Activation and HLA-DO Is Induced by B Lymphocyte in Vivo and in Vitro

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

This information is current as Corinne Roucard, Claire Thomas, Marie-Anne Pasquier, John of September 30, 2021. Trowsdale, Jean-Jacques Sotto, Jacques Neefjes and Marieke van Ham J Immunol 2001; 167:6849-6858; ; doi: 10.4049/jimmunol.167.12.6849

http://www.jimmunol.org/content/167/12/6849 Downloaded from

References This article cites 69 articles, 34 of which you can access for free at: http://www.jimmunol.org/content/167/12/6849.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 30, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. In Vivo and In Vitro Modulation of HLA-DM and HLA-DO Is Induced by B Lymphocyte Activation1

Corinne Roucard,2* Claire Thomas,† Marie-Anne Pasquier,* John Trowsdale,‡ Jean-Jacques Sotto,* Jacques Neefjes,§ and Marieke van Ham§¶

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 Downloaded from 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. The Journal of Immunology, 2001, 167: 6849–6858.

ajor histocompatibility complex class II molecules In B lymphocytes, another MHC class II-like heterodimer, http://www.jimmunol.org/ present Ags derived from exogenous sources to the termed HLA-DO, associates with DM (6). The expression of the M TCR on CD4ϩ T lymphocytes. In the human, MHC DO complex (7, 8) and the murine equivalent H2-O, is unusual in class II molecules are composed of the HLA-DR, -DQ, and -DP. that it seems to be restricted to B lymphocytes and thymic epithe- Shortly after synthesis, the DR␣ and DR␤ chains form a complex lium (9, 10). B lymphocytes have unique features as APCs, in that with the invariant chain (Ii)3 (1), which targets the class II/Ii com- they can take up Ags via a speciﬁc receptor, the B cell receptor plex into the endocytic pathway (2). During transport to the Ag (BCR). Recent studies demonstrated that DO is a negative modu- loading compartments (dubbed MIIC for MHC class II-containing lator of the catalytic activity of DM (11Ð13). The action of DO

compartment), Ii is degraded until only a class II-associated Ii depends on the pH of the compartment in which the DM/DO com- by guest on September 30, 2021 peptide (CLIP) remains bound to the class II peptide binding plex resides. DO effectively blocks DM function at the endosomal groove. In the MIICs, binding of incoming Ag to class II requires pH, while allowing DM action at the more acidic pH of the MIICs the action of HLA-DM that resides in endosomal/lysosomal com- (11, 14, 15). In this way, DO action may skew the class II-peptide partments, as it catalyzes the release of CLIP from the class II loading process toward acidic compartments. This may favor pre- backbone (3, 4). Finally, the class II-peptide complex is trans- sentation of speciﬁc peptides internalized via the BCR, as these ported to the cell surface (5) where interaction with an appropriate preferentially form a complex with class II in the MIICs (16, 17), T cell can occur. 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

*Groupe de Recherche sur les Lymphomes, Institut Albert Bonniot, Domaine de la enhanced class II presentation of fluid phase Ags as opposed to Merci, La Tronche, France; †Molecular Neuropathobiology Laboratory, Imperial Ags internalized via BCR (11). ‡ Cancer Research Fund, London, United Kingdom; Division of Immunology, De- In this work, the expression and regulation of both modulators partment of Pathology, University of Cambridge, Cambridge CB2 1QP, United King- dom; ¤Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, of class II-mediated Ag presentation as well as HLA class II mol- The Netherlands; and ¶Department of Pathology, Unit Experimental Oncopathology, ecules were studied in primary human B lymphocytes isolated Free University Hospital, Amsterdam, The Netherlands from peripheral blood. The expression of DO in primary B lym- Received for publication March 23, 2001. Accepted for publication October 15, 2001. phocytes is much higher than anticipated on the basis of previous The costs of publication of this article were defrayed in part by the payment of page studies with B cell lines. Strikingly, in in vivo low-density acti- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. vated B lymphocytes the expression of both DM and DO is 1 C.R. was supported by grants from Fondation de France and the Association Espoir- strongly reduced compared with high-density resting B lympho- Ise`re Contre le Cancer. M.v.H. was supported by a Pioneer Grant from the Nether- cytes. In vitro B cell stimulation via the BCR or with the phorbol lands 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 Mo- ester PMA induced a reduction in DM and DO expression. Inac- bility of Researchers Network Grant CT960069 from the European Community. tivation of protein kinase C (PKC) with the bisindolylmaleimide 2 Address correspondence and reprint requests to Dr. Corinne Roucard, Groupe de Ro 31-8220 resulted in a significant loss of HLA-DO expression Recherche sur les Lymphomes, Institut Albert Bonniot, Domaine de la Merci, 38706 and, in some cases, of HLA-DM. Lysosomal degradation could be La Tronche, France. E-mail address: [email protected] involved as chloroquine (CQ) reverses the effect of Ro 31-8220. 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 Thus, activation of primary B lymphocytes enable the cells to con- class II compartment; PKC, protein kinase C; MFI, mean fluorescence intensity. trol the efficacy of MHC class II-mediated Ag presentation.

