Vaults During Dendritic Cell Development Up-Regulation of Drug Resistance-Related

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Up-Regulation of Drug Resistance-Related Vaults During Dendritic Cell Development Anouk B. Schroeijers, Anneke W. Reurs, George L. Scheffer, Anita G. M. Stam, Mariska C. de Jong, Thomas Rustemeyer, This information is current as Erik A. C. Wiemer, Tanja D. de Gruijl and Rik J. Scheper of September 23, 2021. J Immunol 2002; 168:1572-1578; ; doi: 10.4049/jimmunol.168.4.1572 http://www.jimmunol.org/content/168/4/1572 Downloaded from

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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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Up-Regulation of Drug Resistance-Related Vaults During Dendritic Cell Development1

Anouk B. Schroeijers,* Anneke W. Reurs,* George L. Scheffer,* Anita G. M. Stam,* Mariska C. de Jong,* Thomas Rustemeyer,* Erik A. C. Wiemer,‡ Tanja D. de Gruijl,† and Rik J. Scheper2*

P-glycoprotein (Pgp) and vaults are associated with multidrug resistance in tumor cells, but their physiological functions are not yet clear. Pgp, the prototypical transmembrane transporter molecule, may also facilitate the migration of skin dendritic cells (DC). Vaults—ribonucleoprotein cell organelles, frequently overexpressed in Pgp-negative drug-resistant tumor cells—have also been associated with intracellular transport processes. Given the pivotal role of DC in dealing with exposure to potentially harmful substances, the present study was set out to examine the expression of Pgp and vaults during differentiation and maturation of DC. Downloaded from DC were obtained from different sources, including blood-derived monocytes, CD34؉ mononuclear cells, and chronic myeloid leukemia cells. Whereas flow cytometric and immunocytochemical analyses showed slightly augmented levels of Pgp, up-regulation of vault expression during DC culturing was strong, readily confirmed by Western blotting, and independent of the source of DC. In further exploring the functional significance of vault expression, it was found that supplementing DC cultures with polyclonal or mAbs against the major vault protein led to lower viabilities of LPS- or TNF-␣-matured monocytes-DC. Moreover, expression of critical differentiation, maturation, and costimulatory molecules, including CD1a and CD83, was reduced and their capacity to http://www.jimmunol.org/ induce Ag-specific T cell proliferative and IFN-␥ release responses was impaired. These data point to a role for vaults in both DC survival and functioning as APC. The Journal of Immunology, 2002, 168: 1572–1578.

endritic cells (DC)3 are recognized as major players in plore this issue. Notably, immune responsiveness might well ben- the regulation of immune responses by directing both eﬁt from such mechanisms. To preserve their sentinel function in D their vigor and quality. The ability of DC to activate immune responses, DC should not readily suffer untoward damage naive T cells depends on their maturation state. Immature DC are from exposure to potentially toxic materials, as often derived from mainly distributed in tissues interfacing with the external environ- microbial and environmental sources. Hence, the expression of

