Ligation of MHC Class II Induces PKC-Dependent Clathrin-Mediated Endocytosis of MHC Class II

cells

Article Ligation of MHC Class II Induces PKC-Dependent Clathrin-Mediated Endocytosis of MHC Class II

1, 1, 2 1 3 Kento Masaki †, Yuhji Hiraki †, Hiroka Onishi , Yuka Satoh , Paul A. Roche , Satoshi Tanaka 4 and Kazuyuki Furuta 1,* 1 Department of Immunobiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Tsushima naka 1-1-1, Kita-ku, Okayama 700-8530, Japan; [email protected] (K.M.); [email protected] (Y.H.); [email protected] (Y.S.) 2 Department of Immunobiology, Faculty of Pharmacy and Pharmaceutical Sciences, Okayama University, Tsushima naka 1-1-1, Kita-ku, Okayama 700-8530, Japan; [email protected] 3 Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] 4 Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Misasagi Nakauchi-cho 5, Yamashina-ku, Kyoto 607-8414, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-86-251-7962 These authors have contributed equally to this work. † Received: 30 June 2020; Accepted: 28 July 2020; Published: 30 July 2020

Abstract:InadditiontoantigenpresentationtoCD4+ Tcells,aggregationofcellsurfacemajorhistocompatibility complex class II (MHC-II) molecules induces signal transduction in antigen presenting cells that regulate cellular functions. We previously reported that crosslinking of MHC-II induced the endocytosis of MHC-II, which was associated with decreased surface expression levels in murine dendritic cells (DCs) and resulted in impaired activation of CD4+ T cells. However, the downstream signal that induces MHC-II endocytosis remains to be elucidated. In this study, we found that the crosslinking of MHC-II induced intracellular Ca2+ mobilization, which was necessary for crosslinking-induced MHC-II endocytosis. We also found that these events were suppressed by inhibitors of Syk and phospholipase C (PLC). Treatments with a phorbol ester promoted MHC-II endocytosis, whereas inhibitors of protein kinase C (PKC) suppressed crosslinking-induced endocytosis of MHC-II. These results suggest that PKC could be involved in this process. Furthermore, crosslinking-induced MHC-II endocytosis was suppressed by inhibitors of clathrin-dependent endocytosis. Our results indicate that the crosslinking of MHC-II could stimulate Ca2+ mobilization and induce the clathrin-dependent endocytosis of MHC-II in murine DCs.

Keywords: MHC-II; dendritic cells; endocytosis; crosslink; PKC

1. Introduction Dendritic cells (DCs) are potent antigen presenting cells (APCs) widely distributed in peripheral tissues. They capture antigens, such as invaded pathogens, and present the antigen-derived peptides with major histocompatibility antigen class II (MHC-II) molecules on the cell surface. Antigen-specific CD4+ T cells recognize the antigen peptide-MHC-II complex (pMHC-II) by T cell receptors (TCRs), which leads to their proliferation and differentiation into effector T cells that initiate an adaptive immune response to the pathogens [1–5]. In addition to their role in antigen presentation, substantial evidence suggests that an aggregation of cell surface MHC-II induces signaling inside the cells by regulating the functioning of APCs, including DCs and B cells [6]. One of the features triggered by MHC-II-induced signaling is the suppression of the potential of antigen presentation in murine bone marrow-derived DCs (BMDCs). MHC-II crosslinking

Cells 2020, 9, 1810; doi:10.3390/cells9081810 www.mdpi.com/journal/cells Cells 2020, 9, 1810 2 of 12 was reported to suppress LPS-induced activation of BMDCs [7]. In addition, we previously reported that crosslinking of MHC-II induces endocytosis and decreases the expression levels of cell surface MHC-II, which results in an attenuation of the potential of antigen presentation [8]. However, the signal transduction and molecular mechanisms that induce endocytosis through MHC-II remain unclear. Clathrin-dependent endocytosis is one of the pathways through which proteins are transported from the cell surface to intracellular compartments by clathrin-coated vesicles. During this process, the GTPase dynamin is required for the endocytic membrane scission process. Many receptors are internalized through clathrin-dependent and dynamin-dependent endocytosis pathways after ligand binding [9]. On the other hand, some cell surface proteins are internalized by clathrin-independent endocytosis pathways [10,11]. In steady-state DCs, it has been reported that pMHC-II is internalized by clathrin-independent and dynamin-independent pathways [12]. However, it is unclear whether crosslinking-induced endocytosis of MHC-II is attributed to increased steady-state clathrin-independent endocytosis or the induction of an alternative endocytosis route. In this study, we aimed to investigate the signal transduction pathways involved in crosslinking- induced MHC-II endocytosis in BMDCs.

2. Materials and Methods

2.1. Materials The following materials were obtained from the sources indicated: anti-mouse MHC-II antibody (clone 11-5.2) from Biolegend (San Diego, CA, USA); 12-O-tetradecanoyl phorbol 13-acetate (TPA), cytochalasin D, dynasore, chlorpromazine, staurosporine, and piceatannol from Sigma-Aldrich (St. Louis, MO, USA), Fura-2/AM from Dojindo (Kumamoto, Japan), 1,2-bis(o-aminophenoxy)ethane- N,N,N0,N0- tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), U-73122, PD98059, SB203580, and SP600125 from Merck Millipore (Darmstadt, Germany), GF 109203X and R406 from Cayman Chemical (Ann Arbor, MI, USA), recombinant murine granulocyte macrophage colony-stimulating factor (GM-CSF) from Peprotech (Rocky Hill, NJ, USA), anti-FcγRII/RIII (clone 2.4G2) antibody and anti-clathrin heavy chain (CHC) antibody from BD Biosciences (San Jose, CA, USA), Alexa Fluor 488-labeled goat anti-mouse IgG1 antibody and Alexa Fluor 546-labeled goat anti-mouse IgG2b antibody from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were commercial products of a reagent grade.

2.2. Mice Speciﬁc-pathogen-free, male B10.BR mice (H-2k) were obtained from Japan SLC (Hamamatsu, Japan), and all mice were kept in a speciﬁc-pathogen-free animal facility at Okayama University. The Committee on Animal Experiments of Okayama University (approval numbers OKU-2015042 (date of approval: 1 April 2015) and OKU-2018087 (date of approval: 1 April 2018)) approved this study.

2.3. Preparation of BMDCs BMDCs were prepared following the standard protocol [13]. Brieﬂy, bone marrow cells obtained from B10.BR mice were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 2-mercaptoethanol (50 µM), penicillin (100 units/mL), and streptomycin (100 µg/mL) in the presence of 10 ng/mL GM-CSF for 7 days. More than 90% of the obtained cells were CD11c+ and MHC-II+ at day 7.

2.4. Crosslinking of Cell Surface MHC-II BMDCs were incubated with an anti-MHC-II antibody (clone 11-5.2, 5 µg/mL) in the presence of an anti-FcγRII/RIII antibody (clone 2.4G2) in FACS staining medium (phosphate-buffered saline (PBS) ( ) containing 2% FBS) for 30 min on ice. The cells were washed and surface MHC-II molecules − were crosslinked with a biotinylated goat anti-mouse IgG antibody (5 µg/mL) in RPMI-1640 medium containing 10% FBS, 2-mercaptoethanol (50 µM), penicillin (100 units/mL), and streptomycin (100 µg/mL) for 30 min at 37 ◦C. After washing with FACS staining medium, the cells were incubated with PE-labeled Cells 2020, 9, 1810 3 of 12 streptavidin for 30 min on ice for detection of the remaining cell surface MHC-II. The fluorescence intensity was measured using a flow cytometer (FACS Calibur, Becton Dickinson, Franklin Lakes, NJ, and Gallios, Beckman Coulter, Brea, CA, USA). The results were presented as a percentage of the fluorescence intensity of the cells placed on ice.

