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of June 18, 2017.
Type I IFN Drives a Distinctive Dendritic
Cell Maturation Phenotype That Allows
Continued Class II MHC Synthesis and
Antigen Processing
Daimon P. Simmons, Pamela A. Wearsch, David H.
Canaday, Howard J. Meyerson, Yi C. Liu, Ying Wang, W.
Henry Boom and Clifford V. Harding
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 188:3116-3126; Prepublished online 27
February 2012;
doi: 10.4049/jimmunol.1101313
http://www.jimmunol.org/content/188/7/3116
The Journal of Immunology
Type I IFN Drives a Distinctive Dendritic Cell Maturation
Phenotype That Allows Continued Class II MHC Synthesis
and Antigen Processing
Daimon P. Simmons,*,1 Pamela A. Wearsch,*,1 David H. Canaday,†,‡,x
Howard J. Meyerson,* Yi C. Liu,* Ying Wang,* W. Henry Boom,†,‡ and
Clifford V. Harding*,‡,{
T
he functions of dendritic cells (DCs) at the nexus of innate and adaptive immunity (1–5) are regulated by their
maturation in response to pathogen-associated molecular
patterns (PAMPs); for example, agonists of TLRs (4, 6–11). When
activated by TLR agonists at a site of infection, DCs mature,
upregulate expression of lymph node homing molecules, and
stimulate Ag-specific T cell responses. TLR-induced DC matu-
*Department of Pathology, Case Western Reserve University/University Hospitals
Case Medical Center, Cleveland, OH 44106; †Division of Infectious Diseases, Case
Western Reserve University/University Hospitals Case Medical Center, Cleveland,
OH 44106; ‡Center for AIDS Research, Case Western Reserve University/University
Hospitals Case Medical Center, Cleveland, OH 44106; xGeriatric Research, Education and Clinical Center, Cleveland Veterans Administration Medical Center, Cleveland, OH 44106; and {Case Comprehensive Cancer Center, Case Western Reserve
University/University Hospitals Case Medical Center, Cleveland, OH 44106
1
D.P.S. and P.A.W. are joint first authors.
Received for publication May 6, 2011. Accepted for publication January 20, 2012.
This work was supported by National Institutes of Health Grants AI034343,
AI035726, and AI069085 (to C.V.H.), AI027243 and HL055967 (to W.H.B.), and
AI073217 and AI077056 (to D.H.C.); the Case Comprehensive Cancer Center (P30
CA43703); and the Case Western Reserve University Center for AIDS Research
(National Institutes of Health Grant AI036219). D.P.S. received partial support from
National Institutes of Health Grants HL083823 and GM007250.
Address correspondence and reprint requests to Dr. Clifford V. Harding, Department
of Pathology, WRB 6522, Case Western Reserve University, 10900 Euclid Avenue,
Cleveland, OH 44106. E-mail address: [email protected]
Abbreviations used in this article: CD11b/c, CD11b and CD11c; DC, dendritic cell;
Flt3L, Flt3 ligand; I-Abb, b-chain of MHC-II I-Ab; IFN-I, type I IFN; LAMP1/2,
LAMP1 and LAMP2; mDC, myeloid dendritic cell; MFI, mean fluorescence intensity; MHC-II, class II MHC; ODN, oligodeoxynucleotide; Pam3CSK4, synthetic
triacylated lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine; PAMP, pathogen-associated
molecular pattern; pDC, plasmacytoid dendritic cell; poly(I:C), polyinosinic:polycytidylic acid; qRT-PCR, quantitative RT-PCR.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101313
ration is characterized functionally by increased potency of DCs
as APCs, resulting from increased expression of peptide–class II
MHC complexes (3, 4, 12) and costimulator molecules (e.g.,
CD80, CD86) (13, 14). The induction of maturation produces a
brief upregulation of Ag endocytosis and processing (15, 16) and
a prolongation of the half-life of peptide–class II MHC complexes
(3, 6) due to decreased ubiquitination, endocytosis, and degradation (17–24). Decreased class II MHC (MHC-II) ubiquitination is
caused by decreased expression of MARCH1, an E3 ubiquitin
ligase expressed primarily in professional APCs (20–25). This
results in prolonged expression of a cohort of pathogen-associated
peptide–MHC-II complexes, driving activation of pathogen-specific T cells in lymph nodes. After the initial TLR-induced burst of
Ag processing, however, Ag endocytosis decreases (26), intracellular localization of MHC-II molecules in Ag processing compartments decreases (27, 28), and the synthesis of MHC-II molecules
and formation of peptide–MHC-II complexes drop dramatically
(29, 30). Decreased synthesis of MHC-II is due to decreased expression of CIITA, which regulates MHC-II gene transcription (6,
29, 31). This mechanism focuses DC Ag presentation on a cohort
of peptide–MHC-II complexes formed at the time of exposure to
PAMPs, resulting in presentation of peptides that are enriched in
pathogen-derived Ag, and it prevents processing and presentation
of Ags later encountered by DCs.
Although the TLR pathways, often involving MyD88, are its
most studied inducers, DC maturation is also driven by type I IFN
(IFN-I), for example, IFN-a subtypes or IFN-b, which signal
through a single IFN-I receptor. IFN-I can induce maturation of
DCs characterized by upregulation of MHC-II and CD86 (32–39)
and drive migration of Langerhans cells (33). IFN-I and TLR/
MyD88 signaling are two semiredundant pathways that drive
DC maturation (40, 41). IFN-I is required for the enhancement of
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Microbial molecules or cytokines can stimulate dendritic cell (DC) maturation, which involves DC migration to lymph nodes and
enhanced presentation of Ag to launch T cell responses. Microbial TLR agonists are the most studied inducers of DC maturation, but
type I IFN (IFN-I) also promotes DC maturation. In response to TLR stimulation, DC maturation involves a burst of Ag processing
with enhanced expression of peptide–class II MHC complexes and costimulator molecules. Subsequently, class II MHC (MHC-II)
synthesis and expression in intracellular vacuolar compartments is inhibited, decreasing Ag processing function. This limits
presentation to a cohort of Ags kinetically associated with the maturation stimulus and excludes presentation of Ags subsequently
experienced by the DC. In contrast, our studies show that IFN-I enhances DC expression of MHC-II and costimulatory molecules
without a concomitant inhibition of subsequent MHC-II synthesis and Ag processing. Expression of mRNA for MHC-II and the
transcription factor CIITA is inhibited in DCs treated with TLR agonists but maintained in cells treated with IFN-I. After
stimulation with IFN-I, MHC-II expression is increased on the plasma membrane but is also maintained in intracellular vacuolar
compartments, consistent with sustained Ag processing function. These findings suggest that IFN-I drives a distinctive DC
maturation program that enhances Ag presentation to T cells without a shutdown of Ag processing, allowing continued sampling
of Ags for presentation. The Journal of Immunology, 2012, 188: 3116–3126.