Materials and Methods PKC inhibition Abs and peptides Freshly purified high-density B lymphocytes were incubated for 4 h with ␣ ␣ the PKC inhibitor bisindolylmaleimide Ro 31-8220 (final concentration 10 The mouse mAbs 5C1 (anti-DM ) (18), DA6-147 (anti-DR cytoplasmic ␮ tail) (19, 20), D1.12 (anti-DR␣) (21), and CerCLIP (22) were previously M; stock solution in DMSO; Calbiochem). Control cells were incubated described. The anti-PKC␣ and anti-PKC␤ mAbs were obtained from BD with equivalent amounts of DMSO, representing less than 0.1% of the total volume. For inhibition of de novo protein synthesis, cells were preincu- Transduction Laboratories (Lexington, KY) and the anti-actin mAb was ␮ obtained from Oncogene Research Products (Cambridge, MA). bated for 1 h with cycloheximide (CHX) (final concentration 20 M; stock The mAbs anti-CD80 labeled with FITC and anti-CD86 labeled with PE solution in ethanol; Sigma-Aldrich) followed by a 4-h incubation in the Ј presence or absence of Ro 31-8220 (10 ␮M). To neutralize the acidic pH were obtained from Immunotech (Marseille, France), and the rabbit F(ab )2 Abs against human IgD labeled with FITC, IgG labeled with FITC, and of endosomal/lysosomal compartments, cells were preincubated with 200 ␮M CQ for 15 min, followed by 4 h with both CQ and Ro 31-8220 (10 IgM labeled with PE were from DAKO (High Wycombe, U.K.). ␮ The rabbit anti-DO␤ serum was generated by immunizing a rabbit with M) or CQ alone. the C-terminal peptide (SGNEVSRAVLLPQSC) of DO␤ (8) conjugated SDS-PAGE and Western blot analysis via glutaraldehyde (Sigma-Aldrich, Poole, U.K.) to keyhole limpet hemo- cyanin (Calbiochem, La Jolla, CA). The DO␤ peptide was synthesized The protein content of cell lysates was quantified using the bicinchoninic using F-moc/tBu strategy, and structure and purity were confirmed by acid protein assay (Pierce, Rockford, IL). SDS-PAGE of cell lysates was HPLC analysis. performed on 10% polyacrylamide gels after boiling of the samples for 10 Analysis of Endoglycosidase H-resistant molecules was conducted and min in reducing Laemmli sample buffer. For Western blot analysis, equal showed that the 5C1 mAb and the rabbit anti-DO␤ serum recognized ma- amounts of proteins were separated by SDS-PAGE and transferred onto ture populations, as DO␤ molecules were all resistant and DM␣ presented polyvinylidene difluoride membranes (Immobilon P; Millipore, Bedford, one level of sensitivity to Endoglycosidase H as reported for mature MHC MA) in 25 mM Tris, 192 mM glycine, 5% methanol, pH 8.3. In the case class II ␣ molecules (data not shown). of transfer of high amounts of protein (i.e., up to 150 ␮g of protein per Downloaded from lane), a modified transfer buffer was used consisting of 10 mM 3(cyclo- Cell lines and culture conditions hexylamino)-1-propane sulfonic acid, 5% methanol, pH 11. Membranes were blocked in 3% skimmed milk in PBS. Ab binding (in 1.5% skimmed The Burkitt’s lymphoma B cell lines Raji (African origin, EBV-positive) milk in PBS) was detected by incubation with secondary HRP-conjugated and Ramos (American origin, EBV-negative) were obtained from the rabbit anti-mouse or swine anti-rabbit Ig Abs (DAKO), followed by ECL American Type Culture Collection (Manassas, VA). The cell lines were detection (Amersham, Arlington Heights, IL). Washes were performed us- routinely grown in RPMI 1640 (Life Technologies, Gaithersburg, MD) ing 0.1% Triton X-100 in PBS. http://www.jimmunol.org/ supplemented with 10% FCS (Life Technologies), penicillin/streptomycin, For semiquantitative analysis of the Western blots, subsaturated auto- and glutamin in the presence of 5% CO2 at 37¡C. radiograms were scanned and the signals were analyzed by densitometry (TINA 2.09 software; Raytest, Straubenhardt, Germany). The different Preparation of B lymphocytes samples of one experiment were loaded on the same gel and were therefore transferred and treated in the same conditions. The autoradiograms for Buffy-coats were obtained from healthy donors from the Ise`re and Savoie actin were used as the reference for protein loading. The lane correspond- Blood Transfusion Center (n ϭ 30; Grenoble, France), the London North ing to the control (high-density cells or control of the experiment) was used Blood Bank (n ϭ 19; London, U.K.) or the Central Laboratory for as the reference and accorded an arbitrary value of 1. The ratio for the other the Blood Transfusion Service (n ϭ 13; Amsterdam, The Netherlands). lanes compared with the control was determined. The corresponding den- Mononuclear cells were isolated by centrifugation on Ficoll-Hypaque gra- sitometric values for DR, DM, and DO were corrected by the obtained

dient (Pharmacia Biotech, Uppsala, Sweden). B lymphocytes were purified by guest on September 30, 2021 ratios. The corrected value for the control was accorded an arbitrary value with anti-CD19 Dynabeads and detached with DETACHaBEAD (Dynal of 100 and the corresponding percentage was calculated for the other con- Biotech, Oslo, Norway), according to the manufacturer’s instructions. B ditions. After this standardization was done for every donor, the values lymphocytes isolated by this method were extensively shown to remain were pooled together and the mean and SD were calculated. quiescent (23). The cell purity and viability was Ͼ99% as determined by cytofluorometric analysis (data not shown). FACS analysis Purification of high- and low-density B lymphocytes Cells (5 ϫ 105 cells per experiment) were washed in PBS containing 0.02% NaN and incubated for 15 min on ice in PBS containing 0.02% NaN ,5% ϩ 3 3 Freshly purified CD19 B lymphocytes were separated on a discontinuous FCS, 100 ␮g/ml human gamma-globulins (Calbiochem). Subsequently Percoll (Pharmacia Biotech) density gradient of 60, 50, 40, and 30% Per- cells were washed, incubated with the unlabeled Abs for 30 min in PBS, coll layers as previously described (24). Lymphocytes obtained from the 0.02% NaN3, and 1% BSA on ice, washed, and incubated with a secondary Ј bottom of the 60% fraction are referred to as high-density B lymphocytes FITC-labeled F(ab )2 of goat anti-mouse Ig (DAKO) or with Abs directly and correspond to cells in a resting state, and lymphocytes collected from labeled with FITC or PE, for 30 min on ice before washes and analysis on the 50Ð60% interface are referred to as low-density B lymphocytes and a FACScan cytometer (BD Biosciences, Moutain View, CA). Ten thousand correspond to cells with a more activated phenotype. Routinely, the amount events were acquired on living cells gated on forward scatter. of cells obtained from the 50Ð60% interface was ϳ10 times less than the amount obtained from the bottom of the 60% fraction. For analysis, 5 ϫ Statistical analysis 106 B lymphocytes were immediately lysed in 1% Nonidet P-40 lysis Two nonparametric tests (Kruskal-Wallis and Mann-Whitney) were used buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, 1 mM for statistical analysis of DR, DM, and DO expression in different condi- 4-(2-aminoethyl)benzene sulfonyl fluoride HCl, 100 ␮M iodoacetamide, tions in Figs. 2, 4, and 6. 10 ␮M leupeptin) for 20 min at 4¡C and centrifuged for 30 min at 10,000 ϫ Ϫ g. The supernatants were stored at 20¡C until further use. Results In vitro activation of high-density B lymphocytes Primary mature B lymphocytes express high amounts of HLA-DO B lymphocytes obtained from the bottom of the 60% fraction, high-density B lymphocytes, were cultured at 1 ϫ 106 cells/ml for 24 h at 37¡Cin5% So far, the function of the modulators of human MHC class II- CO2 in RPMI 1640 supplemented with 10% FCS, and activated with 20 mediated Ag presentation, HLA-DM and HLA-DO, has been stud- ng/ml PMA (Sigma-Aldrich). Alternatively, cells were stimulated using the ␮ Ј ied in immortalized cells (6, 12Ð14). However, an important ques- anti-DR mAb D1.12 (10 g/ml) followed by cross-linking by F(ab )2 of goat anti-mouse IgG (20 ␮g/ml) (Jackson ImmunoResearch Laboratories, tion remains; i.e., how do these modulators regulate the Ag West Grove, PA). Cells were activated by BCR stimulation by addition of presentation process and consequently the composition of the Ј ␮ a F(ab )2 of goat anti-human IgM, Fc5␮ fragment specific(5 g/ml, azide MHC class II antigenic peptide repertoire in the in vivo situation? free; Jackson ImmunoResearch Laboratories). Finally, cells were activated Therefore, the physiological, in vivo expression levels of the var- using a combination of IL-4 (10 ng/ml; Eurocetus, Amsterdam, The Neth- erlands) and anti-CD40 mAb (0.5 ␮g/ml, clone mAb89, azide free; Immu- ious components of MHC class II-dependent Ag presentation were notech). After activation, cells were lysed in a 1% Nonidet P-40 lysis buffer studied in human primary B lymphocytes isolated from the pe- and centrifuged, and supernatant was stored at Ϫ20¡C until further use. ripheral blood of healthy donors. The Journal of Immunology 6851

