Differential regulation of MHC II andinvariant chain expression during maturation

of monocyte-derived dendritic cellsOle J. B. Landsverk,* Anett H. Ottesen,* Axel Berg-Larsen,* Silke Appel,†

and Oddmund Bakke*,†,1

*Centre for Immune Regulation, Department of Molecular Biosciences, University of Oslo, Norway; and †BroegelmannResearch Laboratory, The Gade Institute, University of Bergen, Norway

RECEIVED MARCH 18, 2011; REVISED JANUARY 13, 2012; ACCEPTED FEBRUARY 2, 2012. DOI: 10.1189/jlb.0311150

ABSTRACTDCs are potent initiators of adaptive immune responsestoward invading pathogens. Upon reception of patho-genic stimuli, DCs initiate a complex differentiation pro-gram, culminating in mature DCs with an extreme ca-pacity to activate naıve T cells. During this maturation,DCs reduce the synthesis and turnover of MHC II mole-cules. This allows for a stable population of MHC II, pre-senting peptides captured at the time and place of acti-vation, thus provoking specific immune responses to-ward the activating pathogen. The efficient loading ofantigenic peptides onto MHC II is vitally dependent onthe accessory molecule Ii, which aids in the assemblyof the MHC II �- and �-chains in the ER and directs theirtrafficking to the endocytic compartments, where theyencounter endocytosed antigen. However, Ii plays ad-ditional roles in DC function by influencing migration,antigen uptake, and processing. To examine the bio-synthetic background for diverse Ii functions in DCs, weinvestigated mRNA and protein levels of Ii comparedwith MHC II in human moDCs during maturation usingvarious stimuli. We find that the production of Ii did notcorrelate with that of MHC II and that mature DCs main-tain abundant levels of Ii despite a reduced productionof new MHC II. J. Leukoc. Biol. 91: 729–737; 2012.

IntroductionHuman genes encoding the classical MHC II proteins com-prise the HLA-DR, HLA-DP, and HLA-DQ isotypes, with multi-ple alleles of each [1]. These gene products function to pres-ent peptides derived from internalized protein antigens to theTCR on CD4� T cells [2]. Expression of MHC II is, in gen-eral, restricted to professional antigen presenting cells, and

DCs are the most potent activators of naıve T cells. In an im-mature state, DCs are highly efficient at capturing antigens asa result of a high endocytic activity but generally display lowamounts of MHC II at the cell surface, and these are ex-changed continuously. Inflammatory stimuli trigger an elabo-rate differentiation program, culminating in mature DCs,which down-regulate endocytic activity, up-regulate costimula-tory molecules CD80/86 and LN homing receptors such asCCR7, and display vast amounts of peptide-loaded MHC II onthe cell surface [3, 4]. The increase in surface MHC II iscaused by the activation-induced reduction in the endocytosisand degradation of MHC II and serves to conserve MHC II-presenting peptides acquired at the time and place of activa-tion [5, 6]. Following DC activation, the biosynthesis of newMHC II has been shown to transiently increase followed by agradual down-regulation [7–12]. This is a result of a rapid re-duction in the levels of the CIITA [13], a transcriptional regu-lator required for MHC II expression [14, 15]. CIITA is re-markably selective in its targets and apart from MHC II, onlyregulates 12–13 other genes [16]. One of these genes encodesIi, a type-II transmembrane protein that is translated to fourisoforms in humans. Ii p33 and p41 are distinguished by alter-native splicing of the Ii transcript, where the p41 isoform con-tains an extra exon (exon 6b) [17, 18]. These two isoformsyield two additional protein products (p35 and p43), as a re-sult of an alternative translation initiation site, which adds anN-terminal cytoplasmic extension of 16 residues [17, 19]. Iiassembles into a hetero- or homotrimer in the ER, whichserves as scaffolds for the assembly of MHC II, blocks bindingof endogenous peptides in the ER and Golgi apparatus, anddirects MHC II to the endocytic pathway as a result of twostrong, sorting motifs in its cytoplasmic tail [2]. In endosomes,Ii is degraded rapidly, allowing MHC II to bind antigenic pep-tides and releasing it for transport to the cell surface [20, 21].Ii also modulates the endosomes into which it delivers MHC IIby converging endocytic cargo and delaying the progression

1. Correspondence: Department of Molecular Biosciences, University of Oslo, Pb.1041, Blindern, 0316 Oslo, Norway. E-mail: oddmund.bakke@imbv.uio.no

Abbreviations: �CT�change in threshold cycle, CIITA�MHC II transactiva-tor, DC-SIGN�DC-specific ICAM-3-grabbing nonintegrin, F�forward,Ii�invariant chain (also called CD74), moDC�monocyte-derived DC, qRT-PCR�quantitative real-time RT-PCR, R�reverse

The online version of this paper, found at www.jleukbio.org, includessupplemental information.

Article

0741-5400/12/0091-729 © Society for Leukocyte Biology Volume 91, May 2012 Journal of Leukocyte Biology 729

through the endocytic pathway [22, 23], and the fragment en-coded by exon 6b of Ii p41/43 has an inhibitory function onendosomal proteases [24–28]. Interestingly, Ii seems to alsoplay a role in antigen capture by transiently binding up themotor protein myosin II; this causes a pause in DC motilityafter activation and provides time for enhanced endocytosis ofthe antigen present in the extracellular milieu in the immedi-ate vicinity of the activating stimulus [29]. Thus, Ii serves tofacilitate not only MHC II assembly and transport but also an-tigen uptake and the generation of an endosomal platform forthe slow proteolysis of antigen and ample time for loading ofMHC II. This ultimately leads to the presentation of a diverserepertoire of peptide–MHC II complexes to T cells. Alto-gether, the events, resulting in an immunogenic DC present-ing a pathogen-relevant repertoire of peptides on MHC II, re-quire a stringent regulation of biosynthesis, trafficking, andproteolysis of MHC II and Ii. In this study, we examine howthese events are regulated in maturing human moDCs.

MATERIALS AND METHODS

In vitro culture and activation of moDCsBuffy coats from healthy blood donors were acquired from Ullevål Univer-sity Hospital (Oslo, Norway), and mononuclear cells were isolated by den-sity gradient centrifugation with Lymphoprep (Axis-Shield, Oslo, Norway).moDCs were generated from plastic-adherent or directly isolated mono-cytes (Monocyte Isolation Kit II; Miltenyi Biotec, Auburn, CA, USA) by cul-ture for 6 days in RPMI (Lonza, Verviers, Belgium) containing 100 ng/mlGM-CSF (Immunotools, Friesoythe, Germany) and 20 ng/ml IL-4 (Invitro-gen, Life Technologies, Grand Island, NY, USA), supplemented with 10%FBS (Saveen Werner, Malmö, Uppsala), penicillin/streptomycin, and L-glu-tamine (Lonza, Walkersville, MD, USA). GM-CSF and IL-4 were replen-ished every 2–3 days. On Day 6, moDCs were activated with 100 ng/ml LPS(sc3535; Santa Cruz Biotechnology, Santa Cruz, CA, USA), 100 �g/ml zy-mosan (Z4250; Sigma-Aldrich, St. Louis, MO, USA), or a cocktail consistingof 10 ng/ml TNF-�, 10 ng/ml IL-1� (both from R&D Systems, Minneapo-lis, MN, USA), 10 ng/ml IL-6, and 1 �g/ml PGE2 (Sigma-Aldrich) for indi-cated times.

