This information is current as
of June 17, 2017.
Long-Peptide Cross-Presentation by Human
Dendritic Cells Occurs in Vacuoles by
Peptide Exchange on Nascent MHC Class I
Molecules
Wenbin Ma, Yi Zhang, Nathalie Vigneron, Vincent
Stroobant, Kris Thielemans, Pierre van der Bruggen and
Benoît J. Van den Eynde
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The Journal of Immunology is published twice each month by
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Copyright © 2016 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2016; 196:1711-1720; Prepublished online 20
January 2016;
doi: 10.4049/jimmunol.1501574
http://www.jimmunol.org/content/196/4/1711
The Journal of Immunology
Long-Peptide Cross-Presentation by Human Dendritic Cells
Occurs in Vacuoles by Peptide Exchange on Nascent MHC
Class I Molecules
Wenbin Ma,*,†,‡ Yi Zhang,*,‡,x,{ Nathalie Vigneron,*,†,‡ Vincent Stroobant,*,‡
Kris Thielemans,‖ Pierre van der Bruggen,*,†,‡ and Benoı̂t J. Van den Eynde*,†,‡
C
ross-presentation is the process whereby antigenic
peptides derived from exogenous Ags are presented by
dendritic cells (DCs) on their MHC class I (MHC-I)
molecules. This process is needed for the induction of CD8
T cell responses to Ags derived from tumors or from pathogens that
do not infect DCs (1–3). Cross-presentation is key to the development of vaccines aimed at inducing CD8 responses against
cancer or HIV. A promising vaccine approach pioneered by Melief
et al. (4, 5) is based on synthetic long peptides, which need to be
cross-presented and therefore results in efficient presentation of
the antigenic peptide by DCs only, as opposed to short peptides,
which can load any cell and therefore induce tolerance. Therapeutic vaccines based on long peptides have shown clear promise
in preclinical models (6), and recently showed clinical efficacy in
patients with human papillomavirus–induced neoplasia (7). Contrasting with the advanced clinical development of long-peptide
*Ludwig Institute for Cancer Research, Brussels B-1200, Belgium; †Walloon Excellence in Life Sciences and Biotechnology, Brussels B-1200, Belgium; ‡de Duve Institute,
Université Catholique de Louvain, Brussels B-1200, Belgium; xThe Biotherapy Center,
The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan,
China; {Department of Oncology, The First Affiliated Hospital of Zhengzhou University,
Zhengzhou 450052, Henan, China; and ‖Laboratory of Molecular and Cellular Therapy,
Department of Physiology and Immunology, Vrije Universiteit Brussel, Brussels B-1090,
Belgium
Received for publication July 13, 2015. Accepted for publication December 14, 2015.
This work was supported by grants from the Ludwig Institute for Cancer Research,
the Walloon Region (Programme d’Excellence CIBLES), the Fonds National de la
Recherche Scientifique, the Fondation contre le Cancer, the Fonds Maisin, and the
Walloon Excellence in Life Sciences and Biotechnology Program. N.V. was a postdoctoral researcher with the Fonds National de la Recherche Scientifique.
Address correspondence and reprint requests to Prof. Benoı̂t J. Van den Eynde,
Ludwig Institute for Cancer Research, de Duve Institute, Université Catholique de
Louvain, Avenue Hippocrate 75, Bte. B1.74.03, Brussels B-1200, Belgium. E-mail
address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: DC, dendritic cell; MHC-I, MHC class I; Mo-iDC,
monocyte-derived immature DC; siRNA, small interfering RNA; SPP, signal peptide
peptidase; TAP, transporter associated with Ag processing.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501574
vaccination, relatively little is known about the pathway used for
cross-presentation of long peptides (8–11).