ment where they can capture and process Ags with high efficiency. Pgp and a newly discovered cell organelle, the vault, was studied by guest on September 23, 2021 After Ag internalization, DC leave the peripheral tissues to reach during in vitro DC development from various peripheral blood- the draining lymphoid organs. During this migration, DC undergo derived precursor cells (4, 5). Vaults are evolutionarily highly con- maturation, involving augmented surface expression of MHC, che- served, large ribonucleoprotein particles (6). The particles repre- mokine receptors and costimulatory molecules, and acquire the sent multimeric RNA-protein complexes with one predominant ability to prime T cells (1). component, the major vault protein (MVP). Although the cellular It was only recently that P-glycoprotein (Pgp), a molecule well role of vaults has remained elusive, several findings support the known for its ability to transport a broad spectrum of xenobiotics view that vaults have a transport function by acting as a carrier, out of cells and thereby induce multidrug resistance (MDR) (2), mediating bi-directional nucleo-cytoplasmic exchange as well as was identified as yet another molecule that might play a role in this vesicular transport of compounds, including cytostatic drugs (7Ð migratory process (3). Since the putative contribution of Pgp and, 14). The present results demonstrate that not only Pgp but also possibly, other mechanisms defined earlier in drug resistance stud- vaults are expressed and up-regulated during the differentiation ies, to DC functioning is still unknown, we set out to further ex- and maturation of DC from various sources. Furthermore, endo- cytosis of anti-MVP Abs leads to reduced viabilities and interference with DC maturation and Ag-presenting capacity, thus point- Departments of *Pathology and †Medical Oncology, VU University Medical Center, ing to a critical role of vaults in DC functioning. Amsterdam, The Netherlands; and ‡Department of Hematology, Erasmus University, Rotterdam, The Netherlands Materials and Methods Received for publication February 5, 2001. Accepted for publication December 4, 2001. Monocyte (MO)-derived DC culture The costs of publication of this article were defrayed in part by the payment of page Human peripheral blood mononuclear cells isolated by adherence to plastic charges. This article must therefore be hereby marked advertisement in accordance were (either upon cryopreservation or directly) cultured in IMDM (10 ϫ with 18 U.S.C. Section 1734 solely to indicate this fact. 106 cells/2.5 ml) containing 25 mM HEPES (BioWhittaker, Verviers, Bel- 1 This work was financially supported by the Dutch Cancer Society, Grant VU gium), 10% FCS (Integro, Zaandam, The Netherlands), 2 mM L-glutamine 95-923. (Life Technologies, Paisley, U.K.), 50 IE/ml sodium-penicillin G, 50 ␮ ␮ 2 Address correspondence and reprint requests to Dr. Rik J. Scheper, Department of g/ml streptomycin sulfate, and 50 M 2-ME (Merck, Darmstadt, Ger- Pathology, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, many) (complete medium) supplemented with 100 ng/ml rGM-CSF (Leu- The Netherlands. E-mail address: [email protected] comax, Sandoz, The Netherlands) and 1000 U/ml rIL-4 (CLB, Amsterdam, 3 Abbreviations used in this paper: DC, dendritic cell; CML, chronic myeloid leuke- The Netherlands). After 6 days of culture, a semiadherent cell population mia; DFI, dye fluorescence index; DNR, daunorubicin; MDR, multidrug resistance; was obtained which displayed a veiled DC morphology. These immature MFI, mean fluorescence index; MO, monocyte; MVP, major vault protein; Pgp, P- DC were subsequently cultured for 1 day in the absence or presence of glycoprotein; VER, verapamil. either LPS (25 ng/ml, Escherichia coli; Difco, Detroit, MI) or rTNF-␣ (50

ng/ml; Eurocetus, Amsterdam, The Netherlands) to induce maturation be- between the mean fluorescence of the specific mAb and the mean fluores- fore harvesting. Loosely adherent cells were harvested with 0.5 mM cence of the negative control Abs. EDTA/PBS. Cultures set up for studying vault functions were performed with anti- Immunocytochemistry vault Abs added to the culture medium at the onset, day 4, and day 6 of Cytocentrifuge preparations of cells were air dried, fixed at room temper- MO-DC cultures. Each time the amount of administered Abs was set on 5 ␮ ature in acetone for 10 min, or, in the case of the mAbs MVP-9, MVP-37, g/ml. Polyclonal Abs used were rabbit anti-MVP pabW (15) and rabbit and p193-4, in 3% (w/v) paraformaldehyde/0.4% (w/v) glucose in PBS for IgG (normal rabbit serum; DAKO, Glostrup, Denmark). mAb experiments 10 min. Further immunocytochemical staining procedures were performed were performed with mouse (LRP-56 and MVP-9) and rat (LMR5) mAbs as described before (15). In addition, biotinylated swine anti-rabbit Ig and against MVP, respectively, irrelevant control mouse and rat mAbs pro- streptavidin-biotin-peroxidase complexes (DAKO) were used. Cytospins duced in our laboratory (16, 17). These Abs were dialyzed before use were scored by two independent observers. (Slide-Alyzer Cassette; Pierce, Rockford, IL). Immunoblots CD34-derived DC culture ϩ Postnuclear supernatants from MO-derived mature DC and control cells DC were generated from CD34 cells isolated from peripheral blood of lines SW-1573/2R160 and GLC4/ADR were prepared and analyzed by G-CSF-mobilized patients. Cells bearing CD34 Ag were isolated from immunoblotting following procedures as described previously (15). mononuclear fractions through positive selection by mini-MACS using the CD34ϩ progenitor cell isolation kit according to the manufacturer’s rec- Dye efflux studies ommendations (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34ϩ ␮ progenitors were cryopreserved or seeded for expansion at 105 cells/ml in Cells were incubated in culture medium containing 2 M daunorubicin complete medium in the presence of rGM-CSF (100 ng/ml) and rTNF-␣ (DNR; Sigma-Aldrich, St. Louis, MO) for 60 min at 37¡C, washed in ice-cold medium, and resuspended in medium without or with 2 ␮M (2.5 ng/ml) for 14 days before harvesting. Adherent cells were recovered Downloaded from ␮ using 0.5 mM EDTA/PBS. PSC833 (Novartis, Basel, Switzerland) or 10 M verapamil (VER; Sigma- Aldrich). After 60 min of incubation at 37¡C, cells were washed and im- Chronic myeloid leukemia (CML)-derived DC culture mediately placed on ice for analysis by flow cytometry. Results were calculated as the ratios between the mean fluorescence of DNR retention in PBMC from CML patients were (either upon cryopreservation or directly) the presence of Pgp inhibitors and the mean fluorescence of DNR retention seeded in a 2 ϫ 106 cells/ml concentration and cultured in complete me- in the absence of Pgp inhibitors (dye fluorescence index (DFI)). dium supplemented with rGM-CSF (100 ng/ml), rTNF-␣ (2.5 ng/ml), and