First, the phenotypic characteristics of the isolated primary B expression were higher in Raji than in Ramos as well (Fig. 1B). In lymphocytes were compared with those of two Burkitt’s lym- primary B lymphocytes the total HLA-DR expression was of an phoma B cell lines, Raji and Ramos, which are considered to intermediate level and DM expression was consistently slightly represent different maturation stages of B cell differentiation higher (Fig. 1B). However, in marked contrast, DO expression was (25). Human B lymphocytes purified from peripheral blood greatly enhanced in all primary isolates tested (Fig. 1B). Western were Ͼ80% IgMϩ and IgDϩ and Ͻ10% IgGϩ (Figs. 2A and 1A, blot analysis of increasing protein levels between primary B lym- respectively), demonstrating that they constitute a population of phocytes and the B cell lines demonstrated that for Raji a total of mature B lymphocytes. Ramos had an IgMϩ, IgGϪ, and IgDϪ 100Ð150 ␮g of protein was necessary to obtain a DO signal equiv- phenotype and Raji did not express any surface Ig (Fig. 1A), as alent to that observed in primary B lymphocyte lysates containing described before (25). 5Ð10 ␮g protein (Fig. 1C). Thus, the in vivo DO expression level The costimulatory molecules CD80 and CD86, ligands of CD28 in primary B lymphocytes seems to be much higher than antici- and CTLA-4 on T lymphocytes, are crucial for optimal Ag pre- pated so far from studies using immortalized B cell lines, which is sentation, and their expression levels are increased upon activa- strongly indicative of an important physiological role for DO in tion. CD80 and CD86 were observed at a low level in both primary primary B lymphocytes. B lymphocytes and Ramos (Fig. 1A), suggesting that they were relatively unactivated. In contrast, Raji expressed a high level of Attenuation of DM and DO expression upon B cell activation CD80 and CD86 (Fig. 1A), implying a higher activation status for Next, we investigated whether the components of the MHC class Raji, which is in line with the presence of Fc, C3, and EBV re- II Ag presentation pathway are subject to modulation in vivo via B ceptors on the cells (25). Increased expression of MHC class II cell activation. For this, primary B lymphocytes were subjected to Downloaded from molecules has been correlated with B lymphocyte activation as Percoll gradient centrifugation, which separates the lymphocytes well (26, 27). Indeed, the cell surface expression of HLA-DR was in fractions according to their specific cellular density, because much higher in Raji cells than on Ramos or primary cells (Fig. 1A). activated lymphocytes have a lower density than resting lympho- The expression levels of the various proteins that contribute to cytes (24). Typically, ϳ10% of the B lymphocytes migrated to a the efficiency of MHC class II Ag presentation were examined by low-density fraction and 90% to a higher-density fraction (data not

Western blot analysis of the B cell lines and different isolates of shown). As anticipated, FACS analysis of primary cells isolated http://www.jimmunol.org/ primary B lymphocytes (Fig. 1B). In total, Raji has a far more from the lower-density fractions shows a different scatter pattern abundant HLA-DR expression than Ramos, in agreement with the than for the cells isolated from the higher-density fractions (Table higher cell surface levels of HLA-DR (Fig. 1A). Both DM and DO I). Each fraction contained B lymphocytes that were Ͼ80% of by guest on September 30, 2021

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 Ј (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 IgGϩ 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 CD19ϩ freshly purified B lymphocytes and Raji and Ramos B cell lines. Equal amounts of cellular proteins were analyzed by Western blot (15 ␮g for DO, and 5 ␮g 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␤ serum. The slightly higher m.w. position of DM␣ 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␤ 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). 6852 DOWN-REGULATION OF HLA-DM AND HLA-DO IN HUMAN B LYMPHOCYTES

Table I. Cell surface expression of CD80, CD86, and HLA-DR on cules, we compared the relative amount of DR/CLIP complexes vs high- and low-density (in vivo activated) primary human B lymphocytes total DR complexes that reach the cell surface on Ramos, Raji, and high- and low-density primary B lymphocytes from three donors. FSCa CD80b CD86b HLA-DRb Ramos did not signiﬁcantly express any DR/CLIP at its cell sur- Donor 1 face, while considerable amounts of cell surface exposed DR/CLIP High-density B 287.09 2.1 5.98 753.69 were observed on Raji (Fig. 3). This correlates with the observa- Low-density B 329.7 10.39 13.04 804.82 tion that Ramos expressed relative low amounts of DR, hardly any Donor 2 DO, and only slightly less DM than Raji (Fig. 1B), a situation that High-density B 282.27 1.4 4.84 1027.81 will ensure optimal CLIP removal from the relatively low amount Low-density B 316.19 2.85 9.4 1112.4 Donor 3 of newly synthesized DR/CLIP through an active DM pool that is High-density B 286.45 1.53 6.29 1377.32 only marginally restrained by a minor pool of DO. Low-density B 321.45 5.54 12 1589.28 All primary B lymphocytes expressed clearly detectable levels a Forward scatter. of DR/CLIP at the cell surface (Fig. 3), indicating that in vivo in b Indicates MFI value (after subtraction of the control). The three samples were B lymphocytes a fraction of newly synthesized DR/CLIP routinely acquired with the same parameters after labeling with anti-CD80 mAb-FITC and anti-CD86 mAb-PE or with anti-HLA-DR D1.12 mAb followed by anti-mouse Ig escapes the editing action of DM and DO. The relative amount of Ј F(ab )2-FITC. DR/CLIP varied highly between donors (Fig. 3), possibly due to allelic variation between donors, as the strength of HLA-DR as- IgMϩ and IgDϩ (Fig. 2A) and Ͻ10% IgGϩ (data not shown), sociation with CLIP differs with the DR allele. The ratio of DR/ demonstrating that the majority of both types of B lymphocytes CLIP vs total DR was neither strongly nor consistently affected by Downloaded from were of a mature phenotype. FACS analysis showed that the B in vivo activation of the B lymphocytes (Fig. 3). This correlates lymphocytes obtained from the lower-density fractions expressed with the ﬁnding that B lymphocyte activation down-modulates ex- both more CD80, CD86, and HLA-DR (Table I). Therefore, the B pression of both DM (positively affecting DR/CLIP dissociation) lymphocytes from the lower-density fraction have a more activated and DO (negatively affecting DR/CLIP dissociation) (Fig. 2C)ina phenotype than those migrating in the higher-density fraction. coordinated fashion.