2.5. Measurement of Cytosolic Ca2+ Concentrations Cells were loaded with 6 µM Fura-2/AM in modified Tyrode’s buffer (10 mM HEPES-NaOH, pH 7.3, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% bovine serum albumin (BSA)) containing 2.5 mM probenecid for 45 min at room temperature. Fluorescence intensities were measured with a fluorescence spectrometer (CAF-110, Jasco, Tokyo, Japan) with an excitation wavelength of 340 or 380 nm and an emission wavelength of 510 nm. Cytosolic Ca2+ concentration was expressed as the 340 nm/380 nm ratio.

2.6. Immunofluorescence Microscopy Immunofluorescence microscopy analyses were performed as described previously [14]. BMDCs were seeded on poly-l-lysine coated glass cover slips and allowed to adhere for 30 min at room temperature. The cells were incubated with an anti-MHC-II antibody (clone 11-5.2, 5 µg/mL) for 30 min at 4 ◦C. The cells were washed and then crosslinked with an Alexa Fluor 546-labeled anti-mouse IgG2b antibody (5 µg/mL) for the periods indicated at 37 ◦C. The cells were fixed with 4% paraformaldehyde in PBS for 20 min at 4 ◦C and permeabilized with 0.1% saponin in PBS at room temperature. After the cells were blocked with 2% normal goat serum for 1 h at room temperature, the cells were stained with an anti-CHC antibody for 1 h at 4 ◦C. The cells were washed and stained with an Alexa Fluor 488-labeled anti-mouse IgG1 antibody as the secondary antibody. The cells were visualized using confocal laser scanning microscopy (OLYMPUS FV300 and FV1200, Tokyo, Japan). For quantitative fluorescence analysis, Manders’ overlay coefficients were calculated using ImageJ software with the Coloc2 plugin, using at least 10 cells in each analysis [15].

2.7. Statistical Analysis Data are presented as the mean SEM. Statistical signiﬁcance for comparisons between the two ± groups was determined using the Student’s t-test. Statistical signiﬁcance for comparisons among multiple groups was determined using one-way ANOVA. Additional comparisons were made using Dunnett’s Multiple Comparison test to compare with the control groups, or the Tukey-Kramer Multiple Comparison test for all pairs of column comparisons.

3. Results

3.1. Crosslinking-Induced Intracellular Ca2+ Mobilization is Required for Endocytosis of MHC-II As previously reported [8], MHC-II was spontaneously internalized, and crosslinking of MHC-II enhanced a down-regulation of cell surface MHC-II expression in BMDCs (Figure1A). In this case, we investigated the signaling mechanisms involved in the endocytosis of MHC-II. Since MHC-II crosslinking was reported to induce intracellular Ca2+ mobilization in B cells [16–18], we measured the cytosolic Ca2+ levels after MHC-II crosslinking in BMDCs and found that MHC-II crosslinking induced Ca2+ mobilization (Figure1B). We then examined the e ffects of cytosolic Ca2+ depletion in spontaneous and crosslinking-induced endocytosis of MHC-II using a Ca2+ chelator, BAPTA-AM. While BAPTA-AM treatment did not affect the spontaneous (crosslink ( )) endocytosis of MHC-II, it − inhibited the crosslinking-induced endocytosis of MHC-II (Figure1C). We confirmed that BAPTA-AM treatment did not affect the distribution of MHC-II expression in BMDCs (Figure S1). These results indicate that intracellular Ca2+ mobilization is required for crosslinking-induced endocytosis of MHC-II. CellsCells 20 202020, 9, ,9 x, xFOR FOR PEER PEER REVIEW REVIEW 4 4of of 12 12 resultsCellsresults2020 indicate ,indicate9, 1810 that that intracellular intracellular Ca Ca2+2+ mobilization mobilization is is required required for for crosslinking crosslinking-induced-induced endocytosis endocytosis4 of 12 ofof MHC MHC-II.-II.

2+ FigureFigureFigure 1. 1. IntracelIntracellular Intracellularlular Ca Ca2+2+ mobilization mobilizationmobilization is isis required requiredrequired for forfor crosslinking crosslinking-inducedcrosslinking-induced-induced endocytosis endocytosisendocytosis of ofof major major histocompatibilityhistocompatibilityhistocompatibility complex complex class class II II ( (MHC-II). MHC(MHC-II-II).) (. ( A(A)) )Bone Bone Bone marrow marrow-derived marrow-derived-derived dendritic dendritic cell cells cells s( (BMDCs) BMDCs(BMDCs) )were were were incubatedincubatedincubated withwith with anan an anti-MHC-IIanti anti-MHC-MHC-II- antibodyII antibody antibody for for for 30 30 min30 min min at at 4 at ◦4C. 4 ° C.° ForC. For For crosslinking, crosslinking, crosslinking, the the cellthe cell surfacecell surface surface MHC-II MHC MHC was-II-II wasincubatedwas incubated incubated with with awith biotin-labeled a a biotin biotin-labeled-labeled anti-mouse anti anti-mouse-mouse IgG antibody IgG IgG antibody antibody for 30 minfor for 30 at30 min 37min◦ C.at at The37 37 ° C.° remainingC. The The remaining remaining cell surface cell cell surfaceMHC-IIsurface MHC wasMHC detected-II-II was was detected usingdetected PE-labeled using using PE PE streptavidin.-labeled-labeled streptavidin. streptavidin. (B) BMDCs ( B( wereB) )BMDCs BMDCs crosslinked were were crosslinked with crosslinked an anti-MHC-II with with an an 2+ antiantibodyanti-MHC-MHC and-II-II anantibodyantibody anti-mouse andand IgG anan antibody. antianti-mouse-mouse The cytosolicIgGIgG antibody.antibody. Ca mobilization TheThe cytosoliccytosolic after Ca stimulationCa2+2+ mobilization mobilization was detected afterafter 2+ stimulationusingstimulation Fura-2 was /AM.was detected Barsdetected= 1 min.usingusing Relative Fura Fura-2/AM.-2/AM. changes Bars Bars in = the= 1 1 cytosolicmin. min. Relative Relative Ca concentrationschanges changes in in the the are cytosolic cytosolic expressed Ca Ca as2+2+ concentrationstheconcentrations ratio of fluorescence are are expressed expressed intensity as as atthe the 340 ratio ratio/380 of nm.of fluorescence fluorescence (C) BMDCs intensity wereintensity pretreated at at 340/380 340/380 with nm. vehiclenm. ( C(C) (0.1%)BMDCs BMDCs dimethyl w wereere µ pretreatedsulfoxidepretreated (DMSO)) with with vehicle vehicle or BAPTA-AM (0.1% (0.1% dimethyl dimethyl (50 M) sulfoxide sulfoxide for 30 min ( DMSO(DMSO at 37 )◦))C. )or or The BAPTA BAPTA cell surface-AM-AM (50 (50 MHC-II μ μM)M) for wasfor 30 30 crosslinked min min at at 37 37 with an anti-MHC-II antibody for 30 min. The remaining cell surface MHC-II was detected by flow °C.°C. The The cell cell surface surface MHC MHC-II-II was was crosslinked crosslinked with with an an anti anti-MHC-MHC-II-II antibody antibody for for 30 30 min min. The. The remaining remaining cytometry. Values of ** p < 0.01 are regarded as significant. cellcell surface surface MHC MHC-II-II was was detected detected by by flow flow cytometry cytometry. .Values Values of of ** ** p p < < 0.01 0.01 are are regarded regarded as as significant. significant. 3.2. Intracellular Ca2+ Mobilization Induces Endocytosis of MHC-II 3.2.3.2. Intracellular Intracellular Ca Ca2+2+ Mobilization Mobilization Induces Induces Endocytosis Endocytosis of of MHC MHC-II-II Next, we investigated whether intracellular Ca2+ mobilization accelerates the endocytosis of MHC-II Next, we investigated whether intracellular Ca2+2+ mobilization accelerates the endocytosis of by examiningNext, we theinvestigated effects of awhether reagent intracellular that induces Ca intracellular mobilization Ca2+ mobilization.accelerates the An endocytosis endoplasmic of MHC-II by examining the effects of a reagent that induces intracellular Ca2+2+ mobilization. An MHCreticulum-II by calcium examining ATPase the inhibitor,effects of thapsigargin, a reagent that is knowninduces to intracellular induce Ca2 +Camobilization mobilization. in many An endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, is known to induce Ca2+2+ mobilization endoplasmiccell types and reticulum we confirmed calcium that ATPase thapsigargin inhibitor, induced thapsigargin, intracellular is known Ca to2+ inducemobilization Ca mobilization in BMDCs in many cell types and we confirmed that thapsigargin induced intracellular Ca2+2+ mobilization in in(Figure many2 A).cell We types then and investigated we confirmed the e thatffects thapsigargin of thapsigargin induced on MHC-II intracellular endocytosis Ca mobilization and found that in BMDCs (Figure 2A). We then investigated the effects of thapsigargin on MHC-II endocytosis and BMDCsthapsigargin (Figure treatment 2A). We induced then investigated significant levelsthe effects of endocytosis of thapsigargin of MHC-II on MHC (Figure-II2 B).endocytosis These results and found that thapsigargin treatment induced significant levels of endocytosis of MHC-II (Figure 2B). foundsuggest that that thapsigargin intracellular treatment Ca2+ mobilization induced significant can induce levels MHC-II of endocytosis.endocytosis of MHC-II (Figure 2B). TheseThese results results suggest suggest that that intracellular intracellular Ca Ca2+2+ mobilization mobilization can can induce induce MHC MHC-II-II endocytosis. endocytosis.