rIL-4 (1000 U/ml) for 10 days. These immature CML-derived DC were Double-labeling immunofluorescence http://www.jimmunol.org/ subsequently cultured for 3 days in the absence or presence of LPS (25 For double-labeling experiments, cytospin preparations of immature and ng/ml) to induce maturation. Loosely adherent matured cells were har- mature MO-DC were fixed for 2Ð4 min in methanol (Ϫ20¡C) or 10 min in vested with 0.5 mM EDTA/PBS. acetone at room temperature. After a blocking step with normal goat serum Tumor cell lines (DAKO) for 20 min at room temperature, the cells were incubated simultaneously with anti-MVP (mAb MVP-9 on methanol-fixed spins or The drug-sensitive non-small lung cancer cell SW-1573 and its Pgp over- LRP-56 on acetone-fixed spins) and rabbit polyclonal Abs against TGN-46 expressing multidrug-resistant, doxorubicin-selected (160 nM) subline for labeling the trans-Golgi network, CD63 for labeling lysosomes, MHCI SW-1573/2R160 (18) and the drug-sensitive small lung cancer cell line or MHCII diluted in PBS/0.5% BSA for 60 min at room temperature. All GLC4 and its MVP/vault overexpressing multidrug-resistant, doxorubicin- rabbit polyclonal Abs were kindly provided by J. Neefjes and M. Fer-