The pathway used for cross-presentation appears to vary
according to the nature of the exogenous Ag. In the mouse, two
main cross-presentation pathways have been proposed: cytosolic
and vacuolar. In the cytosolic pathway, the Ag internalized by
endocytosis is transferred to the cytosol, from which it follows the
classical MHC-I processing route. This involves processing by the
proteasome and translocation of the resulting peptide by transporter
associated with Ag processing (TAP) into the endoplasmic reticulum (ER), where it combines with newly synthesized MHC-I
molecules. A proposed variation of the cytosolic pathway involves TAP-mediated transport of the cytosolic peptide back to the
phagosome or the endolysosome, where it would combine with
MHC-I molecules. The latter can be routed to phagosomes or to
endolysosomes by SEC22B or CD74 (invariant chain), respectively
(12, 13). Until recently, the missing piece in the cytosolic pathway
was the transporter in charge of the transfer of Ags from the
endosome to the cytosol. However, recent evidence has implicated
SEC61 in this process (14). In the vacuolar pathway, there is no
need for Ag transfer across membranes, because the whole processing happens in vacuoles, including cleavage of the internalized protein by endolysosomal peptidases and loading of the
resulting peptide onto MHC-I molecules. This pathway is therefore independent from the proteasome and from TAP. Mechanistic
studies of the vacuolar pathway are limited. It is unclear whether
MHC-I molecules used in the vacuolar pathway are recycled from
the cell surface or are newly synthesized (13, 15–19).
Materials and Methods
Cell lines and CTL clones
T2 cells were given by A. Hill (Oxford University), and T1 cells were given
by P. Cresswell (Yale University). We used two gp100209–217-specific CTL
clones: EB81-CTL-606C/2.1 (clone 7) (20), which was used in Figs. 1 and
2A–C, and LB2686-CTL-811/327.4 (given by P. Coulie, de Duve Institute), which was used in Figs. 2D–G, 3, and 4. The RU134 – 42-specific
CTL clone 381/84 was described previously (21). The Melan-A26–35specific CTL clone CTL CP50-549/18 was given by P. Coulie. CTL clone
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Cross-presentation enables dendritic cells to present on their MHC class I molecules antigenic peptides derived from exogenous
material, through a mechanism that remains partly unclear. It is particularly efficient with long peptides, which are used in cancer
vaccines. We studied the mechanism of long-peptide cross-presentation using human dendritic cells and specific CTL clones against
melanoma Ags gp100 and Melan-A/MART1. We found that cross-presentation of those long peptides does not depend on the proteasome or the transporter associated with Ag processing, and therefore follows a vacuolar pathway. We also observed that it makes
use of newly synthesized MHC class I molecules, through peptide exchange in vesicles distinct from the endoplasmic reticulum and
classical secretory pathway, in an SEC22b- and CD74-independent manner. Our results indicate a nonclassical secretion pathway followed
by nascent HLA-I molecules that are used for cross-presentation of those long melanoma peptides in the vacuolar pathway. Our results
may have implications for the development of vaccines based on long peptides. The Journal of Immunology, 2016, 196: 1711–1720.
1712
A10, which recognizes peptide MAGE-A3168–176 presented by HLA-A1,
was derived in-house (22).
Generation of monocyte-derived immature DCs
PBMCs were isolated from whole blood of hemochromatosis patients with
Ficoll gradient (Lymphoprep; Axis-Shield PoC), after approval from the
Commission d’Ethique Biomédicale Hospitalo-Facultaire from the Université Catholique de Louvain institutional review board. Monocytes were
enriched on Percoll as described (23) and isolated by adherence. After
washing away nonadherent cells with PBS, adherent cells were kept in
culture in RPMI 1640 medium supplemented with 10% FCS, 200 U/ml
human IL-4 (made in-house) and 70 ng/ml GM-CSF (Leukine [sargramostim]).
The cultures were fed with fresh medium and cytokines every 2–3 d. The
monocyte-derived immature DCs (Mo-iDCs) were harvested and used on
days 5 to 7 of differentiation.
Abs and reagents
LONG-PEPTIDE CROSS-PRESENTATION PATHWAY
stripped with 50 mM citric acid (pH 3) for 2 min on ice and neutralized
immediately with 150 mM NaH2PO4 (pH 10.5). After washing, cells
were incubated at 37˚C or 20˚C. Aliquots were removed at various
intervals and placed on ice. Recycled HLA-I/Ab complexes were detected with flow cytometry using Alexa-647 conjugated Donkey antimouse IgG.
HLA-class I secretion
For HLA-class I secretion analysis, DCs were first stripped with 50 mM
citric acid for 2 min. After neutralization and wash, cells were incubated at
20˚C or 37˚C in culture medium. Aliquots were removed at various intervals and placed on ice. Reappearance of HLA-class I molecules was
detected with Alexa-633 conjugated mAb W6/32 and analyzed by flow
cytometry.