The Journal of Immunology
T cell responses by TLR agonists such as polyinosinic:polycytidylic acid [poly(I:C)] that do not signal through MyD88 (42,
43). Regulation of DCs by IFN-I contributes to induction of class I
MHC cross-processing and CD8+ responses as well as the induction of CD4+ responses (44–48).
Although IFN-I is important for regulation of DCs and T cell
responses, its regulation of DC MHC-II Ag processing and presentation has not been studied in-depth; important differences
between DC maturation induced by IFN-I versus that by TLR
agonists are not understood. In contrast to TLR-induced DC maturation, our studies show that IFN-I enhances DC expression of
MHC-II and costimulatory molecules without inducing subsequent
inhibition of MHC-II synthesis and Ag processing. Thus, DCs that
undergo maturation in response to IFN-I may be less kinetically
restricted in the acquisition and processing of Ag for presentation
to T cells. This may result in increased opportunity to present Ags
that are encountered at different times and locations.
3117
Flow cytometry
Murine DCs (2 3 105 to 4 3 105/well) were incubated with medium,
Pam3CSK4, LPS, or IFN-b for 48 h. Cells were washed with PBS with
0.1% BSA (Sigma-Aldrich). DCs were incubated for 15 min on ice in Fc
block (anti-CD16, anti-CD32) (BD Biosciences, San Jose, CA) and stained
for an additional 30 min on ice with the following Abs: PE–anti–I-A/I-E
(BD Biosciences), fluorescein–anti-CD80 (eBioscience), PE–anti-CD86
(eBioscience), PE–cyanin7–anti-CD11b (eBioscience), and allophycocyanin–anti-CD11c (eBioscience). Human DCs (2 3 104 to 5 3 104) were
incubated in polypropylene tubes for 16–18 h in the presence of GM-CSF
(200 U/ml) with medium, IFN-a, IFN-b, or Pam3CSK4, washed, and
stained with PE–anti-CD80 (BioLegend, San Diego, CA), PE–Texas red–
anti–HLA-DR (Invitrogen), PE–cyanin7–anti-CCR7 (BD Biosciences),
and Pacific blue–anti-CD86 (BioLegend). Cells were washed with PBS
with 0.1% BSA, fixed in 1% paraformaldehyde (Polysciences, Warrington,
PA), and analyzed on a BD LSR-II flow cytometer (BD Biosciences). Data
were analyzed using Winlist (Topsham, ME) or FlowJo (Tree Star, Ashland, OR). Mean fluorescence intensity (MFI) with isotype control Ab was
subtracted from MFI with specific Ab to determine “specific MFI.”
Endocytosis assay
Reagents and animals
C57BL/6J and C57BL/10ScnJ mice were from The Jackson Laboratory
(Bar Harbor, ME). MyD882/2 mice were from Shizuo Akira. All mice
were housed under specific pathogen-free conditions. CpG-A oligodeoxynucleotide (ODN) 2336 (59-ggG GAC GAC GTC GTG ggg ggG-39)
(lowercase letters indicate nucleotides for which the 39 internucleotide linkage was phosphorothioate-modified; uppercase letters indicate
standard phosphodiester-linked nucleotides) was from Sigma-Aldrich
(St. Louis, MO). Synthetic triacylated lipopeptide N-palmitoyl-S-[2,3-bis
(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]lysyl-[S]-lysine (Pam3CSK4) and ultrapure LPS from Escherichia coli 0111:
B4 were from Invivogen (San Diego, CA). Recombinant human IFN-b was
from Peprotech (Rocky Hill, NJ). Recombinant human IFN-a2a and murine
IFN-a4 and IFN-b were from PBL IFNSource (Piscataway, NJ).
Cells and media
Incubations were carried out at 37˚C with 5% CO2. DCs were grown in
complete RPMI 1640 medium consisting of RPMI with L-glutamine and
glucose, supplemented with 10% heat-inactivated FCS, 50 mM 2-mercaptoethanol, 1 mM sodium pyruvate, and 1% penicillin/streptomycin (all
from Hyclone, Logan, UT). Ag processing assays were done in complete
DMEM medium consisting of DMEM with L-glutamine and glucose
(Hyclone), supplemented with 10% heat-inactivated FCS, 50 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES (Hyclone), and 1%
penicillin/streptomycin.
To make murine DCs, bone marrow cells were isolated from mouse
femurs and tibias. RBCs were lysed with ACK lysis buffer (Lonza, Walkersville, MD). To make DCs derived from Flt3 ligand (Flt3L)-stimulated
murine bone marrow (“Flt3L DCs”), marrow cells were cultured with
Flt3L–Ig fusion protein (1 mg/ml) (Bioexpress, West Lebanon, NH) to
produce a DC culture containing myeloid dendritic cells (mDCs) and
plasmacytoid dendritic cells (pDCs). On day 8, nonadherent cells were
collected. Alternatively, marrow cells were cultured with J558L cellconditioned medium (containing GM-CSF) diluted in complete RPMI
medium to produce “GM-CSF DCs,” which are mDCs (49). On day 7,
nonadherent cells were collected.
Studies with cells from human donors were approved by the University
Hospitals Case Medical Center Institutional Review Board. Blood was
harvested in heparinized syringes. PBMCs were collected as reported (50).
Human mDCs were purified by positive selection for CD1c (BDCA-1)
with a kit (Miltenyi, Auburn, CA) as reported (51). DCs were cultured
in IMDM (Lonza) supplemented with 5% pooled human serum (Gemini
Bioproducts, West Sacramento, CA) and GM-CSF (200 U/ml; Berlex,
Montville, NJ).
The CD4OVA.1 and CD4OVA.2 T hybridoma cell lines were generated
in these studies by incubating splenocytes from OT-II TCR transgenic mice
in vitro with OVA(323–339) peptide (100 nM) for 4 d and immortalizing
proliferating T cells by fusion with BW1100 cells. CD4OVA.1 and
CD4OVA.2 T hybridoma cells were used to detect OVA(323–339)–I-Ab
complexes in Ag processing assays with murine DCs. F9A6 T hybridoma
cells (52) were used to detect Ag85B(97–112)–HLA-DR1 complexes
presented by human DCs.
After 7 d in culture, murine DCs were sorted with CD11c microbeads
(Miltenyi MACS) and incubated with agonists for 24 h. DCs were washed
twice in PBS with 0.1% BSA, incubated on ice for 15 min in Fc block,
incubated 30 min with unconjugated anti–I-Ab or isotype control (BD
Biosciences), washed four times in ice-cold PBS with 0.1% BSA, and
resuspended in complete medium. Duplicate samples were cultured at
37˚C for 0, 45, or 90 min, chilled on ice, and stained with Alexa Fluor
488 rabbit anti-mouse Ab (Invitrogen). Samples were washed, fixed, and
analyzed by flow cytometry as described earlier. Specific MFI was normalized to the level at time 0.