selected (1152 nM) subline GLC4/ADR (19) were used as controls for the nandes Borja from the Dutch Cancer Institute (Amsterdam, The Nether- by guest on September 23, 2021 immunoreactivity of the specific anti-Pgp protein and anti-vault protein lands). Subsequently, anti-MVP mouse mAb binding was detected using Abs. The drug-selected cell lines were cultured in the presence of doxo- FITC-labeled goat anti-mouse Ig (DAKO), and the binding of rabbit poly- rubicin every other week and propagated in DMEM (BioWhittaker) sup- clonal Abs was detected with Texas Red-labeled goat anti-rabbit Ig (Mo- plemented with 10% FCS, 50 U/ml penicillin G, and 50 ␮g/ml lecular Probes, Eugene, OR) (simultaneous incubation for 30 min at room streptomycin. temperature). Cells were mounted with Vectashield (Vector Laboratories, Burlingame, CA). Cells were examined with a MRC-600 confocal micro- Antibodies scope (Bio-Rad, Herfordshire, U.K.), coupled to a Nikon microscope (Melville, NY). Negative controls consisted of simultaneously processed Immunophenotypical analysis of blood precursor cells and DC was per- slides replacing either the mouse mAb LRP-56 and MVP-9 with isotype- formed using unlabeled mAb and mAb directly conjugated to FITC or PE. matched control mAb (mouse IgG2b, anti-chromogranin A; DAKO) or These included anti-CD83/PE (Immunotech, Marseille, France), anti-HLA- replacing the rabbit polyclonal Abs with control polyclonal (anti- DR/FITC, anti-CD80 and anti-CD80/PE, anti-CD14 and anti-CD14/FITC calcitonin; DAKO). For each condition, at least 50 immature and mature (BD Biosciences, San Jose, CA), anti-CD86/PE, anti-CD54PE, anti- DC were evaluated for resulting staining patterns. CD1a/PE (BD PharMingen, San Diego, CA), anti-CD86 (Ancell, Bayport, MN), anti-CD1a and anti-HLA-DR (Monosan; Sanbio, Uden, The In vitro stimulation of specific T cell proliferation and IFN-␥ Netherlands). release For immunophenotypical evaluation of Pgp, MVP and p193 expression, next to the unlabeled MVP-specific mAbs mentioned above under MO-DC After 7 days of culture, viable LPS-matured MO-DC were seeded as stim- culturing, the anti-MVP specific mAb MVP-37 (17) was used, as well as ulator cells in round-bottom 96-well culture plates (Nunclon International, the following mAbs: anti-p193 mAb p193-4 (15), respectively, anti-Pgp Roskilde, Denmark) at 104 viable (trypan blue exclusion) DC per well and mouse mAbs JSB-1 (20) and C494 (21) (Centocor, Leiden, The Nether- pulsed for 2 h with 1 ␮g/ml influenza viral Ag preparation containing three lands), both recognizing internal epitopes, and MRK16 (22), UIC2 (23), major hemagglutinins as outlined elsewhere (26). After discarding the su- 4.17 (24), and 4E3 (25) directed against extracellular epitopes. Except for pernatant, 250 ␮l of fresh medium and 50 ␮l with 105 autologous PBL the latter five Pgp-specific mAbs, all other mAbs mentioned were produced were added per well. Cells were cultured for 6 days, after which 50 ␮lof in our laboratory. Negative isotype-matched control murine mAbs were supernatant was collected for IFN-␥ assays by ELISA, and [3H]thymidine included in every experiment. was added (0.4 ␮Ci/well; Amersham, Aylesbury, U.K.), after which the cells were harvested onto fiberglass filters and [3H]thymidine incorporation Flow cytometric analysis was determined using a flatbed liquid scintillation counter (Wallac, Turku, Finland). Cells were washed in PBS supplemented with 0.1% BSA and 0.02% NaN3 and either fixed and permeabilized with 2% formaldehyde in acetone for Results and Discussion 10 s (intracellular epitope detection) or stained directly after harvesting (extracellular epitope detection). Cells were preincubated for 20 min with Up-regulation of Pgp and vault particles during DC normal goat serum followed by incubation for 45 min with primary mAb. development Binding of unlabeled mAb was revealed by FITC-conjugated goat anti- ␣ mouse Ig (CLB). Fluorescence analysis was performed on a FACScan flow Immature and LPS- and TNF- -matured DC were differentiated in cytometer after acquisition of 10,000 events (BD Biosciences). Results vitro from