Lentivirus production and cell transduction
mRNA preparation and transfection
Surface HLA-A2 reconstitution and peptide affinity estimation
EGFP-, gp100-, and ICP47-encoding cDNAs were first cloned into the
backbone of pST1-A(120) (25). Next, the plasmids were linearized with
SapI, and mRNA was prepared as described (26). Electroporation was
performed in a 4-mm gap electroporation cuvette using gene pulse Xcell
electroporation system (Bio-Rad) with 2.5 million DCs resuspended in
200 ml OPTI-MEM I no phenol red (Life Technologies) containing 10 mg
mRNA, under the following conditions: mode, exponential decay; voltage,
300 V; capacitance, 150 mF; resistance, ‘ V, resulting in a pulse time of
∼10 ms.
For the reconstitution of cell surface HLA-A2, T2 cells were first
stripped with 50 mM citric acid for 2 min. After neutralization and
washing, cells were incubated with b2-microglobulin (2.5 mM) and
short peptides (10 mM) at 18˚C for 20 h. For peptide affinity estimation
after acid-stripping, T2 cells were incubated with b2-microglobulin
(2.5 mM) and serially diluted peptides at 18˚C for 3 h. Next, surface
HLA-A2 was detected with FITC-conjugated conformation-dependent
anti–HLA-A2 murine mAb BB7.2, and analyzed by flow cytometry.
Peptide concentrations were then plotted against the mean fluorescence
intensity of surface HLA-A2. The concentration needed to restore surface
HLA-A2 molecules to 50% of the maximum was calculated (EC50). The
maximum level of HLA-A2 stabilization achieved with each peptide
relative to that obtained with peptide gp100205-217 was also calculated
(relative efficacy).
Cross-presentation assay
CD74, Sec22b silencing
CD74-targeted 3-RNAi mix (CD74HSS190576, CD74HSS190577,
CD74HSS190578, equally mixed) and its control (nontargeting negative
control medium GC 12935-300) stealth small interfering RNA (siRNA)
were from Invitrogen. Sec22b-targeted siRNA (on-target plus SMARTpool siRNA L-011963-00-0005) and its control (non-targeting SiControl)
were from Dharmacon. To knock down CD74, DCs were transfected with
1 mM siRNA on day 3 after differentiation and assayed for their ability of
cross-presentation 72 h after transfection. To knock down Sec22b, DCs
were transfected with 1 mM siRNA on day 4 after differentiation and
assayed for their ability of cross-presentation 48 h after transfection.
Efficacy of knocking down was analyzed by Western blot with cells
collected at the same time as the cross-presentation assay. siRNA were
delivered into cells by electroporation as described above for mRNA
transfection.
HLA-I recycling
Mo-iDCs were first incubated with murine mAb W6/32 (40 mg/ml) at
4˚C. After thorough washing, cells were incubated at 20˚C or 37˚C for
12 min in the presence of 220 mM of primaquine. Next, the cells were
Results
Human Mo-iDCs cross-present the long gp100 peptide along a
vacuolar pathway
We used a 44-aa-long peptide derived from melanocytic protein
gp100 (gp100184–227) to study cross-presentation of long peptides
by hDCs (Fig. 1). After a 2-h incubation with this peptide at 37˚C,
HLA-A2–positive Mo-iDCs stimulated the production of IFN-g
by a CTL clone recognizing the HLA-A*0201–restricted epitope
gp100209–217 comprised in long peptide gp100184–227. We observed no cross-presentation when the long peptide was incubated
with Mo-iDC at 4˚C, or with fixed Mo-iDC (Fig. 1A, B). We
performed a two-step experiment to exclude the possibility that
the long peptide was processed in the extracellular milieu and
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Target cells were pulsed with long peptide (5 mM) for 2–4 h at the indicated temperatures in complete medium, and cocultured with CTL
after washing. CTL activation was evaluated in a cytokine production
assay or a degranulation assay. For IFN-g and TNF production assay,
40,000–50,000 Ag-loaded target cells were cocultured with 10,000–
20,000 CTLs in round-bottom 96-well plates in triplicate. Then IFN-g or
TNF was quantified by ELISA in the supernatant after 18 h. Degranulation assay was done by coculturing target cells with CTLs in the
presence of monensin and FITC-labeled mouse anti-human CD107a and
CD107b Ab at 20˚C. The percentage of FITC-positive T cells among the
CD8+ T cells was determined by FACS after 3 h of coculture. To study
the effect of inhibitors, unless stated otherwise, DCs were treated with
drugs for 30 min before and during the 2 h of pulse with the long
precursor peptides.