Ag presentation assay
Murine DCs (5 3 104 to 10 3 104/well) were incubated for 20–24 h in
complete DMEM medium with IFN-b, Pam3CSK4, LPS, or CpG-A ODN
with or without OVA. Cells were washed and fixed with 1% paraformaldehyde or washed and incubated 4 h with OVA prior to fixation. DCs were
washed, and CD4OVA T hybridoma cells (105/well) were added and incubated for 24 h. Alternatively, human mDCs (104 per well) were incubated for 16–18 h with Pam3CSK4 or IFN-a in the continued presence of
GM-CSF (200 U/ml), washed, and incubated with Mycobacterium tuberculosis Ag85B [purified as described (53)] and F9A6 T hybridoma cells
(50) (105/well) for 24 h. Supernatants (100 ml) were harvested, frozen,
thawed, and used in a CTLL-2 bioassay for IL-2 (54). Alamar blue (15 ml;
Invitrogen, Carlsbad, CA) was added for the last 24 h. Alamar blue reduction was analyzed by reading the difference between OD at 570 nm and
OD at 595 nm on a Bio-Rad Model 680 plate reader.
Quantitative RT-PCR
DCs (3 3 106 to 5 3 106/well) were incubated for 18–24 h with medium,
IFN-a, IFN-b, Pam3CSK4, LPS, or CpG-A ODN. RNA was purified using
the Qiashredder kit and RNEasy Plus kit (Qiagen). RNA yield was quantified by OD, and 1 mg of each sample was used to synthesize cDNA using
the Quantitect Reverse Transcription Kit (Qiagen); 4% of the total cDNA
from each reaction was used in a quantitative PCR with 500 nM of 59 and
39 primer for each gene and SYBR Green detection (Bio-Rad, Hercules,
CA) using the Bio-Rad CFX96 Real Time fluorescence detection system.
All conditions were tested in triplicate. Primer sequences are as follows.
GAPDH: sense 59-AACGACCCCTTCATTGAC-39, antisense 59-TCCACGACATACTCAGCAC-39. Total CIITA mRNA: sense 59-ACGCTTTCTGGCTGGATTAGT-39, antisense 59-TCAACGCCAGTCTGACGAAGG-39.
The b-chain of MHC-II I-Ab (I-Abb): sense 59-AAGATGTTGAGCGGCATCGG-39, antisense 59-GTCAGGAATTCGGAGCAGAG-39. MARCH1:
sense 59-ATGCACGGACAAAGCAATGG-39, antisense 59-GTGTGAAGTCACGGGCAATC-39. Primers were previously described (31, 55) or
designed using Clone Manager Suite v7.11 and Primers Designers v5.11
(Scientific & Educational Software, Cary, NC). A BLAST search was
performed to verify specificity.
Nuclear extracts
DCs were incubated with medium, IFN-b, LPS, or Pam3CSK4 for 20 h at
37˚C. Subsequent steps for purification of nuclei were performed at 4˚C.
Cells were washed in PBS with protease inhibitors [protease inhibitor
mixture (P8340; Sigma-Aldrich) plus 1 mM NaF, 1 mM PMSF, and 10 nM
calyculin A], pelleted, and incubated in 1 ml Nuclei EZ Prep Lysis Buffer
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Materials and Methods
3118
TYPE I IFN AND DC MATURATION
(Sigma-Aldrich) with protease inhibitors for 10 min. After centrifugation
at 500 3 g for 5 min, supernatants were removed, and pellets were resuspended in 1 ml Nuclei EZ Prep Lysis Buffer with protease inhibitors for
10 min to remove residual cytosolic material. Nuclei were collected by
centrifugation at 500 3 g for 5 min, solubilized in 10% glycerol, 2% SDS,
63 mM Tris-Cl, then sonicated and boiled. Protein concentrations were
determined using the BCA assay (Thermo Scientific, Rockford, IL) to
standardize loading for analysis by 12% SDS-PAGE and Western blotting.
Immunoprecipitations
DCs (106 per sample) were incubated for 24 h with medium, IFN-b, LPS,
or Pam3CSK4, washed once with PBS, and then lysed in 1% Triton X-100
with 150 mM NaCl, 25 mM Tris-Cl, pH 7.4, 25 mM NEM, and protease
inhibitor mixture. Samples were centrifuged for 15 min at 14,000 3 g.
Supernatants were precleared for 1 h with 50 ml rec-Protein G Sepharose
4B (Invitrogen). Immunoprecipitations were performed for 90 min at 4˚C
using Protein G Sepharose and 4 mg control Ab (mouse IgG) or Y3P,
specific for I-Ab (56). The beads were washed three times with ice-cold
0.1% Triton X-100 in 150 mM NaCl, 25 mM Tris-Cl, pH 7.4, and then
resuspended in nonreducing sample buffer for analysis by 10% SDS-PAGE
and Western blotting.
Western blotting
Confocal microscopy
DCs (3 3 106 to 5 3 106/well) were incubated for 24 h with medium,
IFN-a, IFN-b, Pam3CSK4, LPS, or CpG-A ODN. Cells were fixed and
permeabilized using a Fix and Perm kit (Invitrogen). After permeabilization, cells were stained with biotinylated anti–I-A/I-E (eBioscience) for
30–60 min, washed with PBS, 0.1% BSA, and incubated with streptavidin–Alexa Fluor 488 (Invitrogen) for 30 min. Cells were washed with PBS,
0.1% BSA, and mixed with Prolong Gold mounting medium with DAPI
(Invitrogen). The resulting cell suspension was placed on glass slides and
covered with a poly-lysine–coated coverslip (BD Biosciences). Images
were captured using a Leica TCS-SP microscope. Visual counts were
performed to determine the number of cells with cell-surface MHC-II
expression, and at least 100 cells were counted per condition.
Imaging cytometry
DCs (3 3 106 to 5 3 106/well) were incubated for 24 h with medium,
IFN-a, IFN-b, Pam3CSK4, LPS, or CpG-A ODN. Cells were surfacestained for 30 min on ice with a combination of PE–cyanin7–antiCD11b and PE–cyanin7–anti-CD11c (eBioscience). Cells were washed,
fixed, permeabilized as for confocal microscopy, and stained for 30 min
with PE–cyanin5–anti–I-A/I-E (eBioscience) and a combination of Alexa
Fluor 488–anti-LAMP1 and Alexa Fluor 488–anti-LAMP2 (eBioscience).