peripheral blood MO. Using this model, first Pgp and were expressed as mean fluorescence indices (MFI) calculated as the ratio MVP expression were analyzed by flow cytometry. Development 1574 ROLE OF VAULTS IN DC DEVELOPMENT of MO-DC was monitored by phenotyping the cells at the different immunoblotted by using anti-Pgp mAb C494 and anti-MVP mAb stages in culture. Characteristically, expression of the MO marker MVP-37. Although a protein band of ϳ170 kDa was readily de- CD14 expression was lost, whereas the DC differentiation marker tectable in the control postnuclear supernatant of Pgp-positive SW- CD1a was up-regulated (Fig. 1A). Expression of other DC differ- 1573/2R160 tumor cells, no Pgp could be identified in mature DC, entiation markers (CD80, CD86, and HLA-DR) was also up-reg- indicating that the expression level of Pgp in mature MO-DC re- ulated (data not shown), and there was de novo expression of the mains below the level of detection by Western blotting (data not DC maturation marker CD83 (Fig. 1A). Immunolabeling of per- shown). High levels of MVP in mature MO-DC were confirmed by meabilized cells with mAb directed against intracellularly situated Western blotting; a clear band of ϳ110 kDa was detected, corre- epitopes of Pgp (mAb JSB-1) and MVP (mAb LRP-56 and sponding to the molecular mass of MVP (Fig. 1C). MVP-9) revealed neither of the proteins in the CD14ϩ MO. Im- In vitro, DC-like cells can be generated from a number of mature MO-DC displayed weak expression of Pgp (MFI, 1.6), sources by culturing hemopoietic precursor cells in the presence of which was slightly increased in fully matured MO-DC (MFI, 1.8). a variety of cytokines and medium supplements (4, 5). To elabo- Clear MVP expression was found in immature MO-DC (MFI, 1.9), rate on the correlation between differentiation stage and expression which was further increased in fully matured MO-DC (MFI, 4.3; of Pgp and vaults that was found in MO-DC, we further evaluated Fig. 1A). their expression in CD34-derived DC and CML-derived DC. Im- Immunocytochemical analysis of cytospin preparations con- munocytochemical analyses showed that also in these cell types firmed these findings, i.e., low, but distinctly augmented levels of cellular differentiation/maturation, as reflected by up-regulated ex- Pgp and robust up-regulation of the MVP protein during the dif- pression of CD1a and CD83, was accompanied by increased ex- ferentiation and maturation of MO-DC (Fig. 1B). With two anti- pression of Pgp and MVP (Table I). Given its prominence in DC Downloaded from Pgp mAbs, JSB-1 and C494, we observed cytoplasmic immuno- propagated from three different sources under different culture reactivity, sometimes in mature MO-DC associated with the conditions, up-regulation of Pgp and vaults seems to reflect a com- plasma membrane. Three different anti-MVP mAbs, i.e., MVP-9, mon developmentally regulated event. Thus, in parallel to func- MVP-37, and LRP-56 (shown in Fig. 1B) displayed the character- tional changes accompanying the differentiation from precursor istic coarsely granular cytoplasmic staining of MVP, which was cells and subsequent activation, as a response to danger-signaling significantly increased in fully mature MO-DC as compared with agents present in the microenvironment (such as TNF-␣ and LPS), http://www.jimmunol.org/ immature MO-DC. In addition, both immature and mature DC may rapidly up-regulate Pgp and vault expression. MO-DC showed parallel overexpression of MVP and a minor vault component, the 193-kDa minor vault protein (p193), indi- MO-DC do not exhibit Pgp-mediated transmembrane transport cating that whole vault particles are up-regulated during MO-DC In chemotherapeutic treatment strategies, the induction or up-reg- differentiation and maturation (15). ulation of transmembrane transporter molecules, such as Pgp, in To further establish overexpression of Pgp and MVP molecules cancerous cells represents a major obstacle. In this view one might in mature MO-DC, postnuclear supernatants were prepared and speculate that expression of the same MDR-related molecules by by guest on September 23, 2021