2+ FigureFigureFigure 2. 2. 2. IntracellularIntracellular Intracellular Ca Ca Ca2+2+ mobilization mobilization induces induces endocytosis endocytosisendocytosis of ofof MHC MHC-II.MHC-II.-II. ( (A(AA)) )BMDCs BMDCsBMDCs were werewere stimulated stimulated stimulated 2+ withwith or or without without thapsigargin thapsigargin and and cytosolic cytosolic CaCa Ca2+2+ mmobilization mobilizationobilization was waswas detected detecteddetected using using Fura Fura-2 Fura-2/AM.-2/AM./AM. Bars Bars = = 11 1 2+ min.min.min. Relative RelativeRelative changes changeschanges in inin the the cytosolic cytosolic Ca Ca2+2+ concentrations concentrationsconcentrations are areare expressed expressedexpressed as asas the thethe ratio ratioratio of ofof fluorescence ﬂuorescence fluorescence intensityintensityintensity at at at 340/380 340 340/380/380 nm. nm. (( B(BB)) )BMDCs BMDCsBMDCs were werewere incubated incubatedincubated with with with an an an anti anti-MHC-IIanti-MHC-MHC-II-II antibody antibodyantibody for for for 30 30 30 min minmin at atat 4 44 °◦ C.°C.C. TheThe cells cells werewere were washed washed washed and and and stimulated stimulated stimulated with with with thapsigargin thapsigargin thapsigargin for 30for for min 30 30 atmin min 37 ◦at C.at 37 The37 ° C.° remainingC. The The remaining remaining cell surface cell cell surfaceanti-MHC-IIsurface anti anti-MHC- antibodyMHC-II-II antibody antibody was detected was was detected detected using a using PE-labeledusing a a PE PE-labeled- anti-mouselabeled anti anti- IgGmouse-mouse antibody. IgG IgG antibody. antibody. Values Values of Values * p < of0.05, of * p* p <**< 0.05, p0.05,< 0.01 ** **pp

3.3.3.3. Crosslinking Crosslinking-Induced-Induced Endocytosis Endocytosis of of MHC MHC-II-II is is Syk Syk and and P Phospholipasehospholipase C C ( PLC(PLC)-)Dependent-Dependent

Cells 2020, 9, 1810 5 of 12

CellsCells 20 202020, ,9 9, ,x x FOR FOR PEER PEER REVIEW REVIEW 55 ofof 1212 3.3. Crosslinking-Induced Endocytosis of MHC-II is Syk and Phospholipase C (PLC)-Dependent ActivationActivation ofof anan immunoreceptorimmunoreceptor tyrosinetyrosine-based-based activationactivation motifmotif (ITAM(ITAM) ) containingcontaining CC-type-type Activation of an immunoreceptor tyrosine-based activation motif (ITAM) containing C-type lectin lectinlectin receptor, receptor, Dectin Dectin-1,-1, was was found found to to induce induce intracellular intracellular Ca Ca2+2+ mobilization, mobilization, which which was was mediated mediated by by receptor, Dectin-1, was found to induce intracellular Ca2+ mobilization, which was mediated by Syk SykSyk tyrosinetyrosine kinasekinase-mediated-mediated PLCγPLCγ activation,activation, inin BMDCsBMDCs [19].[19]. ItIt waswas alsoalso reportedreported thatthat MHCMHC-II-II tyrosine kinase-mediated PLCγ activation, in BMDCs [19]. It was also reported that MHC-II interacted interactedinteracted withwith thethe ITAMITAM-containing-containing adaptadaptoror protein, protein, FcγRγ, FcγRγ, in in BMDCs, BMDCs, and and that that MHC MHC-II-II with the ITAM-containing adaptor protein, FcγRγ, in BMDCs, and that MHC-II crosslinking induces crosslinkingcrosslinking induces induces tyrosine tyrosine phosphorylation phosphorylation of of cellular cellular proteins proteins [7]. [7]. Therefore, Therefore, w wee examined examined the the tyrosine phosphorylation of cellular proteins [7]. Therefore, we examined the contribution of Syk and contributioncontribution ofof SykSyk andand phospholipasephospholipase CC ((PLCPLC) ) usingusing inhibitorsinhibitors againstagainst thesethese molecules.molecules. phospholipase C (PLC) using inhibitors against these molecules. Crosslinking-induced endocytosis of CrosslinkingCrosslinking--inducedinduced endendocytosisocytosis ofof MHCMHC-II-II waswas suppressedsuppressed byby SykSyk inhibitors,inhibitors, (piceatannol(piceatannol andand MHC-II was suppressed by Syk inhibitors, (piceatannol and R406), and a PLC inhibitor (U73122) (Figure3). R406),R406), andand aa PLCPLC inhibitorinhibitor (U73122(U73122)) (Figure(Figure 3).3). WeWe thenthen examinedexamined thethe effecteffect ofof piceatannolpiceatannol andand We then examined the effect of piceatannol and U73122 on intracellular Ca2+ mobilization, and found U73122U73122 onon intracellularintracellular CaCa2+2+ mobilization,mobilization, andand foundfound thatthat bothboth inhibitorsinhibitors suppressedsuppressed CaCa2+2+ that both inhibitors suppressed Ca2+ mobilization induced by MHC-II crosslinking (Figure4A,B). These mmobilizationobilization induced induced by by MHC MHC-II-II crosslinking crosslinking ( (FigureFigure 4A 4A,B,B).). These These results results suggest suggest that that Syk Syk and and PLC PLC results suggest that Syk and PLC are potentially involved in intracellular Ca2+ mobilization and MHC-II areare potentiallypotentially involvedinvolved inin intracellularintracellular CaCa2+2+ mobilizationmobilization andand MHCMHC-II-II endocytosisendocytosis inducedinduced byby endocytosis induced by crosslinking of MHC-II. crosslinkingcrosslinking of of MHC MHC-II-II. .

