors here. This pathway does not require Ii (34) but requires the integrity of MHC class II cytoplasmic domains (30). As an example of this class II recycling pathway, we analyzed the presentation of RNase A to the RNase A 43-56 specific I-A k restricted, TS12 T cell hybridoma (31,32) and observed no influence of PKC activation, indicating in addition that reorganization of the plasma membrane is not responsible for the inhibition of HEL presentation to the 3A9 T cell hybridoma. From our results, we think that the biosynthetic pathway of class II presentation requires a rapid degradation of Ii fragments to be fully functional. In splenic B cells derived from mice lacking H2-M molecules, a similar defect occurs for the presentation of antigenic determinants requiring both Ii expression and MHC class II synthesis (44). In the absence of H2-M which catalyze the exchange of CLIP peptides, Ii processing is normal but MHC class II molecules present at the cell surface are massively loaded with CLIP and do not reach their final SDS stable forms (44). In PKC activated cells in which Ii processing is partially inhibited, we found a higher degree of intracellular retention of MHC class II and Ii molecules. This is probably due to the fact that dileucine motives present in multiple copies in the (␣␤) 3 -Ii 3 and the (␣␤) 3 -(p12Ii) 3 nonamers can retain the complexes in endosomal compartments (13)(14)(15).
The molecular mechanism leading to the reduction of Ii degradation and Ii-dependent antigen presentation after PMA treatment and BCR ligation remains, however, unclear. We looked for a PMA-induced phosphorylation of MHC class II and Ii, but we had no evidence in favor of this hypothesis. As shown previously in human B cells (57), Ii chains can be phosphorylated on serine residues lying in the cytoplasmic domain, and this event may regulate Ii-dependent antigen presentation. We have detected a basal level of 32 P incorporation in p31 and p12 forms of Ii immunoprecipitated with ␣Cyt.Ii Ab, but found no modification of Ii and MHC class II phosphorylation after PMA treatment. 2 We cannot exclude that PMA induced another type of post-translational modification of Ii, or reduced the activity and the expression of proteases involved in Ii degradation (58,59). Many endosomal proteases are synthesized as precursors and need a proteolytic step occurring in the trans-Golgi network and in subsequent compartments to become fully active (60). PMA could also influence the maturation of proteases resulting in a slower degradation and the accumulation of Ii fragments partially associated with class II molecules. It should be noted that inhibitors of protein phosphatases such as okadaic acid can regulate endocytic vesicle fusion in vitro (61) and inhibits endocytosis in Hela cells (62) consistent with other reports showing that PMA can modulate the uptake of transferrin and of fluid phase markers (43). Additional events such as a reorganization of the cytoskeleton perturbing the exocytic traffic of MHC class II-Ii complexes remain also possible.
Using BCR ligation and PKC activation, we observed for the first time a negative regulation of the MHC class II biosynthetic pathway of antigen presentation. Phorbol esters reduce peptide loading and conversion of class II molecules into SDSresistant heterodimers as in cells treated with the leupeptin serine protease inhibitor (23,52). However, the mechanisms of action of PKC stimulation and of vacuolar proteases inhibition differ to a considerable extent. The inhibition of vacuolar proteases affects both the processing of the antigens and the degradation of MHC class II associated Ii. In contrast to leupeptin, which blocks the induction of SDS-stable forms of class II and their transport to the cell surface (52), PKC activation through PMA treatment modulates the presentation of Ii-dependent epitopes. It is tempting to speculate that the sequence of events we describe, i.e. delayed Ii degradation, accumulation of Ii products in endosomal compartments involved in peptide loading of ␣␤ heterodimers, delayed maturation of SDS-resistant ␣␤ dimers, and impaired presentation of Ii-dependent exogenous antigens, corresponds to a selective desensitization of the MHC class II antigen presentation pathway in cells in which many BCR molecules are engaged simultaneously.