ImmunostainingImmature and activated moDCs were harvested, washed three times in cold1� PBS with 0.05% BSA, and stained on ice for 30 min with antibodiestargeting HLA-DR (MHLDR05; Caltag Laboratories, Buckingham, UK),CCR7 (FAB197A; R&D Systems), DC-SIGN (BD 551,545), CD11c (BD559,877; both from BD Biosciences, San Jose, CA, USA), CD86 (sc19617),CD83 (sc19678), CD1a (sc19636), CD20 (sc19990), CD14 (sc7328), andisotype controls IgG1-FITC (sc2855), IgG1-PE (sc2866), IgG1-allophycocya-nin (sc2888), and IgG2b-allophycocyanin (sc2890; all from Santa Cruz Bio-technology). After staining, cells were washed in 1� PBS with 0.05% BSA,three times, and fixed in 3% PFA. All flow cytometry was performed on aFACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA), and data wereanalyzed using FlowJo Software (Tree Star, Ashland, OR, USA).

qRT-PCRTotal RNA was isolated from cells using the RNeasy Kit (Qiagen, Valencia,CA, USA), according to the instructions of the manufacturer. Residualgenomic DNA was removed during the purification process by incubationwith RNase-free DNase (Qiagen). Total RNA (1 �g) was used in a 20-�lcDNA synthesis reaction using Transcriptor First Strand cDNA SynthesisKit, as described by the manufacturer (Roche Diagnostics, Mannheim, Ger-many), using a combination of random hexamers and oligo(dT)18 primers.

cDNA (600 ng) was used in the qRT-PCR reactions, carried out in dupli-cates using SYBR Green technology on a Roche 480 Lightcycler, accordingto the instructions of the manufacturer (Roche Diagnostics). Gene-specificprimers targeting conserved regions common to most allelic variants ofHLA-DR, -DP, and -DQ �-chains were: DRA*0101 F: 5�-cgatcaccaatgtacctcca,R: 5�-acttgcggaaaaggtggtct; DPA1*010301 F: 5�-caacttatgccgcgtttgta, R: 5�-ggatcgttggtggcctga; DQA1*010101 F: 5�-tgggcagtcagtcacagaag, R: 5�-ggctc-ccagtgtttcagaag. Primers targeting Ii isoforms: Ii p33/35 (NM_001025159)F: 5�-atgcacctgctccagaatg, R: 5�- tttcggtggagcgtcagt; Ii p41/43 (NM_004355)F: 5�-atgcacctgctccagaatg, R: 5�-ggcacttggtcagtactttcg. Primers for GAPDH:5�-ccacatcgctcagacaccat, R: 5�-ggcaacaatattccactttaccagagt. Tenfold serial di-lutions of cDNA were used to produce standard curves and determineprimer efficiencies [E�10(�1/slope)]. The relative expression ratio (R) ofthe target genes was then calculated using the equation

R �(Etarget)�Ct target (control-sample)

(Ereference)�Ct reference (control-sample)

qRT-PCR data were analyzed and assembled in excel (Microsoft, Redmond,WA, USA) and Prism (GraphPad Software, San Diego, CA, USA).

Metabolic labeling, immunoprecipitation, and Western blotanalysismoDCs were generated as described, washed, and transferred to 75 cm2

cell culture dishes before activation with 100 ng/ml LPS for 0, 4, or 24 h.Immature and mature cells were pulsed for 2 h in RPMI containing 100�Ci/ml 35S-labeled cysteine/methionine (Perkin Elmer, Waltham, MA,USA); washed; resuspended in RPMI containing 15% FCS, 2 mM L-glu-tamine, 25 U/ml penicillin/streptomycin, 1 mM nonessential amino acid,and 1 mM Na-pyruvate; and chased for indicated times. Bafilomycin A1 (10�M; ALX-380-030; Alexis, Lausen, Switzerland) or 10 �M lactacystin(L6785; Sigma-Aldrich), respectively, was added to cells 2 h before pulseand maintained in medium during all subsequent steps. After chase, cellswere harvested, washed in 1� PBS, and lysed on ice in lysis buffer contain-ing 1% Nonidet P-40, 6 mM CHAPS, 5 mM Tris HCl, 150 mM NaCl2, 5mM EDTA, and protease arrest (Genotech, St. Louis, MO, USA). Immuno-precipitations with 0.1 mg/ml anti-Ii [M-B741 (555538)] or anti HLA-DR[Tu36 (555559); both from BD Biosciences] were performed at 4°C over-night, and immune complexes were captured on protein G Dynabeads (In-vitrogen, Life Technologies), washed three times in lysis buffer, resus-pended in sample buffer containing 2% SDS, 62.5 mM Tris HCL, 10%glycerol, and 0.77 M �2-ME, boiled for 5 min at 95°C, loaded onto 4–20%Tris-HEPES-SDS gels (Pierce, Rockford, IL, USA), and transferred ontoPVDF membranes (Millipore, Billerica, MA, USA). Radiolabeled proteinswere detected directly on film (Amersham Hyperfilm ECL from GE Health-care, Waukesha, WI, USA), and immunolabeling was done with M-B741,TAL.1B5, recognizing the HLA-DR �-chain (ab20181) and anti-actin(ab3280), both from Abcam (Cambridge, MA, USA), in 1� PBS with 5%skim milk (Bio-Rad, Hercules, CA, USA) at 21°C. Goat anti-mouse HRP(Bio-Rad) and Supersignal West Pico chemiluminescent substrate (ThermoScientific, Rockford, IL, USA) were used to detect immunolabeled pro-teins.

Confocal immunofluorescense microscopyDay 6 moDCs were seeded onto poly-l-lysine-coated coverslips and maturedfor 24 h with 100 ng/ml LPS. For detection of N-terminal Ii, the cells werefixed in 3% PFA and incubated with Pin.1, followed by goat anti-mouseIgG Alexa 488 (Invitrogen/Molecular Probes, Life Technologies). HLA-DRwas detected using Alexa Fluor 555-labeled L243. For the detection of C-terminal Ii, the cells were allowed to take up Alexa Fluor 488-labeledM-B741 (1 �g/ml) for 4 h. After that, the cells were fixed and stained forHLA-DR (L243). ER was visualized in living cells by incubation with ER-Tracker Blue-White DPX (Invitrogen/Molecular Probes, Life Technolo-gies); Alexa Fluor-labeled L243 and M-B741 were added to cells 2 h beforeimaging to label surface-exposed, mature HLA-DR and Ii, respectively. Con-focal images were acquired on an Olympus FluoView 1000 inverted micro-

730 Journal of Leukocyte Biology Volume 91, May 2012 www.jleukbio.org

scope equipped with a PlanApo 60/1.10 oil objective (Olympus, Hamburg,Germany), processed with ImageJ, and assembled using Adobe Photoshopand Illustrator (Adobe Systems, San Jose, CA, USA).