FigureFigureFigure 3. 3.3.Effects EffectEffect ofss Syk ofof Syk andSyk phospholipaseandand phospholipasephospholipase C (PLC) CCinhibitors (PL(PLCC) ) inhibitorsinhibitors on crosslinking-induced onon ccrosslinkingrosslinking MHC-II-induced-induced endocytosis. MHCMHC-II-II BMDCsendocytosis.endocytosis. were pre-incubated BMDCsBMDCs werewere with pre (preA)-incubated piceatannol-incubated (50withwithµM), (A(A ()B ) )piceatannol R406piceatannol (indicated (50(50 concentrations), μμM)M), , (B(B) ) R406R406or (C (indicated)(indicated U73122 (10concentrations),concentrations),µM) for 30 min or or at ( C( 37C) )U73122◦ U73122C. The cell(10 (10 μ surfaceμM)M) for for MHC-II30 30 min min at wasat 37 37 crosslinked ° °C.C. The The cell cell with surface surface an anti-MHC-II MHC MHC-II-II was was antibody crosslinked crosslinked for 30withwith min. a an Then anti anti remaining-MHC-MHC-II-II cellantibody antibody surface for for MHC-II 30 30 min min was. .The The detected remaining remaining by flow cell cell cytometry. surface surface MHC MHC Values-II-II ofwas was * p

2+ FigureFigureFigure 4. 4.4.Effects EffectEffect ofss Syk ofof andSykSyk PLC andand inhibitors PLPLCC inhibitorsinhibitors on MHC-II onon crosslinking-induced MHCMHC-II-II crosslinkingcrosslinking intracellular-induced-induced Ca intracellularintracellularmobilization. CaCa2+2+ BMDCsmobilization.mobilization. were pre-incubatedBMDCs BMDCs were were withpre pre-incubated-incubated (A) piceatannol with with ( A( (50A) )piceatannol piceatannolµM) or (B) (50 U73122(50 μ μM)M) (10or or ( µB(BM)) )U73122 U73122 for 30 (10 min(10 μ μM) atM) 37 for for◦ C.30 30 2+ Theminmin cytosolic at at 37 37 ° °C. CaC. The Themobilization cytosolic cytosolic Ca afterCa2+2+ mobilization MHC-IImobilization crosslinking after after MHC MHC was measured-II-II crosslinking crosslinking using Fura-2was was measured /measuredAM. Relative using using changes Fura Fura-- 2+ in2/AM2/AM the cytosolic. .Relative Relative Cachanges changesconcentrations in in the the cytosolic cytosolic are expressed Ca Ca2+2+ concentrations concentrations as the ratio are are of expressed fluorescenceexpressed as as intensitythe the ratio ratio of atof fluorescence 340fluorescence/380 nm. Barsintensityintensity= 1 min. at at 340/380 340/380 Values nm. ofnm. * Barsp Bars< 0.05, = = 1 1 min. **min.p

Cells 2020, 9, 1810 6 of 12 suppressed crosslinking-induced MHC-II endocytosis (Figure5B,C). These results suggest that Ca 2+- dependentCells 2020, 9, PKCx FOR activationPEER REVIEW is involved in MHC-II endocytosis. 6 of 12 Activation of mitogen-activated protein (MAP) kinases is required for the endocytosis of several cell Activation of mitogen-activated protein (MAP) kinases is required for the endocytosis of several surface receptors such as epidermal growth factor receptor (EGFR) [20]. Furthermore, MHC-II-induced cell surface receptors such as epidermal growth factor receptor (EGFR) [20]. Furthermore, MHC-II- signaling has been reported to activate ERK, which is one of the MAP kinases, in BMDCs [7]. Therefore, induced signaling has been reported to activate ERK, which is one of the MAP kinases, in BMDCs we investigated the eﬀect of MAP kinase inhibitors on MHC-II endocytosis. Pretreatment with PD98059 [7]. Therefore, we investigated the effect of MAP kinase inhibitors on MHC-II endocytosis. (a MEK inhibitor), SB203580 (a p38 MAPK inhibitor), or SP600125 (a JNK inhibitor) did not aﬀect Pretreatment with PD98059 (a MEK inhibitor), SB203580 (a p38 MAPK inhibitor), or SP600125 (a JNK crosslinking-induced MHC-II endocytosis (Figure5D–F), which suggests that MAP kinases are not inhibitor) did not affect crosslinking-induced MHC-II endocytosis (Figure 5D–F), which suggests that involved in this process. MAP kinases are not involved in this process.

FigureFigure 5.5. Protein kinasekinase CC (PKC)(PKC) activationactivation inducesinduces MHC-IIMHC-II endocytosis.endocytosis. ((AA)) BMDCsBMDCs werewere incubatedincubated withwith anan anti-MHC-IIanti-MHC-II antibodyantibody forfor 3030 minmin atat 44 ◦°C.C. TheThe cellscells were washedwashed and stimulated with 12-O-12-O- tetradecanoyltetradecanoyl phorbol phorbol 13-acetate 13-acetate (TPA) (TPA (100) nM)(100 andnM) A23187 and A23187 (1 µM) for (130 μ minM) for at 37 30◦C. min The at remaining 37 °C. The cell surfaceremaining anti-MHC-II cell surface antibody anti-MHC was detected-II antibody using was a PE-labeled detected anti-mouse using a PE IgG-label antibody.ed anti (B-,mouseC) BMDCs IgG wereantibody. pre-incubated (B, C) BMDCs with staurosporine were pre-incubated for 15 min with orGF109203X staurosporine for30 for min 15 atmin the or indicated GF109203X concentrations for 30 min ofat 37the◦ C.indicated The cell concentrations surface MHC-II of was 37 °C. crosslinked The cell surface using an MHC anti-MHC-II-II was crosslinked antibody. using (D–F) an BMDCs anti-MHC were- pre-incubatedII antibody. (D with–F) SB203580BMDCs were (10 µ preM)- forincubated 30 min, with PD98059 SB203580 (50 µM) (10 for µM) 15 for min, 30 or min, SP600125 PD98059 (20 µ(50M) µM) for 15for min, 15 min, at 37 or◦ C.SP600125 The cell (20 surface µM) MHC-IIfor 15 min, was at crosslinked 37 °C. The usingcell surface an anti-MHC-II MHC-II was antibody crosslinked for 30 using min. Thean anti remaining-MHC- cellII antibody surface MHC-IIfor 30 wasmin.detected The remaining by flow cytometry.cell surface Values MHC of-II * wasp < 0.05, detected ** p < by0.01 flow are regardedcytometry as. Values significant. of * p < 0.05, ** p < 0.01 are regarded as significant. 3.5. Crosslinking-Induced Endocytosis of MHC-II is a Clathrin-Dependent Response 3.5. Crosslinking-Induced Endocytosis of MHC-II is a Clathrin-Dependent Response Next, we investigated the endocytosis pathway of MHC-II upon its crosslinking. Cytochalasin Next, we investigated the endocytosis pathway of MHC-II upon its crosslinking. Cytochalasin D, which inhibits several endocytosis pathways by inhibiting actin polymerization, suppressed D, which inhibits several endocytosis pathways by inhibiting actin polymerization, suppressed crosslinking-induced MHC-II endocytosis (Figure6). In addition, a dynamin inhibitor, dynasore, and a crosslinking-induced MHC-II endocytosis (Figure 6). In addition, a dynamin inhibitor, dynasore, and clathrin-mediated endocytosis inhibitor, chlorpromazine, suppressed crosslinking-induced MHC-II a clathrin-mediated endocytosis inhibitor, chlorpromazine, suppressed crosslinking-induced MHC- endocytosis (Figure6). This result suggests that crosslinking-induced MHC-II endocytosis is dependent II endocytosis (Figure 6). This result suggests that crosslinking-induced MHC-II endocytosis is on both dynamin and clathrin. dependent on both dynamin and clathrin. Clathrin-dependent endocytosis requires a transient interaction between clathrin and the target Clathrin-dependent endocytosis requires a transient interaction between clathrin and the target protein. The degree of co-localization between internalized MHC-II and the clathrin heavy chain (CHC) protein. The degree of co-localization between internalized MHC-II and the clathrin heavy chain was quantiﬁed by calculating Manders’ overlap coeﬃcient. Increases in co-localization of MHC-II (CHC) was quantified by calculating Manders’ overlap coefficient. Increases in co-localization of with CHC just beneath the cell surface were observed 2 and 5 min after crosslinking. The majority MHC-II with CHC just beneath the cell surface were observed 2 and 5 min after crosslinking. The majority of the MHC-II signal was found inside the cells 10 min after crosslinking and did not overlap with those of CHC (Figure 7A). In agreement with a previous study [21,22], chlorpromazine treatment prevented redistribution of CHC (Figure 7B). In the cells treated with chlorpromazine, most of the MHC-II remained at the cell surface for 2, 5, and 10 min after crosslinking and co-