DAPI (10 mg/ml) was added, and cells were analyzed by imaging flow
cytometry on an Imagestream cytometer (Amnis, Seattle, WA). Data were
analyzed using the IDEA software (Amnis).
Results
IFN-I and TLR agonists induce DC maturation with enhanced
Ag presentation
Although TLR agonists and IFN-I stimulate distinct signaling
pathways, these agents share the capacity to induce DC maturation.
However, it is unclear whether mature DCs induced by these
different agents differ in phenotype and function. Moreover, the Ag
processing and presentation functions of DCs induced by these
different agents have not been studied in-depth. We confirmed that
TLR agonists and IFN-b increased expression of CD80, CD86,
and MHC-II on murine Flt3L DCs, although IFN-b produced
a weaker induction of CD80 and CD86 (Fig. 1A–C). We also
FIGURE 1. TLR agonists and IFN-b induce maturation of DCs. Flt3Lderived DCs (2 3 105/well) were cultured for 48 h with medium, IFN-b
(100 U/ml), Pam3CSK4 (10 nM), or LPS (50 ng/ml). DCs were stained
with anti-CD11b, anti-CD11c, and (A) anti-CD80, (B) anti-CD86, (C) anti–
MHC-II or isotype control Ab. Specific MFI was determined for CD11b+
/CD11c+ cells by flow cytometry (see Materials and Methods). (D)
C57BL/6J DCs (105/well) were cultured for 24 h with medium, IFN-b,
Pam3CSK4, or LPS, washed, and fixed with 1% paraformaldehyde. OVA
(323–339) peptide and CD4OVA T hybridoma cells were added for 24 h.
Supernatants were assessed for IL-2. (E) C57BL/10ScnJ DCs (105/well)
were incubated for 24 h with OVA protein with or without IFN-b or
Pam3CSK4 and fixed. CD4OVA T hybridoma cells were added for 24 h to
detect MHC-II–peptide complexes, and supernatants were assessed for
IL-2. Data represent the means of triplicate samples 6 SDs and are representative of three or more independent experiments. Student t test was
performed to compare results with medium and each other condition. *p ,
0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.
determined that IFN-b, Pam3CSK4, and LPS all enhanced the
ability of DCs to present OVA(323–339) peptide to MHC-II–restricted T hybridoma cells (Fig. 1D). When DCs were exposed to
OVA protein with simultaneous stimulation by Pam3CSK4 or IFNb, we observed an increase in OVA processing and presentation to
MHC-II–restricted T cells (Fig. 1E). C57BL/10J Scn mice that are
deficient in TLR4 signaling were used to avoid maturation of DCs
by possible LPS contamination of the OVA Ag preparations (Fig.
1E); similar results were seen with C57BL/6J DCs, but with a
higher level of background DC maturation (data not shown). Although the DC maturation phenotypes induced by TLR agonists
and IFN-b may differ to some degree, they share the common
features of increased expression of MHC-II and costimulator
molecules as well as increased MHC-II Ag presentation.
Active MHC-II Ag processing is maintained after IFN-I–
induced DC maturation but not TLR-induced DC maturation
When DC maturation is induced by TLR signaling, MHC-II Ag
processing is transiently boosted; but as maturation proceeds,
MHC-II Ag processing is diminished, and efficient Ag presentation is focused on previously created peptide–MHC-II complexes.
Thus, after 20 h of maturation induced by Pam3CSK4 or LPS prior
to the addition of Ag, we observed substantial inhibition of MHCII Ag processing activity by Flt3L DCs (Fig. 2A). In contrast,
when DCs were incubated with IFN-b for 20 h prior to the addition of Ag, active MHC-II Ag processing was maintained (Fig.
2A). Similar results were observed with several concentrations of
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Samples were analyzed by SDS-PAGE and transferred to Immobilon
membranes (Millipore, Billerica, MA). Membranes were blocked using
PBS-T with 5% dry milk with the exception of anti-ubiquitin blots, which
were performed using 3% BSA in PBS-T. Primary Abs were specific for
actin (clone I-19; Santa Cruz Biotechnology, Santa Cruz, CA), C/EBP-b
(clone C-19; Santa Cruz Biotechnology), ubiquitin (biotinylated-P4D1;
Covance, Princeton, NJ), or I-Abb (KL295; American Type Culture Collection). Detection was performed using HRP-conjugated neutravidin
(Invitrogen) or HRP-conjugated secondary Abs (eBioscience) followed by
ECL Western blotting substrate (Thermo Scientific).
The Journal of Immunology
3119
FIGURE 2. TLR agonists and IFN-I induce functionally different states
of DC maturation with persistent MHC-II Ag processing only after IFN-I–
induced maturation. (A) Flt3L DCs (105/well) or (B) GM-CSF DCs (105/
well) were cultured for 20 h with medium, IFN-b (100 U/ml), Pam3CSK4
(10 nM), or LPS (50 ng/ml), washed, incubated with OVA for 4 h, and
fixed. CD4OVA T hybridoma cells were added to detect MHC-II–peptide
complexes, and supernatants were assessed for IL-2. Data represent the
means of triplicate samples 6 SDs in an experiment representative of three
independent experiments. (C) Flt3L DCs (105/well) were cultured for 20 h
with medium, IFN-a (100 U/ml), IFN-b (100 U/ml), Pam3CSK4 (10 nM),
LPS (50 ng/ml), CpG-A ODN (300 nM), or IFN-b (100 U/ml) plus
Pam3CSK4 (10 nM), washed, incubated with OVA (316 mg/ml) for 4 h, and
fixed. CD4OVA T hybridoma cells were added to detect peptide–MHC-II
complexes, and supernatants were assessed for IL-2. Data represent the
means of triplicate samples 6 SDs in an experiment representative of
two or more independent experiments. Significance was determined by
comparing treatment conditions with medium alone at the same Ag concentration using Student t test. *p , 0.05, **p , 0.01, ***p , 0.001,