RESULTS

To examine the regulation of MHC II and Ii during DCmaturation, we used moDCs generated by culture in GM-CSF- and IL-4-containing medium for 6 days as described

[30]. For most of the experiments, we used plastic adherentmonocytes as DC progenitors. The cell cultures containedmainly CD1ahi, CD11cint, DC-SIGNhi, CD14low cells (Supple-mental Fig. 1A), which responded to activating stimuli byup-regulating surface MHC II, CD86, CD83, and CCR7(Supplemental Fig. 1B). To verify that any contaminatingcells carried through in the adherence protocol did not in-fluence the results, key experiments were also performedusing moDCs generated from monocytes isolated with the

hours after LPS stimulation

HLA DQ

HLA DP

HLA DR

0 1 40.1 12 24 48

0.8

0.6

0.7

0.5

0.4

0.3

0.2

0.1

0.0

Ii p41

Ii p33

0.8

0.6

0.4

0.00 1 40.1 12 24 48

1.2

0.2

1.0

Ii total

HLA II total

0 1 40.1 12 24 48

0.8

0.6

0.4

0.0

1.2

0.2

1.0

1.4

1.6

1.8

Rat

io re

lativ

e to

tota

l HLA

II a

nd Ii

hours after LPS stimulation

1000

10

1.0

0.1

0.01

100

0 4 8 24 48

Rel

ativ

e C

CR

7 m

RN

A LPS

NT

10

1.0

0.1

0.01

100

Rel

ativ

e C

D83

mR

NA

0 4 8 24 48

LPS

NT

0 4 8 24 48

1

0

2

3

4

5

Rel

ativ

e β-

actin

mR

NA LPS

NT

A B

Figure 1. Sustained high Ii mRNA levels in moDC after LPS stimulation. Human moDCs were stimulated with 100 ng/ml LPS; the cells were har-vested after indicated times, RNA-extracted, and reverse-transcribed; and specific genes were amplified and quantified relative to reference geneGAPDH. (A, top) Relative amount of HLA-DR, -DP, and -DQ mRNA after activation; (middle) relative amount of Ii p33/35 and Ii p41/43 mRNAafter activation; (bottom) relative amount of MHC II (HLA-DR, -DP, and -DQ) and Ii (p33/35 and p41/43) after LPS-induced maturation. Barsrepresent the ratio of each gene to the total data set (HLA-DR, -DP, and -DQ, Ii p33/35, and Ii p41/43) at t � 0, averaged from eight indepen-dent experiments with sd. (B, top) Relative amount of CCR7 mRNA; (middle) relative amount of CD83 mRNA; (bottom) relative amount of �-ac-tin mRNA, all after LPS-induced maturation. Open bars represent the respective mRNA expression in response to distilled H2O, normalized toGAPDH, relative to initial (t�0) values; NT, not treated. Closed bars represent respective mRNA expression in response to LPS, normalized toGAPDH, relative to initial (t�0) values.

Landsverk et al. Regulation of invariant chain after DC activation

www.jleukbio.org Volume 91, May 2012 Journal of Leukocyte Biology 731

Monocyte Isolation Kit II (Miltenyi Biotec; untouched isola-tion; Supplemental Fig. 2).

Regulation of MHC II and Ii mRNA during moDCmaturationPrevious studies have examined the mRNA levels of singleMHC II isoforms after LPS-induced maturation and found atransient increase immediately after activation, followed by adecrease at later time-points [10, 13]. Although CIITA isknown to regulate Ii and MHC II genes, we wanted to estab-lish whether the kinetics of Ii regulation followed that ob-served for MHC II. To this end, we performed qRT-PCR onhuman moDCs after maturation induced with LPS, a com-monly used activating stimuli for moDCs. All MHC II isotypeswere quantified to estimate the total amount of MHC II and tonegate variation in MHC II isotype preference between differ-ent donors. Primers were designed to target the �-chains ofHLA-DR, -DQ, and -DP, which display less allelic diversity andare restricting to assembly of functional heterodimers and ERexport. Primers targeting Ii were designed to discriminate be-tween the two splice variants, p33/35 and p41/43. Discrimina-tion between the p33/41 and p35/43 isoforms is not possibleby qRT-PCR, as these isoforms result from alternative transla-tion initiation sites on the same mRNAs. As shown in Fig. 1A,we observed an incremental increase of the MHC II mRNAs,up to a peak at 4 h after activation, followed by a similarly slowdecrease to 48 h, where the total MHC II mRNA was reducedto less than one-half of the initial amount. The different MHCII mRNA species were synchronous in their increase and de-crease, and the predominant species in the different donors(HLA-DR) exhibited the most pronounced up- or down-regu-lation. The Ii isoforms, however, displayed a less-pronounced,initial increase and maintained relatively high levels through-out maturation, and Ii p33/35 is the dominant isoform. Theratio of total Ii:total MHC II shifted considerably through thematuration, starting at 2:1 in immature cells and increasing to4:1 after 48 h activation. Thus, the Ii mRNA is in considerableexcess of MHC II mRNA in fully mature moDCs. The analysiswas also performed on moDCs generated from directly iso-lated monocytes with similar results (Supplemental Fig. 3A).

Although the GAPDH values remained consistent in all ofthe samples examined, we wanted to verify that the variableMHC II/Ii mRNA levels were not caused by general increaseand decrease of transcription as a result of maturation. Wetherefore examined the levels of CCR7, CD83, and �-actinmRNAs through the maturation process. CCR7 and CD83 areexpressed by moDCs in response to LPS activation (Supple-mental Fig. 1B), whereas �-actin is a commonly used referencegene for qRT-PCR. As shown in Fig. 1B, CCR7 and to a lesserdegree, CD83 mRNAs are increased following LPS stimulation,thus corroborating the flow cytometry data (Supplemental Fig.1B). Interestingly, �-actin is transiently increased 8–24 h afterLPS stimulation but regains initial levels at 48 h. Altogether,these data suggest that the transcription of Ii is less influencedby LPS activation than MHC II.