FIGURE 1. Pgp and vaults are up-regulated during MO-DC differentiation. A, Flow cytometric evaluation of MO, immature MO-DC, and LPS-matured MO-DC using anti-CD14, CD1a, and CD83 for immunophenotypical analysis and the mAbs JSB-1 and LRP-56 for Pgp and MVP detection, respectively. B, Immunocytochemical detection of Pgp (JSB-1), MVP (LRP-56), and the minor vault protein p193 (p193-4) in cytospin preparations of MO, immature MO-DC, and mature (LPS) MO-DC. C, Western blot analysis of MVP (using mAb MVP-37) levels in postnuclear supernatants of control tumor cell lines, MO, and mature (LPS) MO-DC. The Journal of Immunology 1575

Table I. Up-regulation of Pgp and MVP expression during DC To verify whether, as suggested by these data, Pgp might not be development from different sources in vitroa expressed on the outer plasma cell membrane, we performed flow cytometric analysis on viable, mature DC using four different anti- MDR-Related Pgp mAbs (MRK16, UIC2, 4.17, and 4E3) directed against extra- DC Markers Molecules cellular epitopes. Indeed, no immunoreactivity with surface Pgp CD1a CD83 Pgp MVP was found on mature DC (Fig. 2B). Thus, although Pgp could be detected by immunocytochemical and flow cytometric analyses DC lineage MO ϪϪϪ Ϫ using permeabilized MO-DC (Fig. 1, A and B), no surface-exposed Immature DC Ϯ/ϩϮϪ/ϮϮ/ϩ Pgp could be detected nor could Pgp-dependent trans-plasma Mature DC ϩ/ϩϩ ϩϩ ϩ ϩϩ membrane transport activity be shown. These results confirm those ϩ CD34 cells ϪϪϪ/ϮϪ reported earlier by Randolph et al. (3), Chao et al. (27), and Lau- ϩ ϩ ϩϩ Ϫ Ϯ ϩϩϩϩ CD34 /DC / / / peze et al. (28) in showing that DC cultured from MO do not CML cells ϮϮ/ϩϪ/ϮϮ/ϩ Immature CML/DC ϩϮ/ϩϩ ϩϩ exhibit Pgp-mediated trans-plasma membrane transport, but ex- Mature CML/DC ϩϩϩϩϩ tend those data in revealing distinct intracytoplasmic Pgp expres- MDR control cell line sion. Importantly, in the former study extracellular epitopes of Pgp ϩ ϩϩ Ϫ ϩ SW-1573/2R160 ND ND / / could be detected in epidermal skin-derived DC, as collected from GLC4/ADR ND ND Ϫϩ human skin explant culture media, and were found to be function- a Data are derived from immunostaining of cytospin preparations and are repre- ally associated with their migratory capacity. This suggests that

sentative for at least three different experimental samples each. Staining intensities Downloaded from were scored according to the following scale: Ϫ, negative; Ϯ, weak; ϩ, positive; ϩϩ, Pgp can be differentially expressed in DC depending on environ- strongly positive. ND, not done. mental conditions. Intracellular Pgp expression as well as inactive forms of Pgp have also been observed in tumor cells and in lym- phocytes (29Ð31). Intracellularly, MO-DC-associated Pgp might DC, known to literally scavenge their environment for potentially still exert unknown functions in the vacuolar system, whereas on toxic materials, fulfills an evolutionary conserved defense mech- the cell surface Pgp might be expressed as a truncated product (32) anism that may also protect DC during chemotherapy. Therefore, and/or require functional activation through conformational http://www.jimmunol.org/ we evaluated the functional activity of Pgp in fully mature MO-DC changes under specific environmental conditions (33). Whether by investigating the efflux of the Pgp-transported fluorescent cy- Pgp overexpression in DC may facilitate the release of proinflam- tostatic drug DNR in the absence and presence of established Pgp matory mediators, like recently reported for another transmem- inhibitors, PSC833 and VER (Fig. 2A). Pgp-positive control SW- brane transporter molecule, i.e., multidrug resistance protein 1573/2R160 tumor cells transported the substrate DNR into the (MRP1), is still unknown (34). surrounding medium, showing no retention as reflected by a decrease in mean fluorescence intensity down to the level of unloaded cells. This efflux was inhibited by PSC833 (DFI, 2.0) as Subcellular localization of MVP in MO-DC well as VER (DFI, 1.8). Pgp-negative control SW-1573 tumor The presence and function of vaults in DC have not been investi- by guest on September 23, 2021 cells did not efflux DNR mediated by Pgp as was shown by full gated before. To find clues as to a possible role for vaults in the retention, which was not further affected by the Pgp antagonists. functional and phenotypical development of DC, the subcellular Also, in mature MO-DC, neither PSC833 nor VER affected the localization of the MVP protein was studied in cytospin prepara- mean fluorescence intensity level (DFI, 1.0). tions of MO-derived immature and mature DC with the anti-MVP