DR, DM, and DO total expression levels were compared be- http://www.jimmunol.org/ tween high- and low-density B lymphocytes derived from the same Down-modulation of DM and DO upon in vitro activation of donor (Fig. 2B). In total, 11 of 15 donors presented highly reduced primary B lymphocytes levels of both DM and DO in the low-density B lymphocytes. Semiquantitative analysis of the protein expression levels on four Because in vivo the down-modulation of DO and DM expression representative samples confirmed that the reduction of both DM correlates with activation of the B lymphocytes, freshly isolated and DO expression upon B cell activation was significant ( p ϭ high-density B lymphocytes were cultured for 24 h in the presence 0.029 for both), whereas the total amount of DR was not signifi- of various in vitro stimuli. Soluble anti-IgM Ab (inducing B cell cantly reduced by B cell activation ( p ϭ 0.343) (Fig. 2C). There- proliferation) (28) was used to stimulate B lymphocytes via BCR fore, the expression of DM and DO is highly dependent on the cross-linking. The mAb D1.12, recognizing an epitope near the by guest on September 30, 2021 activation status because they are down-modulated upon B cell peptide binding groove on the DR␣ chain (29), was used to stim- activation. ulate the B lymphocytes via HLA-DR (30, 31). IL-4 and anti- CD40 mAb, a well-known combination for B lymphocyte activa- DM and DO expression and the cell surface deposition of DR/ tion and proliferation, were applied as well (32, 33). Finally, cells CLIP complexes were incubated with PMA, a potent B lymphocyte activator via As variations in the relative expression levels of DM and DO may modulation of PKC (34, 35). All stimuli induced various levels of affect exchange of CLIP for antigenic peptide onto class II mole- B cell homotypic adhesion, except in the control, indicative of

FIGURE 2. A, Analysis of IgM and IgD cell surface expression on primary high- and low-density B lymphocytes. Freshly purified CD19ϩ 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. 1B. 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 ϭ 0.343 (nonsignificant) for DR and p ϭ 0.029 (significant) for both DM and DO when comparing low- to high-density B lymphocytes. The Journal of Immunology 6853

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 do- Downloaded from nors, 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. http://www.jimmunol.org/

effective stimulation/activation (36) (data not shown). The conse- quences 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. 4). The levels of both DM␣ and DO␤ were significantly ( p ϭ 0.03 for both) and consistently decreased upon activation with PMA and soluble anti- by guest on September 30, 2021 IgM Ab, while other in vitro stimuli did not affect their expression (Fig. 4). Similar results were obtained with a rabbit serum against DO␣ (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 ϭ 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. FIGURE 4. Modulation of DR, DM, and DO expression after in vitro The cell surface level of HLA-DR/CLIP complexes was inves- activation. Freshly purified high-density B lymphocytes were cultivated for 24 h in vitro in 1) culture medium only (control), or upon addition of tigated on PMA and BCR-stimulated B lymphocytes (data not various stimuli: 2) PMA, 3) D1.12 anti-HLA-DR mAb, 4) soluble anti-IgM shown). Both the total level of cell surface expressed DR and the Ab, and 5) IL-4 plus anti-CD40 mAb. Subsequently, cells were lysed and total level of DR/CLIP were enhanced upon stimulation, without analyzed by Western blot as before. Semiquantitative analysis from three significantly modifying the ratio of CLIP occupied class II vs total different samples is shown, correlated to the level of the control protein class II, in line with the concomitant diminution of both DM and actin (as described in Fig. 2C). A representative Western blot experiment DO expression. is shown. Statistical analysis revealed p ϭ 0.407 (non significant) for DR and p ϭ 0.03 (significant) for both DM and DO when comparing the PKC modulates DM and DO expression in primary human B conditions PMA and anti-IgM to the other conditions. lymphocytes Addition of PMA initially induces a rapid activation of classical (␣, ␤, and ␥) and novel (␦, ⑀, ⑀Ј, ␩, and ␪) isoforms of the PKC DM and DO was observed after 5 h and is marked at 15 h of BCR family and subsequently depletes the cellular PKC pool upon pro- stimulation (Fig. 5A). Interestingly, a slight reduction of PKC␤ but longed incubation times (34, 35). The most abundantly expressed not of PKC␣ was observed at 15 h of stimulation (Fig. 5A). PKC isoforms in B lymphocytes that can be modulated by phorbol As PMA induced a reduction in DM and DO as well, a similar esters are PKC␣ and PKC␤ (37) and were therefore explored in time course experiment was conducted with PMA. DM and DO our study. started to diminish after 15 h of stimulation (Fig. 5B). Five hours The effect of BCR triggering on DM and DO expression was after PMA addition a drop in the level of PKC␤ became apparent examined in more detail using the soluble anti-IgM Ab in a time and from 15 h onwards PKC␣ decreased as well, until after 24 h course experiment on high-density B lymphocytes. The decrease in both PKC␣ and PKC␤ could hardly be detected (Fig. 5B). Thus, 6854 DOWN-REGULATION OF HLA-DM AND HLA-DO IN HUMAN B LYMPHOCYTES

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␣ and -␤ expression. The cells were analyzed as before. The figure shown is a representative experiment performed on one iso- late for PMA and one for IgM stimulation (n ϭ 5 for PMA and n ϭ 3 for anti-BCR). Downloaded from depletion of both PKC␣ and PKC␤, the major phorbol ester-sen- 43). The coordinated modulation of DM and DO expression at the sitive PKC pools in the B lymphocytes, preceded the decline in protein level is more likely, because it has been shown that DO DM/DO expression, suggesting involvement of PKC in DM and depends on its association with DM for its steady state expression DO modulation. As previously noted, the total expression level of level and endoplasmic reticulum exit (6). Subcellular fractionation DR was not negatively affected during the course of PMA incu- studies of DM-mutant B cell lines confirmed that also in B lym- bation (Fig. 5B), and might be explained by the different behavior phocytes, DO expression (and endoplasmic reticulum egress) de- http://www.jimmunol.org/ of DR molecules as, once charged with a peptide, they acquired a pends on DM expression (data not shown). The mode of DM and more stable structure and were targeted to the cell surface (5). DO regulation was investigated by PKC inhibition studies in the It is interesting to note that, in contrast to PMA stimulation, no presence of CHX, an inhibitor of initiation of protein translation total loss of PKC was observed after BCR stimulation. We cannot (44). In CHX-treated B lymphocytes, inhibition of PKC activity by 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 (39Ð41). The modulation of DM and DO expression seems to be selectively activated by some stimuli, because neither HLA-DR nor CD40 and IL-4 by guest on September 30, 2021 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 ϭ 0.008) and a trend toward reduction in DM (nonsignificant, p ϭ 0.151) (Fig. 6). 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 FIGURE 6. Semiquantitative Western blot analysis of DR, DM, and DO degradation rate expression upon PKC inhibition. High-density B lymphocytes were incubated for 4 h with DMSO (control) or with equal amounts of DMSO con- Modulation of the steady state expression levels of DM and DO taining Ro 31-8220 (final concentration 10 ␮M). Cell lysates were treated occurs either via manipulation of their synthesis rates or via reg- and analyzed as before. The presented results are means of five different ulation of the DM and DO life span. A coordinated transcriptional samples, and a representative Western blot experiment is shown. Statistical DM and DO regulation, without affecting DR expression as well, analysis revealed p ϭ 0.69 (nonsignificant) for DR, p ϭ 0.151 (nonsignif- is unlikely because DMA, DMB, DOA, DRA, and DRB genes are icant) for DM, and p ϭ 0.008 (significant) for DO when comparing the under the control of the same transcriptional regulator, CIITA (42, condition Ro 31-8220 to the control. The Journal of Immunology 6855