To determine whether Ii and MHC II mRNAs were trans-lated effectively into proteins in matured moDCs, we per-formed metabolic labeling and chase experiments, followed by

immunoprecipitation and detection of radiolabeled protein.moDCs were cultured with LPS for 0, 4, or 24 h, followed byincubation in medium containing 35S-labeled cysteine and me-thionine, washed, and chased in unlabeled medium for 0, 1, 2,and 3 h. The translation of Ii mRNAs increased during matu-

IP: Ii (M-B741)

75 -

50 -

37 -

25 -20 -

15 -

NT

2 30 1 2 30 12 30 1

4h LPS 24h LPS

β

αp33

p41

IP: HLA-DR (Tü36)

75 -

50 -

37 -

25 -20 -

15 -

NT

2 30 1 2 30 12 30 1

4h LPS 24h LPS

β

αp33

p41

IP: H

LA-D

R 37 -

25 -

20 -

NT2 30 1 2 30 1

24h LPS

Ii p33

LC

37 -

25 -

20 -

NT2 30 1 2 30 1

24h LPS

LC

DRα

IP: I

i

C

B

A

Figure 2. Expression and half life of HLA-DR and Ii upon LPS-in-duced maturation. moDCs were activated with 100 ng/ml LPS for 0, 4,or 24 h, pulsed with 35S-labeled cysteine/methionine, washed, andchased for 0–3 h. (A) Ii was immunoprecipitated (IP) with M-B741;immune complexes were run on 4–20% gradient HEPES-SDS gels,blotted onto PVDF membranes, and exposed on films. One represen-tative blot of an experiment repeated three times with moDCs fromthree different donors shown. (B) HLA-DR was immunoprecipitatedwith Tu36; immune complexes were run on 4–20% gradient HEPES-SDS gels, blotted onto PVDF membranes, and exposed on films. Onerepresentative blot of an experiment repeated three times with moDCsfrom three different donors shown. (C) Membranes from B (upper)and A (lower), not treated, and 24 h LPS were probed with anti-IiM-B741 (upper) or anti-HLA-DR �-chain antibody TAL.1B5 (lower),and goat anti-mouse IgG HRP, incubated with HRP substrate andchemiluminescence, was detected on films. Representative immuno-blots from an experiment repeated twice are shown. LC, IgG lightchain of the antibody used for immuno-precipitation.

732 Journal of Leukocyte Biology Volume 91, May 2012 www.jleukbio.org

ration, with an elevated protein production apparent after 4 h,maintained for at least 24 h after activation (Fig. 2A). Theseresults were confirmed using sorted moDCs (Supplemental Fig.3B). The amount of HLA-DR produced was increased slightlyafter 4 h, corresponding to the elevated HLA-DR transcripts butquite drastically reduced after 24 h (Fig. 2B). Immunoblottingrevealed that in immature moDCs, a large fraction of HLA-DRwas associated with Ii, whereas in mature moDCs, this fractionwas reduced considerably (Fig. 2C, upper right). This is consis-tent with the mature phenotype of these cells, with elevated sur-face MHC II as assessed by flow cytometry (Supplemental Fig.1B), and demonstrates the longevity of MHC II in mature DCs.However, there is clearly some neosynthesis of HLA-DR from thetranscripts present, and these associate efficiently with the vastsurplus of Ii (Fig. 2C, lower right).

In mature moDCs, Ii is present predominantly in the ERThe levels of newly produced Ii were clearly in excess ofHLA-DR in mature moDCs, and we therefore wanted to exam-ine the localization of this pool of Ii. To this end, we seeded

moDCs onto poly-l-lysine-treated coverslips, induced their mat-uration by adding LPS for 24 h, fixed the cells with parafor-maldehyde, and immunostained for Ii. As shown in Fig. 3A,the majority of Ii was present in a tubular network, corre-sponding to the ER (for ER distribution in mature moDCs,see Supplemental Fig. 4). This is in agreement with other stud-ies, suggesting that to overcome the ER retention motif in hu-man-specific Ii p35, Ii p35-containing trimers need to interactwith the MHC II �-chains [31, 32]. However, when we incu-bated the mature moDCs with antibodies targeting the extra-cellular/luminal domain of Ii, we found that these antibodieswere quite efficiently taken up and targeted to endosomalcompartments (Fig. 3B). This uptake was specific, as isotype-control antibodies were not internalized (data not shown),and the antibodies were internalized to the same compart-ments as antibodies targeting HLA-DR. It was not possible toquantify and correlate the ratio of trafficking Ii:ER-resident Iito define whether the trafficking Ii was associated with MHCII. However, the long (2–4 h) incubation required to achieve

HLA DR

Ii (N-term.)

HLA DR

Ii (C-term.)

A

Bmerge

merge

Figure 3. Ii is localized to the ER in mature moDCs. moDCs were seededonto poly-l-lysine-coated coverslips, matured for 24 h with 100 ng/mlLPS, and (A) fixed in 3% PFA and stained for N-terminal Ii (Pin.1,green) and HLA-DR (L243, red) or (B) allowed to take up fluorescentlylabeled M-B741 (targeting C-terminal Ii, green) for 4 h, fixed, andstained for HLA-DR (L243, red). Confocal immunofluorescence imageswere acquired on an Olympus FV1000. Original scale bars, 20 �m.

IP: Ii (M-B741)

75 -

50 -

37 -

25 -20 -

15 -

DMSO BafilomycinLactacystin

NT LPS

0 30 3 0 30 30 30 3

NT LPS NT LPS

IP: HLA-DR (Tü36)

75 -

50 -

37 -

25 -20 -

15 -

DMSO BafilomycinLactacystin

NT LPS

0 30 3 0 30 30 30 3

NT LPS NT LPS

β

αp33

p41

β

αp33

p41

A

B

Figure 4. Ii traffics to and is degraded in endosomes in mature moDCs.Human moDCs matured for 24 h with LPS or untreated were incu-bated with lactacystin (10 �M), bafilomycin (10 �M), or DMSO (nega-tive control) for 2 h to block proteasomal proteolysis or endosomalacidification, respectively. The cells were then metabolically labeled for2 h with 35S-cysteine/methionine, chased for 0 or 3 h at 37°C, andsubsequently lysed. Ii (A) or HLA-DR (B) was immunoprecipitatedwith M-B741 or Tu36, respectively, run on 4–20% HEPES-SDS gels,and blotted onto PVDF membranes. The experiment was repeatedthree times with independent donors producing similar results; onerepresentative experiment is shown.

Landsverk et al. Regulation of invariant chain after DC activation

www.jleukbio.org Volume 91, May 2012 Journal of Leukocyte Biology 733

0 4 12 480.0

0.2

0.4

0.6

0.8 1.21.00.80.60.40.20.0

0 4 12 48 0 4 12 48

1.0

2.0

0.0

1.5

0.5

DR

DQDP

p41p33

IiHLA II

0 4 12 480.0

0.2

0.4

0.6

0.8 1.21.00.80.60.40.20.0

0 4 12 48 0 4 12 48

1.0

2.0

0.0

1.5

0.5

DR

DQDP

p41p33

IiHLA II

0 4 12 480.0

0.2

0.4

0.6

0.8 1.21.00.80.60.40.20.0

0 4 12 48 0 4 12 48

1.0

2.0

0.0

1.5

0.5

DR

DQDP

p41p33

IiHLA II

hours after LPS stimulation

Rat

io re

lativ

e to

tota

l HLA

II a

nd Ii

hours after Zymosan stimulation

hours after Cytokine stimulation

A

BCD86

FL2-H: PE

CCR7

FL4-H: APC

HLA-DR

FL4-H: APC100 101 102 103 104 100 101 102 103 104

0100 101 102 103 104

48h TNFα, IL-1β, IL-6, PGE2Day 6 GM-CSF/IL-4control

Coun

t

CD86

FL2-H: PE

CCR7

FL4-H: APC

HLA-DR

FL4-H: APC100 101 102 103 104 100 101 102 103 104

0100 101 102 103 104

48h ZymosanDay 6 GM-CSF/IL-4control

Coun

tCo

unt

48h LPSDay 6 GM-CSF/IL-4control

CD86

100 101 102 103 104

FL2-H: PE

CCR7

100 101 102 103 104

FL4-H: APC

HLA-DR

0100 101 102 103 104

FL4-H: APC

Figure 5. mRNA responses in moDCs vary depending on activating stimuli. Human moDCs were stimulated with TNF-�, IL-1�, IL-6, and PGE2