Cells 2020, 9, 1810 7 of 12 of the MHC-II signal was found inside the cells 10 min after crosslinking and did not overlap with those of CHC (Figure7A). In agreement with a previous study [ 21,22], chlorpromazine treatment preventedCells 2020, 9, x redistribution FOR PEER REVIEW ofCHC (Figure7B). In the cells treated with chlorpromazine, most of7 theof 12 Cells 2020, 9, x FOR PEER REVIEW 7 of 12 MHC-II remained at the cell surface for 2, 5, and 10 min after crosslinking and co-localization of MHC-II withlocalization CHC was of hardlyMHC- observed.II with CHC These was results hardly indicate observed. that crosslinking-inducedThese results indicate MHC-II that crosslinking endocytosis- localization of MHC-II with CHC was hardly observed. These results indicate that crosslinking- isinduced clathrin-dependent. MHC-II endocytosis We then is investigatedclathrin-dependent. the eﬀects We ofthen inhibitors investigated against the crosslinking-induced effects of inhibitors induced MHC-II endocytosis is clathrin-dependent. We then investigated the effects of inhibitors signalingagainst crosslinking on co-localization-induced of signaling MHC-II and on co clathrin-localization after crosslinking. of MHC-II and Similar clathrin to the after result crosslinking. presented against crosslinking-induced signaling on co-localization of MHC-II and clathrin after crosslinking. inSimilar Figure to7 ,the an increaseresult presented in co-localization in Figure of7, an MHC-II increase and in clathrin co-localization was observed of MHC after-II and crosslinking clathrin was for Similar to the result presented in Figure 7, an increase in co-localization of MHC-II and clathrin was 2observed min in control-pretreated after crosslinking for cells 2 (Figuremin in control8A). When-pretreated the BMDCs cells ( wereFigure pretreated 8A). When with the BAPTA-AM,BMDCs were observed after crosslinking for 2 min in control-pretreated cells (Figure 8A). When the BMDCs were R406,pretreated and U73122, with BAPTA the co-localizations-AM, R406, and of U73122, MHC-II andthe co clathrin-localizations was not of enhanced MHC-II (Figureand clathrin8B–D). was These not pretreated with BAPTA-AM, R406, and U73122, the co-localizations of MHC-II and clathrin was not resultsenhanced suggested (Figure thats 8B MHC-II–8D). These crosslinking-induced results suggested signaling that MHC induces-II crosslinking the co-localization-induced of sig MHC-IInaling enhancedinduces the (Figure co-localizations 8B–8D). of These MHC -resultsII and clathrin.suggested that MHC-II crosslinking-induced signaling andinduces clathrin. the co-localization of MHC-II and clathrin.

Figure 6. Effect of endocytosis inhibitors on crosslinking-induced MHC-II endocytosis. BMDCs were Figurepre-incubated 6. Effect with of endocytosis cytochalasin inhibitors D (3 µM), on dynasore crosslinking-inducedcrosslinking (100- inducedµM), or chlorpromazineMHC-IIMHC-II endocytosis.endocytosis. (30 µM), BMDCs for 30 were min pre-incubatedpreat 37-incubated °C. Thewith withcell cytochalasinsurface cytochalasin MHC D D-II (3 (3 wasµ µM),M), crosslinked dynasore dynasore (100 (100withµ M),µM), an oranti or chlorpromazine chlorpromazine-MHC-II antibody (30 (30µ M), forµM), for30 for 30min 30 min. miTheatn at 37 °C. The cell surface MHC-II was crosslinked with an anti-MHC-II antibody for 30 min. The 37remaining◦C. Thecell cell surface surface MHC-II MHC-II was was crosslinked detected by with flow an anti-MHC-IIcytometry. Values antibody of for* p 30< 0.05 min. are The regarded remaining as p cellremainingsignificant. surface ce MHC-IIll surface was MHC detected-II was by flowdetected cytometry. by flow Values cytometry of * p.< Values0.05 are of regarded* < 0.05 as are significant. regarded as significant.

Figure 7. Crosslinking-induced endocytosis of MHC-II is clathrin-dependent. (A) BMDCs were Figure 7.7.Crosslinking-induced Crosslinking-induced endocytosis endocytosis of MHC-II of MHC is clathrin-dependent.-II is clathrin-dependent. (A) BMDCs (A) wereBMDCs incubated were incubated with an anti-MHC-II antibody forB 30 min at 4 °C. (B) BMDCs pre-incubated withµ withincubated an anti-MHC-II with an antibodyanti-MHC for-II 30 antibody min at 4 ◦ C.for ( 30) BMDCs min at pre-incubated 4 °C. (B) BMDCs with chlorpromazine pre-incubated (30 withM) forchlorpromazine 30 min at 37 (30C wereµM) for incubated 30 min withat 37 an°C were anti-MHC-II incubated antibody with an for anti 30-MHC min at-II4 antibodyC. The cellsfor 30 were min chlorpromazine ◦(30 µM) for 30 min at 37 °C were incubated with an anti-MHC-II antibody◦ for 30 min washedat 4 °C. and The crosslinked cells were with washed an Alexa and 546-labeledcrosslinked anti-mouse with an Alexa IgG for 546 the-labeled indicated anti time-mouse periods IgG at for 37 theC. at 4 °C. The cells were washed and crosslinked with an Alexa 546-labeled anti-mouse IgG for the◦ Theindicated cells were time fixed, periods permeabilized, at 37 °C. The and cells stained were fixed, with anpermeabilized, anti-clathrin heavyand stained chain (CHC)with an antibody anti-clathrin and indicated time periods at 37 °C. The cells were fixed, permeabilized, and stained with an anti-clathrin anheavy Alexa chain Fluor (CHC) 488-labeled antibody secondary and an antibody.Alexa Fluor bars 488= 2-labeledµm. Co-localization secondary antibody of MHC-II. bars and = CHC2µm.was Co- heavy chain (CHC) antibody and an Alexa Fluor 488-labeled secondary antibody. bars = 2µm. Co- quantifiedlocalization by of calculating MHC-II and Manders’ CHC was coefficients quantified M1 by (green calculating pixels (CHC) Manders’ overlapping coefficients red M1 pixels (green (MHC-II)) pixels localization of MHC-II and CHC was quantified by calculating Manders’ coefficients M1 (green pixels and(CHC) M2 overlapping (red pixels overlapping red pixels green(MHC pixels)-II)) and using M2 ImageJ(red pixels software. overlapping Valuesare green presented pixels) as using the mean ImageJ (CHC) overlapping red pixels (MHC-II)) and M2 (red pixels overlapping green pixels) using ImageJ± SEMsoftware. (n = 10),Values * p < are0.05, presented ** p < 0.01. as the mean ± SEM (n = 10), * p < 0.05, ** p < 0.01. software. Values are presented as the mean ± SEM (n = 10), * p < 0.05, ** p < 0.01.