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. 8). 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. 8). This further supports the idea that DM and DO may be modulated in lysosomal compartments. Together, our data reveal the existence of a mechanism regu- lating 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 In B lymphocytes, binding and subsequent presentation of most antigenic peptides to MHC class II molecules requires the action of Downloaded from 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. Be- http://www.jimmunol.org/ FIGURE 7. Western blot analysis of DR, DM, and DO expression dur- cause the initial state of B cell activation of every individual tested ing inhibition of de novo protein synthesis and inhibition of PKC. High- was unclear and the class II haplotype not known, some hetero- ␮ density B lymphocytes were incubated for 1 h with CHX (20 M), fol- geneity in the results was expected and obtained. Still, comparison ␮ lowed by incubation for 4 h with or without Ro 31-8220 (10 M). Control between immortalized B cell lines and primary B lymphocytes cells were incubated with equivalent amounts of DMSO. The presented invariably demonstrated markedly higher DO expression in pri- results are means of three different samples and a representative analysis of one donor is shown. mary cells. This strongly indicates that the in vivo role of DO on human class II-mediated peptide presentation is more prominent than previously anticipated. Ro 31-8220 led to the same reduction of DO as with Ro 31-8220 Separation of primary B lymphocytes into low- and high-density by guest on September 30, 2021 alone, and again less for DM, in most isolates tested (Fig. 7). Thus, populations revealed reduced expression of both DM and DO in in PKC-mediated regulation of DO expression and, to a lesser extent, vivo activated B lymphocytes. Further in vitro activation of high- of DM, is independent from de novo protein synthesis and may density B lymphocytes showed that down-modulation of DM and involve a post-translational control mechanism. DO could be induced via the BCR stimulation or with the tumor Regulation of DM and DO expression via manipulation of their promoter PMA. PMA thus provides a pharmacological and the life span could involve manipulation of their lysosomal degrada- BCR a physiological modulator of DM and DO expression. This tion, as DM and DO mainly reside in the acidic environment of the effect on DM and DO expression was further explored on the basis MIICs (6, 13). This was investigated by examining PKC-mediated that a long exposure to PMA results in degradation of PKC. The regulation of DO and DM expression in cells treated with the weak latter was previously shown to be able to control expression at the base CQ. CQ raises the lysosomal pH by acting on the Hϩ RNA or protein level (for review see Refs. 47 and 48). In this ATPases (45, 46). Indeed, CQ treatment consistently abrogated the study, modulation of DM and DO expression is proposed to occur