****p , 0.0001.
IFN-b (10–1000 U/ml), Pam3CSK4 (1–100 nM), and LPS (10–100
ng/ml) (data not shown). We conclude that IFN-I induces a distinct
pattern of DC maturation that allows continued MHC-II Ag processing, in contrast to the inhibition of MHC-II Ag processing
after DC maturation induced by TLR agonists. We confirmed
these results in murine GM-CSF DCs and found that incubation
with Pam3CSK4 and LPS inhibited subsequent MHC-II Ag processing (e.g., after 20 h), but MHC-II Ag processing was maintained after 20 h of incubation with IFN-b (Fig. 2B). MHC-II Ag
processing was also maintained in Flt3L DCs after maturation
with IFN-a (Fig. 2C). CpG-A ODN activates TLR9 and drives
FIGURE 3. IFN-I differs from a TLR2 agonist in its ability to induce
human DC maturation without inhibiting MHC-II Ag processing. Human
mDCs (2 3 104 to 5 3 104) were cultured for 16–18 h with medium,
human IFN-a2a (300 U/ml), human IFN-b (300 U/ml), or Pam3CSK4 (10
nM) in the continued presence of GM-CSF (200 U/ml). DCs were stained
with anti–HLA-DR and (A) anti-CD86 or (B) anti-CCR7. Gating was
based on size and selection of HLA-DR–positive events. Data show the
MFI of cells from one representative donor of three independently assessed
donors. (C) Human mDCs (104) were treated with medium, IFN-a2a, or
Pam3CSK4 for 16–18 h, washed, and incubated with Ag85B protein and
F9A6 T hybridoma cells for 24 h, and supernatants were assessed for
IL-2. Data represent the means of triplicate samples 6 SDs from one of
two independent experiments. Significance was determined by comparing
results with IFN-a2a or Pam3CSK4 to the results with medium at the same
concentration of Ag85B using Student t test. ***p , 0.001.
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production of IFN-I, but this TLR agonist also inhibited MHC-II
Ag processing (Fig. 2C). Furthermore, the addition of IFN-b did
not prevent Pam3CSK4 from inhibiting MHC-II Ag processing
(Fig. 2C), indicating that inhibition by TLR agonists is a dominant
effect. DCs treated with TLR agonists expressed higher surface
CD80, CD86, and MHC-II (Fig. 1), indicating that the inhibition is
not due to toxicity and is specific for MHC-II Ag processing. In
these studies, we found that MHC-II Ag processing was inhibited
after maturation driven by IFN-inducing and non-IFN-inducing
TLR agonists, but not after maturation induced by IFN-I alone.
To expand the relevance of our findings, we confirmed these
results in human DCs. In primary human mDCs from peripheral
blood, incubation with IFN-a, IFN-b, and Pam3CSK4 induced
CD86 (Fig. 3A), CCR7 (Fig. 3B), CD80 (data not shown), and
HLA-DR (data not shown). Furthermore, human mDCs that were
preincubated with Pam3CSK4 for 16–18 h prior to Ag exposure
had decreased subsequent MHC-II Ag processing, but MHC-II Ag
processing was maintained after 16–18 h of preincubation with
IFN-a (Fig. 3C). The DCs were viable and responsive to stimulation, as indicated by upregulation of CCR7 induced by all of
3120
TYPE I IFN AND DC MATURATION
these agents (Fig. 3B). We conclude that IFN-I induces a distinct
DC maturation program that maintains MHC-II Ag processing
activity in multiple DC models, whereas maturation driven by
a TLR agonist leads to inhibition of subsequent MHC-II Ag
processing.
MHC-II synthesis is inhibited by TLR agonists but not by IFN-I
resent the means of three independent experiments per condition 6 SDs.
Values for (A)–(D) were normalized for expression of GAPDH, and significance was determined by comparing results with treatment conditions
and medium alone using Student t test. *p , 0.05, **p , 0.01, ***p ,
0.001. (E) Nuclear fractions were prepared from DCs treated for 20 h with
medium, IFN-b (100 U/ml), Pam3CSK4 (10 nM), or LPS (50 ng/ml) and
analyzed by Western blotting (10 mg/ lane) for actin (top panel, loading
control) and C/EBP-b (lower panel).
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FIGURE 4. Expression of mRNA for MHC-II and CIITA is inhibited by
TLR agonists but not IFN-I. (A) Flt3L DCs (4 3 106/well) were cultured in
a time course with IFN-b (200 U/ml) or Pam3CSK4 (10 nM). Total mRNA
was prepared and analyzed by qRT-PCR with primers for MHC-II (I-Abb).
Data represent the means of triplicate samples 6 SDs from one of three
independent experiments. (B and C) Flt3L DCs (3 3 106 to 5 3 106/well)
were cultured for 18–24 h with medium, IFN-a (100 U/ml), IFN-b (100 U/
ml), Pam3CSK4 (10 nM), LPS (50 ng/ml), CpG-A ODN (300 nM), or IFNb (100 U/ml) plus Pam3CSK4 (10 nM). Total mRNA was prepared and
analyzed by qRT-PCR with primers for (B) MHC-II (I-Abb) or (C) CIITA
(sequence shared by types I, III, and IV CIITA). Data represent the means
of two or more independent experiments per condition, as indicated, 6
SDs. (D) Wild-type or MyD882/2 DCs (3 3 106 to 4 3 106/well) were
cultured 24 h with medium, IFN-b (100 U/ml), Pam3CSK4 (10 nM),
poly(I:C), LPS (50 ng/ml), or CpG-A ODN (300 nM). Total mRNA was
prepared and analyzed by qRT-PCR with primers for MHC-II. Data rep-
Because IFN-I–induced DC maturation allowed continued MHCII Ag processing activity, in contrast to DC maturation induced by
TLR agonists, we hypothesized that DC maturation states induced
by these two stimuli would differ in regulation of MHC-II synthesis. Incubation of DCs with Pam3CSK4, LPS, or CpG-A ODN
strongly inhibited MHC-II mRNA (e.g., mRNA for the b-chain of
I-Ab, I-Abb), whereas IFN-b decreased MHC-II mRNA expression only slightly (Fig. 4A, 4B). The difference in inhibition of
MHC-II mRNA was particularly striking at the 24 h time point
(Fig. 4B), which corresponds kinetically with substantial progression to a mature DC phenotype. Addition of IFN-b did not
prevent the inhibition of MHC-II mRNA by Pam3CSK4 (Fig. 4B),
again indicating that the TLR-induced DC maturation response is
dominant over the effects of IFN-I. Transcription of MHC-II
mRNA is dependent on the transcription factor CIITA, which
exists in three different isoforms (types I, III, and IV; expressed
from a single gene with three promoters, pI, pIII, and pIV); type I
is the dominant CIITA form in DCs (31). Our quantitative RT-PCR
(qRT-PCR) analysis targeted a sequence common to all CIITA
isoforms, yielding a measure of total CIITA expression. We found
that TLR agonists Pam3CSK4, LPS, and CpG-A ODN inhibited
DC expression of CIITA mRNA, whereas CIITA expression
remained high after treatment with IFN-a or IFN-b (Fig. 4C).
Treatment with IFN-b did not prevent CIITA mRNA inhibition
by Pam3CSK4 (Fig. 4C). As a control, we found that all of these
reagents induced higher expression of CD86 (data not shown),
suggesting that the cells were viable and responsive to the different stimuli. We conclude that expression of CIITA and MHC-II
is inhibited after TLR-induced DC maturation but not after IFN-I–
induced DC maturation.