Cells 2020, 9, 1810 8 of 12

Cells 2020, 9, x FOR PEER REVIEW 8 of 12

Figure 8. EffectEﬀect of inhibitors on crosslinking-inducedcrosslinking-induced co-localizationco-localization ofof MHC-IIMHC-II andand clathrin.clathrin. BMDCs pre-incubatedpre-incubated withwith ((AA)) controlcontrol (0.1%(0.1% DMSO),DMSO), ((BB)) BAPTA-AMBAPTA-AM (50(50 µμM),M), ((CC)) R406 (3(3 µμM),M), or (D) U73122

(10 μµMM)) for for 30 30 min min at at 37 37 °C.◦C. The The cell cell surface surface MHC MHC-II-II was was crosslinked crosslinked with with antianti-MHC-IIMHC-II antibody. antibody. The Theco-localization co-localization of MHC of MHC-II-II and and CHC CHC was was analyzed analyzed as asdescribed described in in Figure Figure 7.7. Values Values are presented asas the mean ± SEMSEM (n (n = 1010),), * * pp << 0.05,0.05, ** ** pp << 0.01.0.01. ± 4.4 . D Discussioniscussion In thisthis study, study, we we investigated investigated the MHC-IIthe MHC crosslinking-induced-II crosslinking-induced signaling signaling pathway andpathway molecular and mechanismsmolecular mechanisms involved in involved the endocytosis in the endocytosis of MHC-II inof murineMHC-II BMDCs in murine (Figure BMDCs9). There (Figure are 9) various. There reportsare various on the reports function on of the MHC-II-induced function of MHC signals-II- [induced6]. Liang signals et al. demonstrated [6]. Liang et that al. MHC-IIdemonstrated crosslinking that suppressesMHC-II crosslinking Toll-like receptor suppresses (TLR) signaling-induced Toll-like receptor upregulation (TLR) signaling of CD86-induced and CD40 upregulation in murine BMDCs of CD86 [7]. Theyand CD40 investigated in murine the BMDCs mechanism [7]. responsibleThey investigated for the suppressionthe mechanism and responsible found that MHC-IIfor the suppress crosslinkingion inducesand found activation that MHC of ERK-II crosslinking via the ITAM-containing induces activation adapter of protein, ERK via Fc γtheRγ ,ITAM which-containing activates a tyrosineadapter phosphataseprotein, FcγRγ, SHP-1. which They activates also showed a tyrosine that phosphatase an inhibitor ofSHP Syk-1. didThey not also affect showed the MHC-II that an crosslinking- inhibitor of inducedSyk did not suppression affect the of MHC TLR- signaling.II crosslinking In our-induced study, we suppress investigatedion of theTLR MHC-II signaling. crosslinking-induced In our study, we 2+ signalinginvestigate thatd the induces MHC- MHC-IIII crosslinking endocytosis,-induced and signaling found that that Syk-and induces PLC-mediated MHC-II endocytosis, Ca mobilization and found isthat involved Syk-and in PLC MHC-II-mediated endocytosis. Ca2+ mobilization Crosslinking-induced is involved MHC-II in MHC endocytosis-II endocytosis. was not Crosslinking affected by- ERKinduced inhibition. MHC-II These endocytosis results suggestwas not thataffected MHC-II by ERK crosslinking inhibition. activates These multipleresults suggest signaling that pathways MHC-II andcrosslinking that these activates signals multiple act in diff signalingerent ways, pathways which might and that impair these the signals ability act of in antigen-presentation different ways, which in BMDCs.might impair Since the Fc γabilityRγ is knownof antigen to- bepresentation involved in in the BMDCs. activation Since of FcγRγ Syk in is several known cells to be [23 involved], a similar in signalingthe activation pathway of Syk may in several be utilized cells in [23], BMDCs a similar upon signaling MHC-II pathway crosslinking. may Alternatively,be utilized in BMDCs since MHC-II upon isMHC reported-II crosslinking. to interact Alternatively, with other membrane since MHC proteins-II is reported in B cells, to such interact as CD79a with other and CD79bmembrane [17], SCIMPproteins [24 in], B and cells, MPYS such/ STINGas CD79a [25 and]. MHC-II CD79b might [17], SCIMP interact [24], with and adapter MPYS/STING proteins other[25]. MHC than Fc-IIγ mightRγ to induceinteract Sykwith/PLC adapter signaling proteins in BMDCs. other than Further FcγRγ analysis to induce is required Syk/PLC to clarifysignaling the signalin BMDCs. transduction Further 2+ formanalysis MHC-II is required crosslinking to clarify to cytosolic the signal Ca transductionmobilization. form MHC-II crosslinking to cytosolic Ca2+ 2+ mobilization.In this study, we found that MHC-II crosslinking induced Ca mobilization and that inhibition of Ca2+ mobilization by inhibitors suppressed MHC-II endocytosis. These results suggested that Ca2+ mobilization is required for crosslinking-induced MHC-II endocytosis. Although thapsigargin promoted Ca2+ mobilization and MHC-II endocytosis, the effect on MHC-II endocytosis was weaker than that on MHC-II crosslinking. Since crosslinking is known to promote the phosphorylation of various proteins in BMDCs [7], MHC-II crosslinking may induce signaling pathways other than Ca2+ mobilization that result in enhanced MHC-II endocytosis. Cells 2020, 9, 1810 9 of 12 Cells 2020, 9, x FOR PEER REVIEW 9 of 12