FIGURE 8. Western blot analysis of DR, DM, and DO expression after neu- tralization of lysosomal compartments by CQ and PKC inhibition. High-density B lymphocytes were either preincubated for 15 min with CQ (200 ␮M) followed by a 4-h incubation with CQ alone or with Ro 31-8220 (10 ␮M), 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. 6856 DOWN-REGULATION OF HLA-DM AND HLA-DO IN HUMAN B LYMPHOCYTES via a novel mechanism involving the PKC activity and the manip- portant and crucial role, either directly or indirectly, in the main- ulation of lysosomal degradation of both DM and DO. Lysosomes tenance of these modulators of class II-mediated Ag processing. are usually referred to as “dead-end organelles” but have also been What is the physiological rationale and need for regulation of reported to be prone to exocytosis (reviewed in Ref. 49) in certain DM and DO expression during B lymphocyte activation? The an- conditions such as elevated intracellular calcium concentration swer is likely to lie in the need for tight control of class II-mediated (50, 51). Sundler and colleagues (52, 53) reported exocytosis of Ag presentation in B lymphocytes to avoid presentation of unde- preformed lysosomal contents in mouse macrophages induced by sirable Ags. The data presented in this work show that high-density zymosan particles. The same effect was obtained by increasing the B lymphocytes seem to be optimally set for class II loading with lysosomal pH and was further enhanced by PKC activation. How- specific Ag; the observed high expression of DM will ensure ef- ever, down-regulation of PKC inhibited the lysosomal exocytosis fective class II peptide loading, while the high levels of DO will induced by zymosan or increase of lysosomal pH. These data could skew peptide loading to the proper, acidic Ag loading compart- provide a parallel to our results where CQ alone induced down- ments. This presetting of the B lymphocyte before Ag encounter regulation of DM and DO. Is this effect mediated by exocytosis of may be required as the timeframe observed for uptake, and class II lysosomal contents? Another hypothesis could be that CQ, by loading of specific Ag is too short (Ͻ 20 min) (58, 59) for efficient modifying the environment and behavior of the proteins present in up-regulation of DM and DO synthesis at this stage. Moreover, in endo/lysosomal compartments, suppresses the identity of these lymphoid organs, after Ag encounter by the B lymphocyte there compartments. The latter, not recognized by the cellular machin- are strict temporal requirements for the recruitment of T cell help ery, becomes prone to destruction and therefore presents a reduced necessary for B lymphocyte survival and development of an im- Downloaded from half-life. Again, in concordance with Sundler et al. (52, 53), PKC mune response. In this study, B lymphocyte activation results in inhibitor and CQ applied together resulted in an inhibition of the enhanced down-regulation of particularly DM and DO. The ob- observed diminution of DM and DO. Although it seems improb- servation that B lymphocyte activation via the BCR leads to fusion able, because Ro 31-8220 and CQ act in different compartments and acidification of the MIICs is of interest (17, 69), because this (cytosol and lysosomes respectively), we cannot exclude the pos- process is likely to enhance the protease activity in the MIICs. The sibility that the two components act together in an unexpected way latter would not only result in the rapid generation of the desired on DM and DO expression. In conclusion, our results reveal two antigenic peptides but may also lead to an elevated turnover rate of http://www.jimmunol.org/ MIIC resident proteins, as observed in this study for DM and DO. phenomena: 1) a basal activity of PKC is needed to maintain the This process would affect the turnover rate of class II molecules far physiological expression level of DO and DM, and 2) DM/DO can less, as they only reside in the MIICs for a limited time period be down-regulated by acting on PKC activity or on lysosomal pH before being exported to the plasma membrane. Indeed, this is by a mechanism that remains to be elucidated. what we observed. Physiologically, a reduction of DM and DO B lymphocytes become activated after Ag uptake and appropri- expression at this stage of B lymphocyte activation would not af- ate costimulatory signals (reviewed in Refs. 54Ð56). Their most fect presentation of the specific Ag, because this rapid process efficient pathway of Ag uptake is mediated via the BCR and tar- would precede DM and DO degradation. However, it would skew geting of the BCR-Ag complex to the MIICs (57Ð59). Cross-link- by guest on September 30, 2021 the activated B lymphocyte to a state where subsequent class II ing of the BCR leads to activation of signaling cascades initiated loading with unrelated, and therefore less desired, peptides would by ITAM phosphorylation of Ig␣␤ and recruitment of tyrosine be minimized. The observation that, like dendritic cells (70), low- kinases such as Syk and Btk. They subsequently activate extracel- density B lymphocytes express relatively more DR on their cell lular signal-related kinase, phospholipase C, Ras, and phosphati- surface and less intracellularly than high-density cells (Table I vs dylinositol 3 kinase. The resulting effects on B lymphocyte acti- Fig. 2) is in keeping with this scheme. vation and differentiation are therefore due to multiple pathways, In conclusion, we report selective modulation of DM and DO some of them being mediated by PKC (56, 60Ð63). Results from during B lymphocyte activation. Moreover, a novel and tightly several studies indicate a role for PKC isoforms in the B cell im- controlled picture of B lymphocyte-specific regulation of class II- ␤ mune response. PKC -deficient mice develop impaired humoral mediated Ag presentation begins to emerge. The regulation of both immune responses and a reduced sensitivity to BCR stimulation, modulators of class II-mediated Ag presentation may ensure that ␤ pointing to a crucial function of PKC in this pathway, as its they are optimally expressed at a stage where B lymphocytes need absence cannot be compensated for by other members of the PKC to be ready for an efficient and fast processing of specificAgand family (64). Interestingly, the differentiation of B lymphocytes into subsequent class II loading. Ab-secreting plasma cells in mice is accompanied by an increase in PKC␣ and loss of PKC␤ expression (65). Taken together these observations indicate that PKC is an important factor for B lym- Acknowledgments phocyte activation and differentiation. Previous studies have al- We thank Peter Cresswell for providing the CerCLIP mAb; Nuala Mooney, Fre«de«ric Garban, Dominique Charron, and Christian Villiers for helpful ready revealed an impact of signal transduction on the class II discussions and support; Christophe Chiquet for statistical analysis; and processing and presentation pathway (reviewed in Ref. 66), nota- colleagues from our respective laboratories for useful hints and ␣ bly via the BCR (17) and more recently via the Fc R (67, 68). discussions. Signalization via the BCR or via Fc␣R results in fusion and acidification of the subcellular MIICs in which Ag processing and class References II loading occur. PKC is involved in the modification of the MIICs 1. Roche, P. A., M. S. Marks, and P. Cresswell. 1991. Formation of a nine-subunit as PMA/ionomicine stimulation reproduces the effect of BCR complex by HLA class II glycoproteins and the invariant chain. Nature 354:392. stimulation (17). The Ag associated to its receptor is specifically 2. Lotteau, V., L. Teyton, A. Peleraux, T. Nilsson, L. Karlsson, S. L. Schmid, V. Quaranta, and P. A. Peterson. 1990. Intracellular transport of class II MHC targeted to these modified compartments, and Lang et al. (68) pro- molecules directed by invariant chain. Nature 348:600. pose a role for phosphatidylinositol-dependent protein kinase 1 3. Sloan, V. S., P. Cameron, G. Porter, M. Gammon, M. Amaya, E. Mellins, and and protein kinase B␣ in this process. Although we cannot exclude D. M. Zaller. 1995. Mediation by HLA-DM of dissociation of peptides from HLA-DR. Nature 375:802. that other factors are acting in the observed down-regulation of 4. Denzin, L. K., and P. Cresswell. 1995. HLA-DM induces CLIP dissociation from DM and DO, our results support the notion that PKC has an im- MHC class II ␣␤ dimers and facilitates peptide loading. Cell 82:155. The Journal of Immunology 6857

5. Wubbolts, R., M. Fernandez-Borja, L. Oomen, D. Verwoerd, H. Janssen, 31. Mooney, N. A., C. Grillot-Courvalin, C. Hivroz, L. Y. Ju, and D. Charron. 1990. J. Calafat, A. Tulp, S. Dusseljee, and J. Neefjes. 1996. Direct vesicular transport Early biochemical events after MHC class II-mediated signaling on human B of MHC class II molecules from lysosomal structures to the cell surface. J. Cell lymphocytes. J. Immunol. 145:2070. Biol. 135:611. 32. Valle, A., C. E. Zuber, T. Defrance, O. Djossou, M. De Rie, and J. Banchereau. 6. Liljedahl, M., T. Kuwana, W. P. Fung-Leung, M. R. Jackson, P. A. Peterson, and 1989. Activation of human B lymphocytes through CD40 and interleukin 4. Eur. L. Karlsson. 1996. HLA-DO is a lysosomal resident which requires association J. Immunol. 19:1463. with HLA-DM for efﬁcient intracellular transport. EMBO J. 15:4817. 33. Martin, I., T. Bonnefoix, C. Roucard, P. Perron, A. Lajmanovich, A. Moine, ϩ 7. Trowsdale, J., and A. Kelly. 1985. The human HLA class II ␣ chain gene DZ␣ D. Leroux, J. J. Sotto, and F. Garban. 1999. Role of autologous CD4 T cell is distinct from genes in the DP, DQ and DR subregions. EMBO J. 4:2231. clones in human B non-Hodgkin’s lymphoma: aborted activation and G1 block- 8. Tonnelle, C., R. DeMars, and E. O. Long. 1985. DO␤: a new ␤ chain gene in ade induced by cell-cell contact. Eur. J. Immunol. 29:3188. HLA-D with a distinct regulation of expression. EMBO J. 4:2839. 34. Krug, E., and A. H. Tashjian, Jr. 1987. Time-dependent changes in protein kinase 9. Karlsson, L., C. D. Surh, J. Sprent, and P. A. Peterson. 1991. A novel class II C distribution and disappearance in phorbol ester-treated human osteosarcoma MHC molecule with unusual tissue distribution. Nature 351:485. cells. Cancer Res. 47:2243. 10. Douek, D. C., and D. M. Altmann. 1997. HLA-DO is an intracellular class II 35. Mond, J. J., N. Feuerstein, C. H. June, A. K. Balapure, R. I. Glazer, K. Witherspoon, molecule with distinctive thymic expression. Int. Immunol. 9:355. and M. Brunswick. 1991. Bimodal effect of phorbol ester on B cell activation: im- 11. Liljedahl, M., O. Winqvist, C. D. Surh, P. Wong, K. Ngo, L. Teyton, plication for the role of protein kinase C. J. Biol. Chem. 266:4458. P. A. Peterson, A. Brunmark, A. Y. Rudensky, W. P. Fung-Leung, and 36. Kansas, G. S., and T. F. Tedder. 1991. Transmembrane signals generated through L. Karlsson. 1998. Altered antigen presentation in mice lacking H2-O. Immunity MHC class II, CD19, CD20, CD39, and CD40 antigens induce LFA-1-dependent 8:233. and independent adhesion in human B cells through a tyrosine kinase-dependent pathway. J. Immunol. 147:4094. 12. Denzin, L. K., D. B. Sant’Angelo, C. Hammond, M. J. Surman, and P. Cresswell. 1997. Negative regulation by HLA-DO of MHC class II-restricted antigen pro- 37. Setterblad, N., I. Onyango, U. Pihlgren, L. Rask, and G. Andersson. 1998. The cessing. Science 278:106. role of protein kinase C signaling in activated DRA transcription. J. Immunol. 161:4819. 13. van Ham, S. M., E. P. M. Tjin, B. F. Lillemeier, U. Gruneberg, 38. Sidorenko, S. P., C. L. Law, S. J. Klaus, K. A. Chandran, M. Takata, T. Kurosaki, K. E. van Meijgaarden, L. Pastoors, D. Verwoerd, A. Tulp, B. Canas, D. Rahman,