Because TLR agonists exerted a dominant inhibitory effect on
MHC-II mRNA expression, we tested the hypothesis that this
inhibition is downstream of MyD88 signaling. Poly(I:C) activates
MyD88-independent signaling through TLR3. Treatment with
poly(I:C) resulted in downregulation of MHC-II, suggesting that
the TRIF pathway can also mediate inhibition of MHC-II mRNA
transcription (Fig. 4D). Furthermore, Pam3CSK4 and CpG-A
ODN 2336 did not inhibit MHC-II synthesis in MyD882/2 DCs,
whereas poly(I:C) did (Fig. 4D). Notably, LPS inhibition of
MHC-II mRNA was largely reversed in MyD882/2 DCs, suggesting that LPS drives this inhibition primarily through MyD88
(Fig. 4D).
We have previously demonstrated that IFN-g–induced CIITA
expression is inhibited by TLR2 agonists in macrophages because
of the induction of C/EBP transcription factors (57). To explore
whether a similar mechanism may be used during DC maturation,
we treated DCs with agonists for 20 h, prepared nuclear extracts,
The Journal of Immunology
3121
and performed Western blots for C/EBP-b. LPS and Pam3CSK4
strongly induced the expression of both C/EBP-b isoforms, LAP
and LIP, whereas IFN-b induced only low levels of C/EBP-b (Fig.
4E). Taken together, these results suggest that TLR signaling by
the MyD88 and TRIF pathways in DCs induces C/EBP and
thereby inhibits CIITA and MHC-II synthesis. In contrast, IFN-I
induces only low levels of C/EBP-b, thereby allowing continued
synthesis of CIITA and MHC-II.
Regulation of peptide–MHC-II stability during DC maturation
FIGURE 5. Stability and endocytosis of MHC-II molecules in IFN-b–
treated DCs. (A) DCs (3 3 106 to 4 3 106/well) were cultured with medium, IFN-b (100 U/ml), Pam3CSK4 (10 nM), or LPS (50 ng/ml) for 24 h.
Total mRNA was prepared and analyzed by qRT-PCR with primers
for MARCH1. Data represent the means of three or more independent
experiments 6 SDs. Significance was determined for comparison of
agonists to medium or TLR agonists to IFN-b using Student t test. *p ,
0.05, ***p , 0.001. (B) DCs were treated with agonists for 24 h, cell
extracts were prepared, and immunoprecipitations were performed using
control (“C”) or anti-MHC class II (“Y3P”) Abs. Samples were analyzed
by SDS-PAGE followed by immunoblotting with anti-ubiquitin (top panel,
8 3 106 cells per lane) or anti–MHC-II (KL295, bottom panel, 5 3 105
cells/lane). Data are representative of two independent experiments. (C)
Flt3L DCs were cultured with medium, IFN-b (100 U/ml), Pam3CSK4 (10
nM), or LPS (50 ng/ml) for 24 h. DCs were pulsed with OVA(323–339)
peptide for 4 h, washed, and chased for varying amounts of time prior to
fixation. CD4OVA T hybridoma cells were added to detect peptide–MHCII complexes, and supernatants were assessed for IL-2. Data represent the
means of triplicate samples 6 SDs in an experiment representative of two
independent experiments. (D) MHC-II endocytosis was followed by the
loss of Ab bound to surface MHC-II molecules. After 24 h treatment with
medium, IFN-b (100 U/ml), or LPS (50 ng/ml), DCs were stained with
unconjugated anti–MHC-II Ab, washed, and then returned to culture.
Aliquots were removed at 0, 45, or 90 min, placed on ice, and stained with
Alexa Fluor 488-conjugated secondary Ab. Duplicate samples were analyzed by flow cytometry, and the specific MFI was normalized to the 0-h
time point.
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Maturation of DCs by TLR signaling involves decreased MHC-II
synthesis and subsequent Ag processing, but IFN-I does not inhibit
MHC-II synthesis and Ag processing (Figs. 2–4). We considered
whether differences in the regulation of Ag processing function
by IFN-I and TLRs might involve functions other than MHC-II
synthesis; for example, endocytosis of Ag or MHC-II or effects
on the stability of peptide–MHC-II complexes. We did not observe significant differences in Ag uptake or degradation after
treatment of DCs with IFN-I or TLR agonists within the time
frame and conditions of these experiments (data not shown), so
we focused our attention on MHC-II endocytosis and stability.
MHC-II targeting for degradation is driven in part by ubiquitination of MHC-II by MARCH1, an E3 ubiquitin ligase that is
expressed in immature DCs and downregulated during DC maturation in response to TLR agonists such as LPS (20, 22, 23, 25).
Because endogenous MARCH1 expression is difficult to detect
by Western blotting with available Abs (21), we performed
qRT-PCR for MARCH1 mRNA. Relative to immature DCs (incubated in normal medium), expression of MARCH1 mRNA
was inhibited in DCs that were matured with IFN-b, LPS, or
Pam3CSK4 for 24 h, although the degree of inhibition was less
with IFN-b than with Pam3CSK4 or LPS (Fig. 5A). Coincident
with the changes in MARCH1 expression, ubiquitination of
MHC-II molecules was drastically reduced in DCs matured with
IFN-b, Pam3CSK4, or LPS (Fig. 5B). Furthermore, MHC-II
degradation products observed by Western blotting of total cell
lysates were reduced for all matured cells versus immature DCs
(data not shown).
To evaluate the stability of peptide–MHC-II complexes expressed on the plasma membrane, DCs were treated with medium,
IFN-b, LPS, or Pam3CSK4 for 24 h, pulsed with OVA(323–
339) peptide, washed, and returned to culture for 0–31 h. Using
CD4OVA T hybridoma cells and IL-2 production as a readout, we
observed that peptide–MHC-II complexes were highly stable in
DCs after maturation with LPS or Pam3CSK4 (apparent t1/2 .31 h),
whereas these complexes declined rapidly in immature DCs or
DCs matured with IFN-b (apparent t1/2 ,7 h) (Fig. 5C). In parallel with the changes in stability of peptide–MHC-II complexes, MHC-II endocytosis was inhibited by LPS but not IFN-b
(Fig. 5D), suggesting that some MHC-II endocytosis proceeds
independent of MHC-II ubiquitination in DCs matured with IFN-I.
Taken together, these data indicate that MARCH1 expression and
MHC-II ubiquitination are decreased by all of these agonists,
whereas endocytosis of MHC-II and the short half-life of peptide–
MHC-II complexes are maintained after treatment with IFN-b,
distinct from the effects of the TLR agonists. Although MARCH1
expression and MHC-II ubiquitination are associated with MHC-II
turnover in immature DCs, they may not fully predict MHC-II
stability and Ag presentation phenotypes in DCs matured by different stimuli. Overall, these results indicate that peptide–MHC-II
complexes in IFN-I–matured DCs have a relatively short half-life,
similar to that of immature DCs and distinct from the much longer
half-life seen in TLR-matured DCs.