FigureFigure 9.9. Schematic overview of MHC-IIMHC-II crosslinkingcrosslinking inducedinduced signaling,signaling, whichwhich inducesinduces MHC-IIMHC-II 2+ endocytosisendocytosis in in BMDCs. BMDCs. MHC-II MHC crosslinking-II crosslinking induces induces Syk and PLC-mediatedSyk and PLC cytosolic-mediated Ca cytosolicmobilization. Ca2+ 2+ Activationmobilization. of PKC,Activation which isof onePKC, of thewhich molecules is one activated of the bymolecule cytosolics activated Ca mobilization, by cytosolic induces Ca2+ endocytosismobilization, of induces MHC-II. endocytosis These signal of transductionsMHC-II. These induce signal colocalization transductions of induce MHC-II colocalization with clathrin, of which results in an induction of clathrin-dependent and dynamin-dependent MHC-II endocytosis. MHC-II with clathrin, which results in an induction of clathrin-dependent and dynamin-dependent MHC-II endocytosis. Crosslinking-induced MHC-II endocytosis was inhibited by PKC inhibitors. In addition, endocytosis of MHC-II was promoted by some PKC activators without MHC-II crosslinking. These results suggest In this study, we found that MHC-II crosslinking induced Ca2+ mobilization and that inhibition that PKC may play an important role in the process. Crosslinking of MHC-II was reported to induce of Ca2+ mobilization by inhibitors suppressed MHC-II endocytosis. These results suggested that Ca2+ activation of PKCβ in human B cells [26,27], and of PKCδ in human monocyte-derived dendritic mobilization is required for crosslinking-induced MHC-II endocytosis. Although thapsigargin cells [28,29]. These PKC isoforms may also be activated in BMDCs by MHC-II crosslinking and regulate promoted Ca2+ mobilization and MHC-II endocytosis, the effect on MHC-II endocytosis was weaker the induction of MHC-II endocytosis. Several PKC isoforms are reported to be involved in ligand-induced than that on MHC-II crosslinking. Since crosslinking is known to promote the phosphorylation of endocytosis of some membrane proteins. Activation of PKCβII is required for ligand-induced endocytosis various proteins in BMDCs [7], MHC-II crosslinking may induce signaling pathways other than Ca2+ of dopamine receptor D3 [30], and activation of PKCα is necessary for the down-regulation of TCRs in mobilization that result in enhanced MHC-II endocytosis. antigen-activated T cells. These PKC isoforms may be activated by MHC-II crosslinking and may be Crosslinking-induced MHC-II endocytosis was inhibited by PKC inhibitors. In addition, involved in endocytosis of MHC-II in BMDCs. Further analysis is required to determine which PKC endocytosis of MHC-II was promoted by some PKC activators without MHC-II crosslinking. These isoforms could be activated upon crosslinking. results suggest that PKC may play an important role in the process. Crosslinking of MHC-II was Induction of clathrin-dependent endocytosis is regulated by PKC for several proteins. Endocytosis of reported to induce activation of PKCβ in human B cells [26,27], and of PKCδ in human monocyte- TCRs induced by ligand binding is clathrin-dependent [31,32]. In this case, PKC-mediated phosphorylation derived dendritic cells [28,29]. These PKC isoforms may also be activated in BMDCs by MHC-II of Ser 163 of β-arrestin-1 regulates the TCR endocytosis [33,34]. Activation of PKC was reported to induce crosslinking and regulate the induction of MHC-II endocytosis. Several PKC isoforms are reported clathrin-dependent endocytosis of the glutamate transporter, GLT-1, and the human organic cation to be involved in ligand-induced endocytosis of some membrane proteins. Activation of PKCβII is transporter (hOAT). In this case, a ubiquitin E3 ligase, Nedd 4-2, is activated by PKCs. The activated required for ligand-induced endocytosis of dopamine receptor D3 [30], and activation of PKCα is Nedd 4-2 ubiquitinates GLT-1 and hOAT, which results in their endocytosis [35,36]. Although MHC-II necessary for the down-regulation of TCRs in antigen-activated T cells. These PKC isoforms may be is known to be ubiquitinated at Lys 225, a K225R point-mutation did not aﬀect crosslinking-induced activated by MHC-II crosslinking and may be involved in endocytosis of MHC-II in BMDCs. Further MHC-II endocytosis [8], which suggests that direct ubiquitination of MHC-II is not involved in analysis is required to determine which PKC isoforms could be activated upon crosslinking. crosslinking-induced MHC-II endocytosis. Induction of clathrin-dependent endocytosis is regulated by PKC for several proteins. We examined the molecular mechanisms of crosslinking-induced MHC-II endocytosis using Endocytosis of TCRs induced by ligand binding is clathrin-dependent [31,32]. In this case, PKC- several endocytosis inhibitors. Crosslinking-induced endocytosis was suppressed by chlorpromazine mediated phosphorylation of Ser 163 of β-arrestin-1 regulates the TCR endocytosis [33,34]. Activation and dynasore, which indicates that crosslinking-induced MHC-II endocytosis is clathrin and dynamin- of PKC was reported to induce clathrin-dependent endocytosis of the glutamate transporter, GLT-1, dependent. The signaling inhibitors, BAPTA-AM, R406, and U73122, also suppressed co-localization and the human organic cation transporter (hOAT). In this case, a ubiquitin E3 ligase, Nedd 4-2, is of MHC-II and clathrin. Crosslinking-induced signals likely promote the transition of an MHC-II to activated by PKCs. The activated Nedd 4-2 ubiquitinates GLT-1 and hOAT, which results in their the clathrin-dependent endocytosis pathway. endocytosis [35,36]. Although MHC-II is known to be ubiquitinated at Lys 225, a K225R point- In steady-state DCs, MHC-II is reported to internalize via clathrin-independent and dynamin- mutation did not affect crosslinking-induced MHC-II endocytosis [8], which suggests that direct independent pathways [12]. We found that BAPTA-AM treatment did not affect the steady-state endocytosis ubiquitination of MHC-II is not involved in crosslinking-induced MHC-II endocytosis. ofMHC-II. Theseresults suggestthatcrosslinkingdoesnot simply acceleratetheexistingendocytosis pathway We examined the molecular mechanisms of crosslinking-induced MHC-II endocytosis using but induces an alternative endocytosis pathway. We previously found that crosslinking-induced MHC-II several endocytosis inhibitors. Crosslinking-induced endocytosis was suppressed by chlorpromazine endocytosis was inhibited by methyl-β-cyclodextrin (MCD) treatment, which raises the possibility that lipid and dynasore, which indicates that crosslinking-induced MHC-II endocytosis is clathrin and dynamin-dependent. The signaling inhibitors, BAPTA-AM, R406, and U73122, also suppressed co-

Cells 2020, 9, 1810 10 of 12 rafts could be involved in the process [8]. Although it is well known that MCD blocks clathrin-independent endocytosis, disruption of lipid rafts is also reported to inhibit the clathrin-dependent endocytosis of some proteins. For example, endocytosis of B cell receptors (BCRs) after crosslinking is suppressed by disruption of lipid rafts, even though endocytosis of BCRs is clathrin-dependent in B cells [37,38]. MCD treatment may disrupt the association of signaling molecules in the lipid raft, which results in inhibition of intracellular signal transduction triggered by MHC-II ligation. In summary, we investigated the molecular mechanisms that could potentially regulate crosslinking- induced MHC-II endocytosis. Our results suggested that MHC-II crosslinking-induced intracellular Ca2+ mobilization is required to induce MHC-II endocytosis, and that crosslinking-induced MHC-II endocytosis is clathrin and dynamin-dependent. Since MHC-II endocytosis was suppressed by PKC inhibitors and promoted by PKC activators, intracellular Ca2+ mobilization by MHC-II crosslinking may induce activation of PKCs. This activation results in induction of MHC-II endocytosis. The present study suggested that intracellular Ca2+ mobilization could be one of the factors determining the cell surface expression levels of MHC-II in DCs.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/8/1810/s1. Figure S1: Effect of crosslinking and BAPTA-AM on endocytosis of MHC-II. Author Contributions: Conceptualization, K.M., Y.H., P.A.R., S.T., and K.F. Investigation, K.M., Y.H., H.O., and Y.S. Writing—original draft preparation, K.M., Y.H., and K.F. Writing—review and editing, P.A.R. and S.T. Supervision, S.T. and K.F. Project administration, K.F. Funding acquisition, P.A.R. and K.F. All authors have read and agreed to the published version of the manuscript. Funding: The JSPS KAKENHI (grant number 25860049, 15K18864, 17K08273), Takeda Science Foundation, Ryobi Teien Memory Foundation, Koyanagi Foundation, Smoking Research Foundation, Suzuken Memorial Foundation and the Intramural Research Program of the National Institutes of Health funded this research. Acknowledgments: The authors thank Assisted Reproductive Technology (ART) Center, Okayama University for assistance in flow cytometry experiments. The authors thank Division of Instrumental Analysis, Okayama University for assistance in confocal microscopic analysis. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Mellman, I.; Steinman, R.M. Dendritic cells: Specialized and regulated antigen processing machines. Cell 2001, 106, 255–258. [CrossRef] 2. Banchereau, J.; Steinman, R.M. Dendritic cells and the control of immunity. Nature 1998, 392, 245–252. [CrossRef] [PubMed] 3. Trombetta, E.S.; Mellman, I. Cell biology of antigen processing in vitro and in vivo. Annu. Rev. Immunol. 2005, 23, 975–1028. [CrossRef][PubMed] 4. Roche, P.A.; Furuta, K. The ins and outs of MHC class II-mediated antigen processing and presentation. Nat. Rev. Immunol. 2015, 15, 203–216. [CrossRef][PubMed] 5. Inaba, K.; Turley, S.; Iyoda, T.; Yamaide, F.; Shimoyama, S.; Reis e Sousa, C.; Germain, R.N.; Mellman, I.; Steinman, R.M. The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. J. Exp. Med. 2000, 191, 927–936. [CrossRef][PubMed] 6. Al-Daccak, R.; Mooney, N.; Charron, D. MHC class II signaling in antigen-presenting cells. Curr. Opin. Immunol. 2004, 16, 108–113. [CrossRef] 7. Liang, B.; Workman, C.; Lee, J.; Chew, C.; Dale, B.M.; Colonna, L.; Flores, M.; Li, N.; Schweighoffer, E.; Greenberg, S.; et al. Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II. J. Immunol. 2008, 180, 5916–5926. [CrossRef] 8. Furuta, K.; Ishido, S.; Roche, P.A. Encounter with antigen-specific primed CD4 T cells promotes MHC class II degradation in dendritic cells. Proc. Natl. Acad. Sci. USA 2012, 109, 19380–19385. [CrossRef] 9. Wolfe, B.L.; Trejo, J. Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis. Traffic 2007, 8, 462–470. [CrossRef] 10. Doherty, G.J.; McMahon, H.T. Mechanisms of endocytosis. Annu. Rev. Biochem. 2009, 78, 857–902. [CrossRef] Cells 2020, 9, 1810 11 of 12