Lentiviral vectors were derived from pCCLsin.PPT.hPGK.GFP (27).
Control pTM895 was generated by inserting, in the backbone pCCLsin.
PPT.hPGK.GFP, an IRES from TMEV (28) and the eGFP coding sequence downstream of the PGK promoter. The ICP47-coding cDNA was
cloned from pBJ1-Neo (gifted by H.G. Rammensee, University of
T€ubingen) into pTM895 upstream the eGFP-IRES sequence. pTM945
vector, which was used to deliver peptides into the ER in a TAPindependent manner, was generated by inserting in the backbone of
the same vector: a CMV promoter, a multicloning site, an IRES from
TMEV and an mCherry coding sequence (28). The coding sequences of
IL-2 signal sequence-peptide fusion polypeptide were cloned into the
multicloning site of pTM945. Vectors pTM897, pTM895, and pTM945
were provided by Prof. Thomas Michiels (de Duve Institute, Brussels,
Belgium). Lentiviral particles were produced upon cotransfection of
HEK-293T cells with vector plasmid, and plasmids pMDLg/pRRE#54,
pRSV-Rev and pMD2.VSVG (provided by Luigi Naldini, Ospedale San
Raffaele, Milan, Italy) (27). Helper lentiviral particles SIVmac were
produced upon cotransfection of HEK-293T cells with envelope plasmid
pMD2.VSVG and SIVmac package construct pSIV3+ (gifted by Prof.
Cimarelli, Ecole Normale Supérieure de Lyon, France) (23). To transduce Mo-iDCs, cells were incubated with both viral particles on day 4 of
differentiation. Next, cells were harvested and analyzed on day 7.
T2 cells were transduced with lentiviral particles by spin infection at
2,400 rpm and 32˚C for 90 min in the presence of 8 mg/ml of polybrene.
Forty-eight hours after transduction, mCherry-positive cells were sorted,
seeded to 2.5 105 cells/ml, and cultured for 24 or 48 h. Crosspresentation assay was then performed.
Fluorochrome-conjugated secondary Abs were obtained from Life Technologies. Alexa-633 (Life Technologies) conjugated and nonconjugated murine mAb W6/32 were produced in-house. FITC-conjugated mouse antihuman CD107a/CD107b, and FITC-conjugated anti-HLA-A2 murine mAb
BB7.2 were from BD Pharmingen. Mouse anti-human b-actin AC15 and
mouse anti-human SEC22B were from Sigma-Aldrich. Rabbit anti-human
CD74 polyclonal Ab Matilda was given by P. Cresswell (24). All inhibitors
were obtained from Sigma-Aldrich. Long peptide gp100184–227 was given
by P. Cresswell. The other peptides were synthesized in-house.
The Journal of Immunology
loaded directly onto surface HLA-A*0201 molecules in our
experimental setup. We first incubated the long peptide for 2 h at
37˚C with HLA-A*0201–negative Mo-iDCs. We then collected
and incubated the supernatant with HLA-A*0201–positive
Mo-iDCs in a cross-presentation assay performed at 4˚C or 37˚C.
If the long peptide was processed in the extracellular milieu
during the first incubation, the supernatant of this first step
should activate the CTL when loaded at 4˚C onto HLA-A*0201positive DC in the second step. This was not the case (Fig. 1C).
This result therefore excluded the processing of the long peptide
1713
by extracellular proteases. To strengthen this conclusion further,
we blocked endocytosis by inhibiting actin polymerization with
cytochalasin B (Supplemental Fig. 1A). We observed a dosedependent inhibition of cross-presentation of the long peptide.
Because cytochalasin B inhibits endocytosis but not proteolysis,
this result further confirmed that cross-presentation of the long
peptide in our system depends on endocytosis and involves an intracellular processing step.