(top), 100 �g/ml zymosan A1 (middle), or 100 ng/ml LPS (bottom). (A) RNA was extracted after 0, 4, 12, and 48 h, reverse-transcribed, and spe-cific genes were amplified and quantified relative to reference gene GAPDH. (Left) Relative amount of HLA-DR, -DP, and -DQ mRNAs after acti-vation; (middle) relative amount of Ii p33/35 and Ii p41/43 mRNA; (right) relative amount of total MHC II (HLA-DR, -DP, and -DQ) and

(continued on next page)

734 Journal of Leukocyte Biology Volume 91, May 2012 www.jleukbio.org

a satisfactory signal from an internalized anti-Ii antibody sug-gests that at least the fraction trafficking via the cell surface issmall compared with the fraction retained in the ER or traf-ficking via a direct route to endosomes.

Ii traffics to and is degraded in endosomes in maturemoDCsTo examine in more detail the fraction of Ii-entering endo-somes, we performed new metabolic labeling and chase exper-iments using bafilomycin to block endosomal acidification andreduce endosomal proteolysis. We also included lactacystin inthese experiments to exclude any degradation of Ii via an ER-associated protein-degradation pathway, potentially induced byaccumulated Ii in the ER. As shown in Fig. 4A, bafilomycinimpaired the degradation of Ii p33/p41 in immature and ma-ture moDCs, whereas in cells treated with lactacystin, the halflife of Ii p33/p41 seemed more similar to control (DMSO)-treated cells. Thus, in mature DCs, a fraction of Ii traffics toendosomes, despite the reduced availability of MHC II in theER. However, in cells treated with bafilomycin, the Ii immuno-precipitation also revealed associated MHC II �- and �-chains,and conversely, the HLA-DR immunoprecipitations revealedassociated Ii p33 and p41 (Fig. 4B), so it seems that at least afraction of Ii reaching endosomes is associated with MHC II.

MHC II genes are differentially regulated dependingon the activating stimuliThe qRT-PCR results clearly showed that the MHC II and Iitranscripts displayed a different regulation in response to LPSstimulation, and we wanted to assess whether stimulation withother activating stimuli produced similar results. We thereforestimulated cells with zymosan, which targets a panel of PRRs,including TLR2 and C-type lectins, and a cytokine cocktailconsisting of TNF-�, IL-6, IL-1�, and PGE2, commonly used tomature moDCs in vitro for immunotherapy [33]. As presentedin Fig. 5A, the mRNA levels of MHC II and Ii were stronglyinfluenced by the type of activating stimuli. Zymosan produceda stronger response than LPS alone, with a more pronounceddecrease in MHC II transcripts at earlier time-points (down50% at 12 h) and a stronger correlation between the regula-tion of Ii and MHC II mRNA levels throughout maturation.Stimulation with the cytokine cocktail produced a reduced re-sponse at the mRNA level, with a low initial elevation of MHCII transcripts and a delayed decrease. The Ii transcripts re-mained high even 48 h after activation (down 28% comparedwith initial levels). However, up-regulated surface levels ofHLA-DR and CD86, as determined by flow cytometry (Fig.5B), showed that these moDCs acquired a mature phenotype.Interestingly, the phenotypes correlated very poorly with theMHC II mRNA levels. Zymosan, which gave a particularlystrong mRNA response, resulted in a lower increase in surface

HLA-DR than both the cytokine cocktail and LPS and pro-nounced less expression of the costimulatory molecule CD86.These results demonstrate the complexity of DC maturationand the elaborate interplay of signals required to achieve acomplete activation of these cells.

DISCUSSION

Ii is vital to achieve the presentation of diverse repertoires ofpeptides derived from an endocytosed antigen on MHC II.This is a result of its function in MHC II assembly and traffick-ing but also, its effects on cell migration [29] and the endo-cytic pathway [34]. Furthermore, Ii has been shown to influ-ence the function of other immune molecules, such as CD1a,CD1d, and FcRn, and also to act as a cell surface receptor formacrophage migration inhibitory factor [35–38]. Thus, thecontrol of Ii biosynthesis influences multiple functions in anti-gen presentin cells. In DCs, the uptake and processing of anti-gen and loading of antigenic peptides are strongly dependenton the activation status, and it has been shown that upon acti-vation, DCs transiently up-regulate the biosynthesis of MHC IIbefore reducing it dramatically [9, 10, 39]. As a result of theirmutual dependence on the CIITA for transcription [40], it hasbeen assumed that Ii is regulated similarly as MHC II; how-ever, our results presented here show that this is not alwaysthe case. To our knowledge, this is the first study to examinethe mRNA levels of all MHC II and Ii isotypes after activationof DCs. We found that the MHC II and Ii mRNA levels do notexhibit a synchronized behavior after LPS-induced maturation.Despite a similar increase up to 4 h after activation, the IimRNA species do not achieve an equally high increase at 4 hand maintain relatively high levels even 48 h after activation(Fig. 1). This seeming paradox might reflect differences in theregulator elements controlling the binding of CIITA to thepromoter regions of the different genes. The transcription ofCIITA is rapidly shut down upon LPS-induced activation ofDCs, with an eightfold reduction in CIITA protein within 24 h[13]. The gene encoding Ii contains unique variations in cis-acting elements [41–43], which might influence the on–offrate and potentially the half life of transacting factors. Thismight consequently contribute to a sustained, high productionof Ii relative to MHC II mRNAs, despite a reduced availabilityof new CIITA. MHC II and Ii might also be subject to differen-tial regulation post-transcription. It has been shown that thestability of Ii and MHC II mRNAs increases following activa-tion of PKC [44, 45], but no destabilizing or microRNA targetelements have, so far, been identified in the 3�-untranslatedregion of MHC II or Ii transcripts [46]. However the sustainedpresence of Ii mRNA is achieved, we show that the transcriptsare functional in mature moDCs. Metabolic pulse and chaseexperiments indicate that translation of the Ii mRNAs is main-

total Ii (p33/35 and p41/43) during maturation of moDCs. Bars represent the ratio of each gene to the total data set (HLA-DR, -DP, and -DQ, Iip33/35, and Ii p41/43) at t � 0, averaged from three (top), two (middle), or eight (bottom) independent experiments with sd. (B) HumanmoDCs were stimulated with TNF-�, IL-1�, IL-6, and PGE2 (top), 100 �g/ml zymosan A1 (middle), or 100 ng/ml LPS (bottom) and analyzed byflow cytometry. The histograms show up-regulation of HLA-DR, CCR7, and CD86 on gated moDCs in immature (dashed lines), 48 h-stimulatedcells (solid lines), or controls (dotted lines). FL-4/2-H, Fluorescence 4/2-height; APC, allophycocyanin.