3122
TLR stimulation drives cell-surface localization of MHC-II,
whereas IFN-I produces MHC-II localization both at the cell
surface and in intracellular compartments
FIGURE 6. IFN-I induces less translocation of MHC-II to the cell
surface than TLR agonists. (A–E) Flt3L DCs (3 3 106 to 4 3 106) were
cultured for 24 h with medium, IFN-a (100 U/ml), IFN-b (100 U/ml),
Pam3CSK4 (10 nM), LPS (50 ng/ml), CpG-A ODN (300 nM), or IFN-b
(100 U/ml) plus Pam3CSK4 (10 nM). DCs were washed, fixed, permeabilized, and labeled with anti–MHC-II. Images shown are representative of three or more independent experiments. (F) Quantification of the
proportion of cells with punctate expression of MHC-II. Data represent the
means of three or more independent experiments 6 SDs. Treatment conditions were compared with medium alone to determine significance using
Student t test. ***p , 0.001, ****p , 0.0001.
positive vacuolar MHC-II Ag processing compartments, whereas
DCs treated with IFN-I continue to express MHC-II in these compartments, albeit at reduced levels in some cells.
To determine the extent of translocation of MHC-II from the vacuole to the cell surface, we assessed MHC-II colocalization with DC
surface markers CD11b and CD11c (CD11b/c). Colocalization of
MHC-II with CD11b/c was lowest in untreated cells (Fig. 7), increased
in cells treated with IFN-b (Fig. 7) or IFN-a (data not shown), and
was highest in cells treated with Pam3CSK4 (Fig. 7), LPS (Fig. 7), or
CpG-A ODN (data not shown). We conclude that DC maturation
by IFN-I is characterized by 1) sustained MHC-II intracellular localization in the setting of continued MHC-II synthesis and endocytosis and 2) higher MHC-II surface levels in parallel with decreased
degradation (Fig. 8). These unique features of MHC-II expression in
IFN-I–matured DCs may allow for continued access of MHC-II to
newly processed Ag in endocytic compartments in conjunction with
high levels of cell surface MHC-II and Ag presentation function.
Discussion
IFN-I shares with TLR agonists the ability to initiate DC maturation
as defined by enhanced expression of MHC-II, CD80, CD86, and
enhanced Ag presentation function in T cell assays. In our studies,
however, IFN-I and TLR agonists differed in their regulation of Ag
processing function subsequent to maturation due to differences in
MHC-II expression and localization in DCs. Expression of CIITA
and MHC-II mRNA was maintained in IFN-I–matured DCs but not
TLR-matured DCs. Similarly, MHC-II Ag processing function was
maintained after stimulation with IFN-I but was inhibited 20 h after
TLR stimulation. Intracellular MHC-II, required for active intracellular MHC-II Ag processing, was maintained after DC maturation in response to IFN-I but not after stimulation of DCs with TLR
agonists. Fig. 8 summarizes the unique phenotype of IFN-I–matured DCs in comparison with those of immature DCs and TLR
agonist-matured DCs. Together, these data establish that DC maturation by TLR agonists produces a subsequent shutdown of MHCII Ag processing, whereas DC maturation induced by IFN-I allows
continued MHC-II Ag processing function.
DC maturation is often considered an all-or-nothing program
in which DCs are either immature or become mature, but these
studies and others show that different stimuli result in different
DC maturation states. One issue is whether some different stimuli
induce differentiation toward qualitatively different phenotypes.
Another issue is whether DC maturation must be all-or-nothing (the
“binary model”) or whether some stimuli (e.g., IFN-I) may induce
a partially mature phenotype that reflects incomplete progression
along a standard maturation pathway. This partially mature phenotype may exhibit some but not all of the features induced by
agents (e.g., TLR agonists) that drive more complete DC maturation. Even if maturation is all-or-nothing at the single-cell level,
some stimuli may induce DC populations with an intermediate
phenotype that are composed of a mixture of immature and fully
mature cells. In contrast, our results suggest that, for some stimuli,
maturation is a continuum rather than a binary state; for example,
IFN-I provides a partial maturation signal for DCs that results in
increased Ag processing and expression of MHC-II and costimulatory molecules without the subsequent shutdown that occurs
with TLR stimulation. It appears that this reflects at least in part an
intermediate state of maturation of some cells, as IFN-I induced
DCs to express MHC-II simultaneously in vacuolar compartments
and on the cell surface, distinct from both immature and TLRmatured phenotypes. Moreover, in dose-response studies, IFN-I
failed to induce the full TLR-driven maturation phenotype even
at high concentrations at which other IFN-I–induced changes (e.g.,
costimulator expression, MHC-II expression) were at plateau,
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During MHC-II Ag processing, MHC-II molecules are loaded with
peptides in endocytic vacuolar compartments and then translocated
to the cell surface for MHC-II Ag presentation. Immature DCs
express MHC-II molecules in intracellular compartments, but
MHC-II expression is shifted to the cell surface after DC maturation (28). Given the different MHC-II regulation seen during DC
maturation driven by TLR stimulation versus that by IFN-I, we
assessed whether these stimuli differ in regulation of MHC-II
localization. Immature DCs (unstimulated, medium control) primarily expressed MHC-II in intracellular vacuoles, which appeared as punctate structures by fluorescence microscopy (Fig.
6A). After incubation with IFN-b (Fig. 6B, 6C) or IFN-a (data not
shown), many DCs continued to express MHC-II in intracellular
vacuoles, although more DCs expressed MHC-II at the cell surface, and some DCs expressed MHC-II simultaneously at the cell
surface and in vacuolar structures (Fig. 6C). In contrast, incubation with Pam3CSK4 (Fig. 6D) or LPS (Fig. 6E) resulted in DCs
that expressed MHC-II predominantly on the cell surface with few
noticeable punctate structures. Quantification of the percentage of
cells with punctate MHC-II staining showed that intracellular
MHC-II localization was maintained more in DCs matured with
IFN-I than in DCs matured with TLR agonists (Fig. 6F).