and E. A. Clark. 1996. Protein kinase C␮ (PKC␮) associates with the B cell Downloaded from et al. 1997. HLA-DO is a negative modulator of HLA-DM-mediated MHC class antigen receptor complex and regulates lymphocyte signaling. Immunity 5:353. II peptide loading. Curr. Biol. 7:950. 39. Katsuta, H., S. Tsuji, Y. Niho, T. Kurosaki, and D. Kitamura. 1998. Lyn-mediated 14. Kropshofer, H., A. B. Vogt, C. Thery, E. A. Armandola, B. C. Li, down-regulation of B cell antigen receptor signaling: inhibition of protein kinase C G. Moldenhauer, S. Amigorena, and G. J. Hammerling. 1998. A role for activation by Lyn in a kinase-independent fashion. J. Immunol. 160:1547. HLA-DO as a co-chaperone of HLA-DM in peptide loading of MHC class II 40. Feng, X., K. P. Becker, S. D. Stribling, K. G. Peters, and Y. A. Hannun. 2000. molecules. EMBO J. 17:2971. Regulation of receptor-mediated protein kinase C membrane trafficking by auto- 15. van Ham, M., M. van Lith, B. Lillemeier, E. Tjin, U. Gruneberg, D. Rahman, phosphorylation. J. Biol. Chem. 275:17024. L. Pastoors, K. van Meijgaarden, C. Roucard, J. Trowsdale, et al. 2000. Modu- 41. Hansra, G., P. Garcia-Paramio, C. Prevostel, R. D. Whelan, F. Bornancin, and lation of the major histocompatibility complex class II-associated peptide reper- http://www.jimmunol.org/ P. J. Parker. 1999. Multisite dephosphorylation and desensitization of conven- toire by human histocompatibility leukocyte antigen (HLA)-DO. J. Exp. Med. tional protein kinase C isotypes. Biochem. J. 342:337. 191:1127. 42. Chang, C. H., S. Guerder, S. C. Hong, W. van Ewijk, and R. A. Flavell. 1996. 16. Mitchell, R. N., K. A. Barnes, S. A. Grupp, M. Sanchez, Z. Misulovin, Mice lacking the MHC class II transactivator (CIITA) show tissue-specific im- M. C. Nussenzweig, and A. K. Abbas. 1995. Intracellular targeting of antigens pairment of MHC class II expression. Immunity 4:167. internalized by membrane immunoglobulin in B lymphocytes. J. Exp. Med. 181: 43. Taxman, D. J., D. E. Cressman, and J. P. Ting. 2000. Identification of class II 1705. transcriptional activator-induced genes by representational difference analysis: 17. Siemasko, K., B. J. Eisfelder, E. Williamson, S. Kabak, and M. R. Clark. 1998. discoordinate regulation of the DN␣/DO␤ heterodimer. J. Immunol. 165:1410. Cutting edge: signals from the B lymphocyte antigen receptor regulate MHC 44. Oleinick, N. L. 1977. Initiation and elongation of protein synthesis in growing class II-containing late endosomes. J. Immunol. 160:5203. cells: differential inhibition by cycloheximide and emetine. Arch. Biochem. Bio- 18. Sanderson, F., M. J. Kleijmeer, A. Kelly, D. Verwoerd, A. Tulp, J. J. Neefjes, phys. 182:171. H. J. Geuze, and J. Trowsdale. 1994. Accumulation of HLA-DM, a regulator of 45. de Duve, C., T. de Barsy, B. Poole, A. Trouet, P. Tulkens, and F. Van Hoof. 1974. by guest on September 30, 2021 antigen presentation, in MHC class II compartments. Science 266:1566. Lysosomotropic agents. Biochem. Pharmacol. 23:2495. 19. Guy, K., V. Van Heyningen, B. B. Cohen, D. L. Deane, and C. M. Steel. 1982. 46. Seglen, P. O., B. Grinde, and A. E. Solheim. 1979. Inhibition of the lysosomal Differential expression and serologically distinct subpopulations of human Ia an- pathway of protein degradation in isolated rat hepatocytes by ammonia, methyl- ␣ ␤ tigens detected with monoclonal antibodies to Ia and chains. Eur. J. Immunol. amine, chloroquine and leupeptin. Eur. J. Biochem. 95:215. 12:942. 47. Dempsey, E. C., A. C. Newton, D. Mochly-Rosen, A. P. Fields, M. E. Reyland, 20. Gruneberg, U., T. Rich, C. Roucard, S. Marieke van Ham, D. Charron, and P. A. Insel, and R. O. Messing. 2000. Protein kinase C isozymes and the regu- ␣ J. Trowsdale. 1997. Two widely used anti-DR monoclonal antibodies bind to lation of diverse cell responses. Am. J. Physiol. 279:L429. an intracellular C-terminal epitope. Hum. Immunol. 53:34. 48. Hershey, J. W. 1989. Protein phosphorylation controls translation rates. J. Biol. 21. Carrel, S., R. Tosi, N. Gross, N. Tanigaki, A. L. Carmagnola, and R. S. Accolla. Chem. 264:20823. 1981. Subsets of human Ia-like molecules defined by monoclonal antibodies. 49. Andrews, N. W. 2000. Regulated secretion of conventional lysosomes. Trends Mol. Immunol. 18:403. Cell Biol. 10:316. 22. Denzin, L. K., N. F. Robbins, C. Carboy-Newcomb, and P. Cresswell. 1994. 50. Rodriguez, A., P. Webster, J. Ortego, and N. W. Andrews. 1997. Lysosomes Assembly and intracellular transport of HLA-DM and correction of the class II behave as Ca2ϩ-regulated exocytic vesicles in fibroblasts and epithelial cells. antigen-processing defect in T2 cells. Immunity 1:595. J. Cell Biol. 137:93. 23. Funderud, S., B. Erikstein, H. C. Asheim, K. Nustad, T. Stokke, H. K. Blomhoff, ϩ 51. Martinez, I., S. Chakrabarti, T. Hellevik, J. Morehead, K. Fowler, and H. Holte, and E. B. Smeland. 1990. Functional properties of CD19 B lympho- N. W. Andrews. 2000. Synaptotagmin VII regulates Ca2ϩ-dependent exocytosis cytes positively selected from buffy coats by immunomagnetic separation. Eur. of lysosomes in fibroblasts. J. Cell Biol. 148:1141. J. Immunol. 20:201. 52. Sundler, R. 1997. Lysosomal and cytosolic pH as regulators of exocytosis in 24. Dagg, M. K., and D. Levitt. 1981. Human B-lymphocyte subpopulations. I. Differ- mouse macrophages. Acta Physiol. Scand. 161:553. entiation of density-separated B lymphocytes. Clin. Immunol. Immunopathol. 21:39. 53. Tapper, H., and R. Sundler. 1995. Protein kinase C and intracellular pH regulate 25. Magrath, I. T., C. B. Freeman, P. Pizzo, J. Gadek, E. Jaffe, M. Santaella, zymosan-induced lysosomal enzyme secretion in macrophages. J. Leukocyte C. Hammer, M. Frank, G. Reaman, and L. Novikovs. 1980. Characterization of Biol. 58:485. lymphoma-derived cell lines: comparison of cell lines positive and negative for 54. Campbell, K. S. 1999. Signal transduction from the B cell antigen-receptor. Curr. Epstein-Barr virus nuclear antigen. II. Surface markers. J. Natl. Cancer Inst. Opin. Immunol. 11:256. 64:477. 55. Hodgkin, P. D., and A. Basten. 1995. B cell activation, tolerance and antigen- 26. Noelle, R., P. H. Krammer, J. Ohara, J. W. Uhr, and E. S. Vitetta. 1984. Increased presenting function. Curr. Opin. Immunol. 7:121. expression of Ia antigens on resting B cells: an additional role for B-cell growth 56. DeFranco, A. L. 1997. The complexity of signaling pathways activated by the factor. Proc. Natl. Acad. Sci. USA 81:6149. BCR. Curr. Opin. Immunol. 9:296. 27. Mond, J. J., E. Seghal, J. Kung, and F. D. Finkelman. 1981. Increased expression 57. Brown, B. K., C. Li, P. C. Cheng, and W. Song. 1999. Trafficking of the Ig␣/Ig␤ of I-region-associated antigen (Ia) on B cells after cross-linking of surface Ig. heterodimer with membrane Ig and bound antigen to the major histocompatibility J. Immunol. 127:881. complex class II peptide-loading compartment. J. Biol. Chem. 274:11439. 28. Mond, J. J., and M. Brunswick. 1991. Proliferative assays for B cell function. In 58. Aluvihare, V. R., A. A. Khamlichi, G. T. Williams, L. Adorini, and Current Protocols in Immunology. I. J. E. Coligan, A. M. Kruisbeek, D. H. M. S. Neuberger. 1997. Acceleration of intracellular targeting of antigen by the Margulies, E. M. Shevach, and W. Strober, eds. Wiley, New York, p. 3.10.1. B-cell antigen receptor: importance depends on the nature of the antigen-antibody 29. Fu, X. T., and R. W. Karr. 1994. HLA-DR ␣ chain residues located on the outer interaction. EMBO J. 16:3553. loops are involved in nonpolymorphic and polymorphic antibody-binding 59. Cheng, P. C., C. R. Steele, L. Gu, W. Song, and S. K. Pierce. 1999. MHC class epitopes. Hum. Immunol. 39:253. II antigen processing in B cells: accelerated intracellular targeting of antigens. 30. Lane, P. J., F. M. McConnell, G. L. Schieven, E. A. Clark, and J. A. Ledbetter. J. Immunol. 162:7171. 1990. The role of class II molecules in human B cell activation: association with 60. Francois, D. T., I. M. Katona, C. H. June, L. M. Wahl, N. Feuerstein, phosphatidylinositol turnover, protein tyrosine phosphorylation, and prolifera- K. P. Huang, and J. J. Mond. 1988. Anti-Ig-mediated proliferation of human B tion. J. Immunol. 144:3684. cells in the absence of protein kinase C. J. Immunol. 140:3338. 6858 DOWN-REGULATION OF HLA-DM AND HLA-DO IN HUMAN B LYMPHOCYTES