FIGURE 2. MO-DC do not exhibit Pgp-mediated transmembrane transport. A, No effect of PSC833 and VER on efflux of DNR from LPS-matured MO-DC. SW-1573 (Pgp negative) and SW-1573/2R160) (Pgp positive) were included as control cell lines. Each graph shows the flow profiles of the measured fluorescence (on a log scale) in DNR-loaded cells (filled histograms) and unloaded cells (open histograms). Representative results of five independent analyses are shown. B, Lack of surface epitope detection of Pgp by the mAbs MRK-16, UIC2, 4.17, and 4E3 on viable mature (LPS) MO-DC compared with SW-1573/2R160-positive control cells. Each graph shows the flow profile of staining (on a log scale) conducted with specific mAbs (filled histograms) and isotypic control mAb (open histograms). Representative results of two independent analyses are shown. 1576 ROLE OF VAULTS IN DC DEVELOPMENT mouse mAb LRP-56 and MVP-9 using confocal laser scan mi- tion. In contrast, MO-DC cultured with polyclonal anti-MVP croscopy (Fig. 3). Similar to what was seen by conventional im- showed reduced expression of each these markers in seven indi- munocytochemistry, a typical cytoplasmic, granular MVP staining vidual experiments as compared with the control cultures (Fig. was observed in immature DC and an even stronger MVP staining 4B). Along with this reduced expression of distinctive surface mol- in mature DC. No distinct colocalization could be observed of the ecules, the addition of polyclonal anti-MVP during MO-DC cul- MVP protein with MHCII or MHCI, distributed along the path- ture led to a reduced viability of the mature MO-DC population ways for presentation of exogenous and endogenous protein Ag, when compared with cultures with control serum (Fig. 4C; paired respectively (Fig. 3A). Thus, no direct association with Ag pre- t test, p ϭ 0.007, n ϭ 7). MO-DC cultured with the anti-MVP mAb senting routes was revealed. Furthermore, the MVP protein did not mixture also remained low in their expression of DC markers, but colocalize with anti-TGN-46, specific for the trans-Golgi network the slight decrease of viability under this condition did not reach (Fig. 3A). Clear partial colocalization was found for MVP and statistical significance (Fig. 4C; p ϭ 0.310, n ϭ 4). Culturing CD63, a lysosomal marker (Fig. 3B). Most likely, in the lysosomal MO-DC in the presence of any of the individual, single-epitope degradative compartment the MVP protein, along with many other confined, mouse anti-MVP mAb had no detectable effects on ex- proteins, is ultimately destroyed. pression of costimulatory membrane molecules or viabilities (data not shown). Inhibitory effects of anti-MVP Abs on MO-DC maturation and After subsequent experiments with dextran-FITC were unsuc- function cessful in revealing clear effects on intracellular uptake of exog- Next, during both differentiation and LPS-induced maturation, enously administered Ag (data not shown), we explored the Ag- MO-DC were cultured in the absence or continuous presence of presenting capacities of mature MO-DC, generated in the presence Downloaded from anti-MVP polyclonal or an oligoclonal mix of three different anti- of anti-MVP or control polyclonal Abs, respectively, anti-MVP, MVP mAbs or nonspecific control sera. Staining of the internal- and control oligoclonal mAb mixes. When the respective DC were ized Abs was conducted on cytospin preparations, applying only pulsed with influenza virus Ag and used to stimulate autologous T biotinylated secondary step reagent and the streptavidin-biotin- cells, highly significant suppression of resulting T cell responses peroxidase complex. In all MO-DC cultured with Abs, cytoplas- was observed. This suppression was observed for both polyclonal mic immunostaining was observed (Fig. 4A), revealing uptake of and monoclonal anti-MVP Abs and revealed by both proliferation http://www.jimmunol.org/ the administered Abs, irrespective of their specificity, through their (paired t test, p Ͻ 0.001 for anti MVP polyclonal Abs (n ϭ 7) and strong endocytotic capacity. Strikingly, the internalization of the p ϭ 0.003 (n ϭ 4) for anti-MVP monoclonal mixture; Fig. 4D) and anti-MVP polyclonal serum had a distinct effect on the phenotypic IFN-␥ release assays (paired t test, p ϭ 0.001 for anti-MVP poly- characteristics of MO-DC. When examining cytospin preparations, clonal and p ϭ 0.005 for anti-MVP monoclonal mixture; Fig. 4E). typically CD1a, CD86, CD83, and CD54 increased upon matura- Thus, in the absence of gross effects on Ag uptake as reflected by by guest on September 23, 2021