Landsverk et al. Regulation of invariant chain after DC activation

www.jleukbio.org Volume 91, May 2012 Journal of Leukocyte Biology 735

tained after LPS-induced maturation (Fig. 2A). The half life ofthe Ii produced, however, seems to transiently decrease 4 h afterstimulation. This correlates with the elevated biosynthesis ofMHC II (Figs. 1 and 2B), 4 h after stimulation, indicating that Iiis more efficiently exported out of the ER in the presence of in-creased levels of MHC II. The sustained production and turnoverof Ii, 24 h after DC activation, show that despite the reduced pro-duction of new MHC II, the export of Ii from the ER is main-tained. Some of this Ii is clearly associated with MHC II, althoughthe total amount of MHC II associated with Ii is drastically re-duced (Fig. 2). This is likely a result of a reduction in the turn-over of MHC II caused by the down-regulation of ubiquitin li-gases acting on MHC II, leading to more stable MHC II at thecell surface in mature moDCs [47–49].

In humans, the two Ii mRNAs yield two additional isoforms,which contain an N-terminal ER retention motif [50, 51]. ThisER retention can be released upon assembly with the MHC II�-chain and/or serine phosphorylation [31, 52–55]. In maturemoDCs, we found that Ii was strongly present in a reticularnetwork corresponding to the ER, indicating that a large frac-tion of Ii is retained there as a result of a lack of availableMHC II. However, MHC II association does not seem to be aprerequisite for p35/43 phosphorylation and forward trans-port, at least in Hela cells [54], and the similar half life of Iiin immature and mature moDCs suggests that some Ii can traf-fic to endosomes without MHC II. Whether this fraction con-sists of trimers lacking these isoforms, trimers releasedthrough phosphorylation, or trimers associated with other sug-gested Ii-interacting proteins, such as CD1 molecules, CD44,or the neonatal FcR, needs further investigation.

The activation of DCs in vivo most often involves a panel ofpathogenic compounds and cytokines. We therefore examinedwhether we could elicit a stronger response through stimulationwith zymosan, which targets multiple PRRs, including TLR2 andC-type lectins, and a cytokine cocktail commonly used in DC im-munotherapy [33]. Surprisingly, we found that zymosan did notelicit a very profound phenotypic maturation, with less increasein surface MHC II, CCR7, and CD86 relative to LPS-maturedmoDCs. However, the mRNA levels of Ii and MHC II showed astronger response and a more synchronous up- and down-regula-tion. In contrast, the cytokine cocktail induced a strong pheno-typic maturation of moDCs but less profound mRNA responsesthan for LPS or zymosan stimulation. These results clearly exem-plify the complex nature of DC maturation and suggest that de-pending on the pathogen and the cytokine milieu at the timeand place of activation, the surface phenotype and MHC II pro-duction are subject to variable regulation. It is of interest to notethat the inflammatory cocktail used for maturing moDCs in vitrofor use in cancer immunotherapy gives a relatively weak transcrip-tional response. Our metabolic pulse and chase experiments onLPS-matured cells indicate that if MHC II mRNAs are present,they will be translated. This could indicate that despite a clearlymature cell surface phenotype, these cells might exchange theMHC II molecules produced and peptide loaded at the time andplace of activation. Young et al. [56] showed that plasmacytoidDCs and conventional DCs from mice differentially regulatedMHC II production and turnover following activation and thatsustained production and turnover of MHC II lead to inefficient

presentation of exogenous antigens but sustained ability to pres-ent endogenous antigens. Depending on the mechanism involvedin targeting tumor antigen to MHC II, this could be detrimentalto establishing a tumor-specific immune response. The poor re-sults from various clinical trials might thereby, in some cases, bethe result of exchange of tumor peptide-loaded MHC II with en-dogenous peptide-loaded MHC II after transfer back into pa-tients.

Altogether, our results show that MHC II biosynthesis doesnot necessarily correlate with up-regulated surface MHC II,costimulatory molecules, and LN homing receptors. We findthat Ii is present in a large surplus relative to MHC II in thesteady-state and that this surplus is increased after DC activa-tion with LPS. The dominant fraction of this Ii is present inthe ER, but transport to endosomes is maintained despite areduced biosynthesis of MHC II in mature DCs. Thus, the pre-sumption that all CIITA-controlled genes are equally affectedupon maturation is not valid, and a large pool of functional Iiis maintained in mature moDCs.

AUTHORSHIP

O.J.B.L. designed and performed experiments, analyzed thedata, and wrote the manuscript. A.H.O. and A.B-L. performedexperiments. SA. and O.B. designed experiments, analyzeddata, and revised manuscript.

ACKNOWLEDGMENTS

This work was supported by the Norwegian Research Counciland the Norwegian Cancer Society.

REFERENCES

1. Trowsdale, J., Ragoussis, J., Campbell, R. D. (1991) Map of the humanMHC. Immunol. Today 12, 443–446.

2. Cresswell, P. (1994) Assembly, transport, and function of MHC class IImolecules. Annu. Rev. Immunol. 12, 259–293.

3. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J.,Pulendran, B., Palucka, K. (2000) Immunobiology of dendritic cells.Annu. Rev. Immunol. 18, 767–811.

4. Reis e Sousa, C. (2006) Dendritic cells in a mature age. Nat. Rev. Immu-nol. 6, 476–483.

5. Van Niel, G., Wubbolts, R., Stoorvogel, W. (2008) Endosomal sorting ofMHC class II determines antigen presentation by dendritic cells. Curr.Opin. Cell Biol. 20, 437–444.

6. Villadangos, J. A., Schnorrer, P., Wilson, N. S. (2005) Control of MHCclass II antigen presentation in dendritic cells: a balance between creativeand destructive forces. Immunol. Rev. 207, 191–205.

7. Kampgen, E., Koch, N., Koch, F., Stoger, P., Heufler, C., Schuler, G., Ro-mani, N. (1991) Class II major histocompatibility complex molecules ofmurine dendritic cells: synthesis, sialylation of invariant chain, and anti-gen processing capacity are down-regulated upon culture. Proc. Natl.Acad. Sci. USA 88, 3014–3018.

8. Villadangos, J. A., Cardoso, M., Steptoe, R. J., van Berkel, D., Pooley, J.,Carbone, F. R., Shortman, K. (2001) MHC class II expression is regulatedin dendritic cells independently of invariant chain degradation. Immunity14, 739–749.

9. Cella, M., Engering, A., Pinet, V., Pieters, J., Lanzavecchia, A. (1997) In-flammatory stimuli induce accumulation of MHC class II complexes ondendritic cells. Nature 388, 782–787.