To characterize further the differences between populations
of DCs after treatment with different stimuli, we used imaging flow
cytometry quantitatively to determine colocalization of MHC-II
with lysosomal markers LAMP1 and LAMP2 (LAMP1/2). Colocalization of MHC-II with LAMP1/2 was highest in untreated
cells (Fig. 7), persisted at slightly lower levels in cells treated
with IFN-b (Fig. 7) or IFN-a (data not shown), and was substantially decreased by Pam3CSK4 (Fig. 7), LPS (Fig. 7), or
CpG-A ODN (data not shown). Notably, DC maturation driven by
TLR agonists also resulted in a coalescence of LAMP1/2 staining
(Fig. 7). These observations suggest that DCs induced to mature
with TLR agonists no longer express MHC-II in the LAMP1/2-
TYPE I IFN AND DC MATURATION
The Journal of Immunology
3123
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FIGURE 7. Intracellular MHC-II is decreased by TLR agonists more than IFN-b. DCs (3 3 106 to 4 3 106) were incubated for 22–24 h with medium,
IFN-b (100 U/ml), Pam3CSK4 (10 nM), or LPS (50 ng/ml). Cells were surface-stained with anti-CD11b, fixed, permeabilized, labeled with anti–MHC-II
and LAMP1/2, and analyzed with an Amnis Imagestream imaging flow cytometer at 360 magnification. Gating was based on single cells that were in focus
and expressed MHC-II and CD11b. (A) Left panels, Brightfield, MHC-II (red), LAMP1/2 (green), and merged MHC-II and LAMP1/2 images from the
median colocalization bin. Right panels, Brightfield, MHC-II (red), CD11b/c (green), and merged MHC-II and CD11b/c images from the median
colocalization bin. (B) Amnis IDEA software was used to calculate colocalization of MHC-II with LAMP1/2 or CD11b/c in cells with regions of bright
detail for those markers. Upper panels, Distribution of colocalization scores in DC populations for MHC-II and LAMP1/2. Lower panels, Distribution of
colocalization scores in DC populations for MHC-II and CD11b/c. Results represent one of four independent experiments with similar results.
3124
TYPE I IFN AND DC MATURATION
further suggesting that IFN-I and TLR agonists drive qualitatively
different states of DC maturation. Alternatively, this difference in
maturation phenotypes may represent a kinetic difference in the
induction of maturation of DCs; MyD88 signaling may rapidly
drive maturation, resulting in downregulation of MHC-II Ag
processing at early time points, whereas IFN-I induces maturation
over a longer time course. Although some of these distinctions
remain to be determined, we propose that IFN-I induces a state of
partial maturation that is qualitatively distinct from the maturation
state driven by TLR stimulation.
Young and colleagues (22) demonstrated that pDCs and mDCs
differ in the regulation of MHC-II after TLR9 stimulation. In
some ways, the maturation response of mDCs to IFN-I stimulation
(this study) is similar to the maturation response of pDCs to TLR9
stimulation (22). Unlike mDCs stimulated with TLR ligands, both
mDCs treated with IFN-I and pDCs treated with CpG DNA express higher levels of MHC-II at the surface without a concomitant shutdown of MHC-II synthesis (Fig. 4 and Ref. 22) or MHCII Ag processing (Fig. 3 and Ref. 22). In contrast to the observations with pDCs, however, we noted a more significant reduction in MARCH1 expression and MHC-II ubiquitination after
maturation of mDCs with IFN-I (Fig. 5). Thus, there are both
parallels and differences between these systems.
The unique DC maturation state induced by IFN-I may be
important in pathological situations that are driven primarily by
IFN-I (e.g., certain viral infections) or where there is sequential
exposure to IFN-I and then TLR agonists (Fig. 8). In the course
of infection, some DCs may be exposed to IFN-I before they
encounter PAMPs (e.g., TLR agonists) and Ags expressed by
pathogens, and it may be important to avoid premature shutdown
of Ag processing before DCs are exposed to pathogens. DCs
generally encounter PAMPs at the time they encounter pathogen
Ags, as both are contained within the pathogen, so PAMP signaling can be coordinated with production of a final cohort of
peptide–MHC-II complexes that will provide presentation of
pathogen Ags, followed by shutdown of Ag processing. In contrast, pathogen-induced IFN-I is a host-derived molecule that may
reach and stimulate DCs that have not been exposed to pathogen
Ags; if Ag processing shutdown were to occur in this scenario, it
would focus Ag presentation on an antigenic repertoire missing
pathogen-derived Ags, indicating the need for broader kinetic
sampling relative to the time of IFN-I exposure. Accordingly, our
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FIGURE 8. Model for MHC-II synthesis, degradation, localization, and function for different DC maturation states induced by medium, IFN-I, or TLR
agonists. Efficient Ag processing depends on MHC-II synthesis and MHC-II localization to endocytic Ag processing compartments, as is observed for
immature and IFN-I–treated cells. Intracellular MHC-II in immature and IFN-I–matured DCs results from synthesis and internalization of MHC-II. Efficient Ag presentation is supported by high MHC-II expression at the cell surface, as is observed for IFN-I and TLR agonist matured cells. Because of
decreased MARCH1 activity, cells treated with either IFN-I or TLR agonists exhibit reduced MHC-II degradation and increased surface expression. In the
case of IFN-I–matured cells, we propose that the enhanced surface expression may be achieved in part by recycling of MHC-II molecules that, in the
absence of MARCH1-mediated ubiquitination, are not sorted for degradation in lysosomes. Physiological interpretations are indicated below the horizontal
line. In the absence of any stimuli, immature cells continue to sample Ag and rapidly turn over the peptide–MHC-II complexes that are expressed at the cell
surface. In the case of TLR agonists, stimulation occurs at the time of direct exposure of DCs to microbes. Thus, microbial Ags will be processed at the time
of the TLR stimulus, leading to a cohort of peptide–MHC-II complexes that are presented for a prolonged period after DC maturation, loss of Ag processing
function, and MHC-II stabilization. In contrast, IFN-I induced DC maturation is not necessarily linked with microbial Ag encounter. Thus, continued Ag
processing by IFN-I–matured DCs may enable them to continue to sample for microbial Ags to ensure presentation of these Ags, even if their sampling is
not kinetically linked with the IFN-I stimulus.
The Journal of Immunology
results demonstrate that IFN-I initiates DC maturation without
inhibiting Ag processing, allowing DCs to both increase MHC-II
expression and continue to process Ags at sites of infection. The
MyD88-driven maturation pathway is dominant over the IFN-I–
induced maturation program, as we observed that simultaneous
signaling by IFN-I and a MyD88-dependent TLR agonist drives
shutdown of MHC-II Ag processing. Thus, if DC maturation is
initiated by exposure to IFN-I, subsequent exposure to PAMPs
may drive further maturation to produce a final cohort of pathogen-associated peptide–MHC-II complexes, followed by shutdown of Ag processing, focusing presentation on Ags encountered
near the time of TLR stimulation.
3125
20.
21.
22.
23.
Acknowledgments
We thank Leola Jones for technical assistance. Paul Roche kindly provided
protocols and advice. J558L cells were a kind gift from Ira Mellman
(Genentech), courtesy of David Gray (University of Edinburgh).
24.
25.
Disclosures
The authors have no financial conflicts of interest.
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TYPE I IFN AND DC MATURATION