FIGURE 3. Subcellular localization of MVP in MO-DC. A, Separate subcellular distribution of MVP/vaults (g, green) and TGN, MHCII, or MHCI (r, red) in immature and mature (LPS) MO-DC. B, Partial colocalization of MVP/vaults (g) and the lysosomal marker CD63 (r) in LPS-matured MO-DC, indicated by arrows. The Journal of Immunology 1577 Downloaded from http://www.jimmunol.org/

FIGURE 4. Inhibitory effect of anti-MVP Abs on MO-DC maturation and induction of influenza virus-specific T cell responses. A, Immunocytochemical evaluation of internalized rabbit sera by antirabbit labeling only in immature and mature MO-DC. B, Immunocytochemical evaluation of MO-DC cultured in the presence of anti-MVP compared with control cultures showed reduced CD1a, CD86, CD83, and CD54 expression by mature MO-DC. C, Reduced viability (trypan blue exclusion) of mature MO-DC cultured in the presence of anti-MVP polyclonal antiserum (n ϭ 7) and mAbs (n ϭ 4) as compared by guest on September 23, 2021 with control cultures containing irrelevant control Abs. D, Result in C is reflected in concomitant reduced percentage of influenza virus-Ag-induced T cell proliferation by mature MO-DC cultured in the presence of anti-MVP polyclonal and monoclonal antisera as compared with control cultures containing irrelevant Abs (control median Ag-specific T cell proliferation index, 3.4, n ϭ 7 and n ϭ 4, respectively), as well as (in E) in reduced IFN-␥ production (control median Ag-specific IFN-␥ release, 165 pg/ml) under these conditions.

intracellular dextran-FITC staining, the latter results support the this involves a nucleo-cytoplasmic shuttle of signal transduction view that intracellular targeting of vault particles with Abs binding (7, 14) or association with microtubular functions (9, 11) remains the major vault protein interferes with appropriate handling and to be seen. The present findings point to a role of vaults in DC presentation of exogenously administered Ag. Interestingly, the functioning, more specifically in their capacity to induce Ag-spe- lack of significant effects with the individual mAbs argues against cific T cell proliferative and IFN-␥ release responses. Obviously, interference with functionally relevant epitopes. Most likely, there- future studies should also clarify whether vault particles might also fore, this blockade results from events secondary to this binding, play roles in other DC functions, e.g., migratory behavior and CTL e.g., from aggregation of vault particles. activation. To further address all of these questions, we are cur- rently developing MVP gene knockout mice. Concluding remarks Taken together, we propose that vaults act as components in the Acknowledgments cascade of events regulating DC effector function. Since their dis- We thank Guus Rimmelzwaan for providing influenza viral Ag, Sinead covery, the occurrence and abundance of vaults in eukaryotic cells Lougheed for technical assistance, and Jacques Neefjes and Mar Fernandes have argued for an important general function. From studies in the Borja for technical assistance and helpful advice regarding the confocal laser scan microscopy experiments, and for useful comments on this slime mold Dictyostelium discoideum, it is known that disruption manuscript. of MVP results in a phenotype incapable of growing under nutri- tional stress (35). From studies in tumor cell lines, vaults have References been implied in drug resistance (12, 15, 16, 36, 37). The present 1. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, findings first demonstrate that, even more so than Pgp, vault par- B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu. ticles are increased during the differentiation of DC and that their Rev. Immunol. 18:767. 2. Germann, U. A. 1996. P-glycoprotein: a mediator of multidrug resistance in numbers are further increased upon maturation. An attractive hy- tumour cells. Eur. J. Cancer 32A:927. pothesis is that this reflects a fundamental response to stress sig- 3. Randolph, G. J., S. Beaulieu, M. Pope, I. Sugawara, L. Hoffman, R. M. Steinman, and W. A. Muller. 1998. A physiologic function for p-glycoprotein (MDR-1) nals and that in mature DC vaults may serve as protection of their during the migration of dendritic cells from skin via afferent lymphatic vessels. integrity during migration to the draining lymph node. Whether Proc. Natl. Acad. Sci. USA 95:6924. 1578 ROLE OF VAULTS IN DC DEVELOPMENT

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