10. Wilson, N. S., El-Sukkari, D., Villadangos, J. A. (2004) Dendritic cells con-stitutively present self antigens in their immature state in vivo and regu-late antigen presentation by controlling the rates of MHC class II synthe-sis and endocytosis. Blood 103, 2187–2195.

11. Pure, E., Inaba, K., Crowley, M. T., Tardelli, L., Witmer-Pack, M. D., Ru-berti, G., Fathman, G., Steinman, R. M. (1990) Antigen processing byepidermal Langerhans cells correlates with the level of biosynthesis of

736 Journal of Leukocyte Biology Volume 91, May 2012 www.jleukbio.org

major histocompatibility complex class II molecules and expression ofinvariant chain. J. Exp. Med. 172, 1459–1469.

12. Rescigno, M., Citterio, S., Thery, C., Rittig, M., Medaglini, D., Pozzi, G.,Amigorena, S., Ricciardi-Castagnoli, P. (1998) Bacteria-induced neo-bio-synthesis, stabilization, and surface expression of functional class I mole-cules in mouse dendritic cells. Proc. Natl. Acad. Sci. USA 95, 5229–5234.

13. Landmann, S., Muhlethaler-Mottet, A., Bernasconi, L., Suter, T., Wald-burger, J. M., Masternak, K., Arrighi, J. F., Hauser, C., Fontana, A., Reith,W. (2001) Maturation of dendritic cells is accompanied by rapid tran-scriptional silencing of class II transactivator (CIITA) expression. J. Exp.Med. 194, 379–391.

14. LeibundGut-Landmann, S., Waldburger, J. M., Krawczyk, M., Otten, L. A.,Suter, T., Fontana, A., Acha-Orbea, H., Reith, W. (2004) Mini-review:specificity and expression of CIITA, the master regulator of MHC class IIgenes. Eur. J. Immunol. 34, 1513–1525.

15. Wright, K. L., Ting, J. P. (2006) Epigenetic regulation of MHC-II andCIITA genes. Trends Immunol. 27, 405–412.

16. Krawczyk, M., Seguin-Estevez, Q., Leimgruber, E., Sperisen, P., Schmid,C., Bucher, P., Reith, W. (2008) Identification of CIITA regulated geneticmodule dedicated for antigen presentation. PLoS Genet. 4, e1000058.

17. O’Sullivan, D. M., Noonan, D., Quaranta, V. (1987) Four Ia invariantchain forms derive from a single gene by alternate splicing and alternateinitiation of transcription/translation. J. Exp. Med. 166, 444–460.

18. Strubin, M., Berte, C., Mach, B. (1986) Alternative splicing and alterna-tive initiation of translation explain the four forms of the Ia antigen-asso-ciated invariant chain. EMBO J. 5, 3483–3488.

19. Strubin, M., Long, E. O., Mach, B. (1986) Two forms of the Ia antigen-associated invariant chain result from alternative initiations at two in-phase AUGs. Cell 47, 619–625.

20. Hsing, L. C., Rudensky, A. Y. (2005) The lysosomal cysteine proteases inMHC class II antigen presentation. Immunol. Rev. 207, 229–241.

21. Watts, C. (2004) The exogenous pathway for antigen presentation on ma-jor histocompatibility complex class II and CD1 molecules. Nat. Immunol.5, 685–692.

22. Vascotto, F., Lankar, D., Faure-Andre, G., Vargas, P., Diaz, J., Le Roux, D.,Yuseff, M. I., Sibarita, J. B., Boes, M., Raposo, G., Mougneau, E., Glaichenhaus,N., Bonnerot, C., Manoury, B., Lennon-Dumenil, A. M. (2007) The actin-basedmotor protein myosin II regulates MHC class II trafficking and BCR-driven anti-gen presentation. J. Cell Biol. 176, 1007–1019.

23. Landsverk, O. J., Barois, N., Gregers, T. F., Bakke, O. (2011) Invariantchain increases the half-life of MHC II by delaying endosomal matura-tion. Immunol. Cell Biol. 89, 619–629.

24. Ogrinc, T., Dolenc, I., Ritonja, A., Turk, V. (1993) Purification of thecomplex of cathepsin L and the MHC class II-associated invariant chainfragment from human kidney. FEBS Lett. 336, 555–559.

25. Bevec, T., Stoka, V., Pungercic, G., Dolenc, I., Turk, V. (1996) Major histocom-patibility complex class II-associated p41 invariant chain fragment is a stronginhibitor of lysosomal cathepsin L. J. Exp. Med. 183, 1331–1338.

26. Mihelic, M., Dobersek, A., Guncar, G., Turk, D. (2008) Inhibitory frag-ment from the p41 form of invariant chain can regulate activity of cys-teine cathepsins in antigen presentation. J. Biol. Chem. 283, 14453–14460.

27. Fineschi, B., Sakaguchi, K., Appella, E., Miller, J. (1996) The proteolytic environ-ment involved in MHC class II-restricted antigen presentation can be modulatedby the p41 form of invariant chain. J. Immunol. 157, 3211–3215.

28. Lennon-Dumenil, A. M., Roberts, R. A., Valentijn, K., Driessen, C., Overk-leeft, H. S., Erickson, A., Peters, P. J., Bikoff, E., Ploegh, H. L., Wolf Bry-ant, P. (2001) The p41 isoform of invariant chain is a chaperone for ca-thepsin L. EMBO J. 20, 4055–4064.

29. Faure-Andre, G., Vargas, P., Yuseff, M. I., Heuze, M., Diaz, J., Lankar, D.,Steri, V., Manry, J., Hugues, S., Vascotto, F., Boulanger, J., Raposo, G.,Bono, M. R., Rosemblatt, M., Piel, M., Lennon-Dumenil, A. M. (2008)Regulation of dendritic cell migration by CD74, the MHC class II-associ-ated invariant chain. Science 322, 1705–1710.

30. Sallusto, F., Lanzavecchia, A. (1994) Efficient presentation of soluble an-tigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregu-lated by tumor necrosis factor �. J. Exp. Med. 179, 1109–1118.

31. Khalil, H., Brunet, A., Saba, I., Terra, R., Sekaly, R. P., Thibodeau, J.(2003) The MHC class II � chain cytoplasmic tail overcomes the invari-ant chain p35-encoded endoplasmic reticulum retention signal. Int. Im-munol. 15, 1249–1263.

32. Khalil, H., Brunet, A., Thibodeau, J. (2005) A three-amino-acid-longHLA-DR� cytoplasmic tail is sufficient to overcome ER retention of in-variant-chain p35. J. Cell Sci. 118, 4679–4687.

33. Jonuleit, H., Kuhn, U., Muller, G., Steinbrink, K., Paragnik, L., Schmitt,E., Knop, J., Enk, A. H. (1997) Pro-inflammatory cytokines and prosta-glandins induce maturation of potent immunostimulatory dendritic cellsunder fetal calf serum-free conditions. Eur. J. Immunol. 27, 3135–3142.