61. Guinamard, R., N. Signoret, I. Masamichi, M. Marsh, T. Kurosaki, and 66. Siemasko, K., and M. R. Clark. 2001. The control and facilitation of MHC class J. V. Ravetch. 1999. B cell antigen receptor engagement inhibits stromal cell- II antigen processing by the BCR. Curr. Opin. Immunol. 13:32. derived factor (SDF)-1␣ chemotaxis and promotes protein kinase C (PKC)-in- 67. Shen, L., M. van Egmond, K. Siemasko, H. Gao, T. Wade, M. L. Lang, M. Clark, duced internalization of CXCR4. J. Exp. Med. 189:1461. J. G. van De Winkel, and W. F. Wade. 2001. Presentation of ovalbumin inter- 62. Xie, H., and T. L. Rothstein. 1995. Protein kinase C mediates activation of nu- nalized via the immunoglobulin-A Fc receptor is enhanced through Fc receptor clear cAMP response element-binding protein in B lymphocytes stimulated ␥-chain signaling. Blood 97:205. through surface Ig. J. Immunol. 154:1717. 68. Lang, M. L., L. Shen, H. Gao, W. F. Cusack, G. A. Lang, and W. F. Wade. 2001. 63. Krappmann, D., A. Patke, V. Heissmeyer, and C. Scheidereit. 2001. B-cell re- Fc␣ receptor cross-linking causes translocation of phosphatidylinositol-depen- ceptor- and phorbol ester-induced NF-␬B and c-Jun N-terminal kinase activation dent protein kinase 1 and protein kinase B ␣ to MHC class II peptide loading-like in B cells requires novel protein kinase C’s. Mol. Cell. Biol. 21:6640. compartments. J. Immunol. 166:5585. 64. Leitges, M., C. Schmedt, R. Guinamard, J. Davoust, S. Schaal, S. Stabel, and 69. Siemasko, K., B. J. Eisfelder, C. Stebbins, S. Kabak, A. J. Sant, W. Song, and A. Tarakhovsky. 1996. Immunodeficiency in protein kinase c␤-deficient mice. M. R. Clark. 1999. Ig␣ and Ig␤ are required for efficient trafficking to late en- Science 273:788. dosomes and to enhance antigen presentation. J. Immunol. 162:6518. 65. Mischak, H., W. Kolch, J. Goodnight, W. F. Davidson, U. Rapp, S. Rose-John, 70. Cella, M., A. Engering, V. Pinet, J. Pieters, and A. Lanzavecchia. 1997. Inflam- and J. F. Mushinski. 1991. Expression of protein kinase C genes in hemopoietic matory stimuli induce accumulation of MHC class II complexes in dendritic cells. cells is cell type- and B cell-differentiation stage specific. J. Immunol. 147:3981. Nature 388:782. Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021