11. Mayor, S.; Pagano, R.E. Pathways of clathrin-independent endocytosis. Nat. Rev. Mol. Cell Biol. 2007, 8, 603–612. [CrossRef][PubMed] 12. Walseng, E.; Bakke, O.; Roche, P.A. Major histocompatibility complex class II-peptide complexes internalize using a clathrin- and dynamin-independent endocytosis pathway. J. Biol. Chem. 2008, 283, 14717–14727. [CrossRef][PubMed] 13. Inaba, K.; Inaba, M.; Romani, N.; Aya, H.; Deguchi, M.; Ikehara, S.; Muramatsu, S.; Steinman, R.M. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 1992, 176, 1693–1702. [CrossRef][PubMed] 14. Furuta, K.; Walseng, E.; Roche, P.A. Internalizing MHC class II-peptide complexes are ubiquitinated in early endosomes and targeted for lysosomal degradation. Proc. Natl. Acad. Sci. USA 2013, 110, 20188–20193. [CrossRef] 15. Manders, E.M.M.; Verbeek, F.J.; Aten, J.A. Measurement of co-localization of objects in dual-colour confocal images. J. Microsc. 1992, 169, 375–382. [CrossRef] 16. Nashar, T.O.; Drake, J.R. Dynamics of MHC class II-activating signals in murine resting B cells. J. Immunol. 2006, 176, 827–838. [CrossRef] 17. Jin, L.; Stolpa, J.C.; Young, R.M.; Pugh-Bernard, A.E.; Refaeli, Y.; Cambier, J.C. MHC class II structural requirements for the association with Igalpha/beta, and signaling of calcium mobilization and cell death. Immunol. Lett. 2008, 116, 184–194. [CrossRef] 18. Mooney, N.A.; Grillot-Courvalin, C.; Hivroz, C.; Ju, L.Y.; Charron, D. Early biochemical events after MHC class II-mediated signaling on human B lymphocytes. J. Immunol. 1990, 145, 2070–2076. 19. Xu, S.; Huo, J.; Lee, K.G.; Kurosaki, T.; Lam, K.P. Phospholipase Cgamma2 is critical for Dectin-1-mediated Ca2+ flux and cytokine production in dendritic cells. J. Biol. Chem. 2009, 284, 7038–7046. [CrossRef] 20. Vergarajauregui, S.; San Miguel, A.; Puertollano, R. Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization. Traffic 2006, 7, 686–698. [CrossRef] 21. Ivanov, A.I. Pharmacological inhibition of endocytic pathways: Is it specific enough to be useful? Methods Mol. Biol. 2008, 440, 15–33. [PubMed] 22. Wang, L.H.; Rothberg, K.G.; Anderson, R.G. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 1993, 123, 1107–1117. [CrossRef][PubMed] 23. Humphrey, M.B.; Lanier, L.L.; Nakamura, M.C. Role of ITAM-containing adapter proteins and their receptors in the immune system and bone. Immunol. Rev. 2005, 208, 50–65. [CrossRef][PubMed] 24. Draber, P.; Vonkova, I.; Stepanek, O.; Hrdinka, M.; Kucova, M.; Skopcova, T.; Otahal, P.; Angelisova, P.; Horejsi, V.; Yeung, M.; et al. SCIMP, a transmembrane adaptor protein involved in major histocompatibility complex class II signaling. Mol. Cell. Biol. 2011, 31, 4550–4562. [CrossRef] 25. Jin, L.; Waterman, P.M.; Jonscher, K.R.; Short, C.M.; Reisdorph, N.A.; Cambier, J.C. MPYS, a novel membrane tetraspanner, is associated with major histocompatibility complex class II and mediates transduction of apoptotic signals. Mol. Cell. Biol. 2008, 28, 5014–5026. [CrossRef] 26. Leveille, C.; Castaigne, J.G.; Charron, D.; Al-Daccak, R. MHC class II isotype-specific signaling complex on human B cells. Eur. J. Immunol. 2002, 32, 2282–2291. [CrossRef] 27. Guo, W.; Castaigne, J.G.; Mooney, N.; Charron, D.; Al-Daccak, R. Signaling through HLA-DR induces PKC beta-dependent B cell death outside rafts. Eur. J. Immunol. 2003, 33, 928–938. [CrossRef] 28. Bertho, N.; Blancheteau, V.M.; Setterblad, N.; Laupeze, B.; Lord, J.M.; Drenou, B.; Amiot, L.; Charron, D.J.; Fauchet, R.; Mooney, N. MHC class II-mediated apoptosis of mature dendritic cells proceeds by activation of the protein kinase C-delta isoenzyme. Int. Immunol. 2002, 14, 935–942. [CrossRef] 29. Lokshin, A.E.; Kalinski, P.; Sassi, R.R.; Mailliard, R.B.; Muller-Berghaus, J.; Storkus, W.J.; Peng, X.; Marrangoni, A.M.; Edwards, R.P.; Gorelik, E. Differential regulation of maturation and apoptosis of human monocyte-derived dendritic cells mediated by MHC class II. Int. Immunol. 2002, 14, 1027–1037. [CrossRef] 30. Zhang, X.; Sun, N.; Zheng, M.; Kim, K.M. Clathrin-mediated endocytosis is responsible for the lysosomal degradation of dopamine D3 receptor. Biochem. Biophys. Res. Commun. 2016, 476, 245–251. [CrossRef] 31. Bonefeld, C.M.; Rasmussen, A.B.; Lauritsen, J.P.; von Essen, M.; Odum, N.; Andersen, P.S.; Geisler, C. TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism. J. Immunol. 2003, 171, 3003–3009. [CrossRef][PubMed] Cells 2020, 9, 1810 12 of 12

32. Monjas, A.; Alcover, A.; Alarcon, B. Engaged and bystander T cell receptors are down-modulated by diﬀerent endocytotic pathways. J. Biol. Chem. 2004, 279, 55376–55384. [CrossRef][PubMed] 33. Von Essen, M.; Nielsen, M.W.; Bonefeld, C.M.; Boding, L.; Larsen, J.M.; Leitges, M.; Baier, G.; Odum, N.; Geisler, C. Protein kinase C (PKC) alpha and PKC theta are the major PKC isotypes involved in TCR down-regulation. J. Immunol. 2006, 176, 7502–7510. [CrossRef][PubMed] 34. Fernandez-Arenas, E.; Calleja, E.; Martinez-Martin, N.; Gharbi, S.I.; Navajas, R.; Garcia-Medel, N.; Penela, P.; Alcami, A.; Mayor, F., Jr.; Albar, J.P.; et al. Beta-Arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism. EMBO J. 2014, 33, 559–577. [CrossRef] 35. Vina-Vilaseca, A.; Bender-Sigel, J.; Sorkina, T.; Closs, E.I.; Sorkin, A. Protein kinase C-dependent ubiquitination and clathrin-mediated endocytosis of the cationic amino acid transporter CAT-1. J. Biol. Chem. 2011, 286, 8697–8706. [CrossRef] 36. Garcia-Tardon, N.; Gonzalez-Gonzalez, I.M.; Martinez-Villarreal, J.; Fernandez-Sanchez, E.; Gimenez, C.; Zafra, F. Protein kinase C (PKC)-promoted endocytosis of glutamate transporter GLT-1 requires ubiquitin ligase Nedd4-2-dependent ubiquitination but not phosphorylation. J. Biol. Chem. 2012, 287, 19177–19187. [CrossRef] 37. Lajoie, P.; Nabi, I.R. Regulation of raft-dependent endocytosis. J. Cell. Mol. Med. 2007, 11, 644–653. [CrossRef] 38. Stoddart, A.; Dykstra, M.L.; Brown, B.K.; Song, W.; Pierce, S.K.; Brodsky, F.M. Lipid rafts unite signaling cascades with clathrin to regulate BCR internalization. Immunity 2002, 17, 451–462. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).