34. Landsverk, O. J., Bakke, O., Gregers, T. F. (2009) MHC II and the endocyticpathway: regulation by invariant chain. Scand. J. Immunol. 70, 184–193.

35. Leng, L., Metz, C. N., Fang, Y., Xu, J., Donnelly, S., Baugh, J., Delohery,T., Chen, Y., Mitchell, R. A., Bucala, R. (2003) MIF signal transductioninitiated by binding to CD74. J. Exp. Med. 197, 1467–1476.

36. Sloma, I., Zilber, M. T., Vasselon, T., Setterblad, N., Cavallari, M., Mori,L., De Libero, G., Charron, D., Mooney, N., Gelin, C. (2008) Regulationof CD1a surface expression and antigen presentation by invariant chainand lipid rafts. J. Immunol. 180, 980–987.

37. Jayawardena-Wolf, J., Benlagha, K., Chiu, Y. H., Mehr, R., Bendelac, A.(2001) CD1d endosomal trafficking is independently regulated by an in-trinsic CD1d-encoded tyrosine motif and by the invariant chain. Immunity15, 897–908.

38. Ye, L., Liu, X., Rout, S. N., Li, Z., Yan, Y., Lu, L., Kamala, T., Nanda,N. K., Song, W., Samal, S. K., Zhu, X. (2008) The MHC class II-associatedinvariant chain interacts with the neonatal Fc � receptor and modulatesits trafficking to endosomal/lysosomal compartments. J. Immunol. 181,2572–2585.

39. Young, L. J., Wilson, N. S., Schnorrer, P., Mount, A., Lundie, R. J., LaGruta, N. L., Crabb, B. S., Belz, G. T., Heath, W. R., Villadangos, J. A.(2007) Dendritic cell preactivation impairs MHC class II presentation ofvaccines and endogenous viral antigens. Proc. Natl. Acad. Sci. USA 104,17753–17758.

40. Reith, W., LeibundGut-Landmann, S., Waldburger, J. M. (2005) Regula-tion of MHC class II gene expression by the class II transactivator. Nat.Rev. Immunol. 5, 793–806.

41. Krawczyk, M., Peyraud, N., Rybtsova, N., Masternak, K., Bucher, P., Bar-ras, E., Reith, W. (2004) Long distance control of MHC class II expres-sion by multiple distal enhancers regulated by regulatory factor X com-plex and CIITA. J. Immunol. 173, 6200–6210.

42. Moore, B. B., Cao, Z. A., McRae, T. L., Woo, C. H., Conley, S., Jones,P. P. (1998) The invariant chain gene intronic enhancer shows homologyto class II promoter elements. J. Immunol. 161, 1844–1852.

43. Zhu, L., Jones, P. P. (1990) Transcriptional control of the invariant chaingene involves promoter and enhancer elements common to and distinctfrom major histocompatibility complex class II genes. Mol. Cell. Biol. 10,3906–3916.

44. Shih, N. Y., Soesilo, I., Floyd-Smith, G. (1997) Stabilization of invariantchain mRNA by 12-O-tetradecanoylphorbol-13-acetate is blocked by IFN-�in a murine B lymphoma cell line. J. Interferon Cytokine Res. 17, 747–755.

45. Shih, N-Y., Floyd-Smith, G. (1995) Invariant chain (CD74) gene regula-tion: enhanced expression associated with activation of protein kinase C�in a murine B lymphoma cell line. Mol. Immunol. 32, 643–650.

46. Asirvatham, A. J., Gregorie, C. J., Hu, Z., Magner, W. J., Tomasi, T. B.(2008) MicroRNA targets in immune genes and the Dicer/Argonauteand ARE machinery components. Mol. Immunol. 45, 1995–2006.

47. De Gassart, A., Camosseto, V., Thibodeau, J., Ceppi, M., Catalan, N.,Pierre, P., Gatti, E. (2008) MHC class II stabilization at the surface of hu-man dendritic cells is the result of maturation-dependent MARCH Idown-regulation. Proc. Natl. Acad. Sci. USA 105, 3491–3496.

48. Walseng, E., Furuta, K., Bosch, B., Weih, K. A., Matsuki, Y., Bakke, O.,Ishido, S., Roche, P. A. (2010) Ubiquitination regulates MHC class II-peptide complex retention and degradation in dendritic cells. Proc. Natl.Acad. Sci. USA 107, 20465–21470.

49. Walseng, E., Furuta, K., Goldszmid, R. S., Weih, K. A., Sher, A., Roche,P. A. (2010) Dendritic cell activation prevents MHC class II ubiquitina-tion and promotes MHC class II survival regardless of the activation stim-ulus. J. Biol. Chem. 285, 41749–41754.

50. Lotteau, V., Teyton, L., Peleraux, A., Nilsson, T., Karlsson, L., Schmid,S. L., Quaranta, V., Peterson, P. A. (1990) Intracellular transport of classII MHC molecules directed by invariant chain. Nature 348, 600–605.

51. Lamb, C. A., Yewdell, J. W., Bennink, J. R., Cresswell, P. (1991) Invariantchain targets HLA class II molecules to acidic endosomes containing in-ternalized influenza virus. Proc. Natl. Acad. Sci. USA 88, 5998–6002.

52. Arunachalam, B., Lamb, C. A., Cresswell, P. (1994) Transport propertiesof free and MHC class II-associated oligomers containing different iso-forms of human invariant chain. Int. Immunol. 6, 439–451.

53. Anderson, H. A., Bergstralh, D. T., Kawamura, T., Blauvelt, A., Roche,P. A. (1999) Phosphorylation of the invariant chain by protein kinase Cregulates MHC class II trafficking to antigen-processing compartments.J. Immunol. 163, 5435–5443.

54. Kuwana, T., Peterson, P. A., Karlsson, L. (1998) Exit of major histocom-patibility complex class II-invariant chain p35 complexes from the endo-plasmic reticulum is modulated by phosphorylation. Proc. Natl. Acad. Sci.USA 95, 1056–1061.

55. O’Kelly, I., Butler, M. H., Zilberberg, N., Goldstein, S. A. (2002) Forwardtransport. 14-3-3 binding overcomes retention in endoplasmic reticulumby dibasic signals. Cell 111, 577–588.

56. Young, L. J., Wilson, N. S., Schnorrer, P., Proietto, A., ten Broeke, T.,Matsuki, Y., Mount, A. M., Belz, G. T., O’Keeffe, M., Ohmura-Hoshino,M., Ishido, S., Stoorvogel, W., Heath, W. R., Shortman, K., Villadangos,J. A. (2008) Differential MHC class II synthesis and ubiquitination con-fers distinct antigen-presenting properties on conventional and plasmacy-toid dendritic cells. Nat Immunol. 9, 1244–1252.

KEY WORDS:CD74 · Ii translation/transport/half life/degradation · DC maturation

Landsverk et al. Regulation of invariant chain after DC activation

www.jleukbio.org Volume 91, May 2012 Journal of Leukocyte Biology 737