To determine whether long-peptide cross-presentation followed
a cytosolic or a vacuolar pathway, we tested the effect of proteasome
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FIGURE 1. Human Mo-iDCs cross-present the long gp100 peptide in the vacuolar pathway (A, left panel) HLA-A*0201–positive Mo-iDCs can stimulate
gp100209–217-specific CTL clone 7 to produce IFN-g after a 2-h pulse with long peptide gp100184–227 at 37˚C but not at 4˚C. As controls (right panel), MoiDCs pulsed with short epitope peptide gp100209–217 stimulate CTL clone 7 at both 37˚C and 4˚C. (B) Fixed Mo-iDCs cannot cross-present the long gp100
peptide. Mo-iDCs were fixed with 0.004% glutaraldehyde for 45 s and then pulsed for 2 h with the long gp100 peptide (left panel) or the final epitope (right
panel). CTL was added and the production of IFN-g was measured after an overnight incubation. (C) The processing of the long gp100 peptide is not
performed by extracellular peptidases. The long gp100 peptide was first incubated with HLA-A*02012 DCs at 37˚C for 2 h. Next, the supernatant was
collected and pulsed onto HLA-A*0201+ DCs at 37˚C or 4˚C for 2 h. After washing, the supernatant-pulsed DCs were assayed for their ability to stimulate
IFN-g production by gp100209–217-specific CTL clone 7. (D) Proteasome inhibition by epoxomicin (0.5 mM) does not block cross-presentation of the long
gp100 peptide (left panel), whereas it blocks endogenous presentation of the same epitope by DCs transfected with gp100 mRNA (right panel). (E) MoiDCs (both HLA-A*0201+ and HLA-B*51+) were transfected with ICP47 or EGFP mRNA by electroporation. Three hours after transfection, they were
incubated with long peptide gp100184–227 and tested for their ability to stimulate CTL 7 two hours later (left panel). As a control for TAP inhibition by
ICP47, endogenous presentation of the TAP-dependent epitope derived from ubiquitous self-protein RU1 was tested with an RU134–42/HLA-B51–specific
CTL clone (right panel) (21). Values are means 6 SD of triplicates from a representative experiment repeated at least three times. ND, not detected.
1714
and TAP inhibition. We observed that cross-presentation of the
long gp100 peptide was not reduced by inhibition of the proteasome with epoxomicin (Fig. 1D) or MG132 (Supplemental
Fig. 1B), whereas endogenous presentation of the same gp100
peptide introduced by mRNA electroporation was blocked by
epoxomicin (Fig. 1D). TAP blockade with viral inhibitor ICP47,
which was introduced into the cells by mRNA electroporation,
did not inhibit the cross-presentation of the long gp100 peptide
either, whereas endogenous presentation of an ubiquitous selfpeptide (RU134–42) was blocked by ICP47 (Fig. 1E). We concluded that cross-presentation of the long peptide occurred in the
vacuolar pathway.
Cross-presentation of the long gp100 peptide is favored by
suboptimally-loaded HLA-I molecules
that the effect of (Z-LL)2-ketone on ICP47-transfected DC was
not caused by toxicity or inhibition of the secretion of HLA-I or
costimulatory signals. This was further confirmed by analyzing the
expression of costimulatory molecules such as CD80 and CD86,
and the production of cytokines such as IL-10 and IL-12 by
(Z-LL)2-ketone treated cells (Supplemental Fig. 2). That (Z-LL)2ketone efficiently reduced the number of suboptimally loaded
HLA-A2 molecules in TAP-deficient cells was confirmed by the
decreased expression of HLA class I—which are mainly HLA-A2
in these cells—at the surface of T2 cells treated with (Z-LL)2ketone (Fig. 2D). Thus, these results supported the second model
positing that increased cross-presentation observed after TAP inhibition resulted from increased abundance of suboptimally
loaded HLA-A*0201 molecules available for peptide exchange.
The cross-presented epitope is seemingly not delivered into the
ER, otherwise it would be able to load empty HLA-A*0201
molecules in the ER and cross-presentation should be increased by
(Z-LL)2-ketone. It follows that peptide exchange on suboptimally
loaded HLA-A*0201 molecules should occur in a post-ER compartment. In line with this, the cross-presentation of long peptide
gp100184–227 by T2 cells was also inhibited dramatically by
(ZLL)2-ketone (Fig. 2E), indicating that, like in DC, it relied on
suboptimally loaded HLA-A*0201 molecules.
To determine whether suboptimally loaded HLA-A*0201
molecules were also needed for cross-presentation of long peptides in TAP-competent cells, we took advantage of the fact that
ERAP, an aminopeptidase that trims antigenic peptides in the ER,
is required to produce an optimal repertoire of stable peptide–
MHC-I complexes (35). We observed that ERAP inhibition
by L-leucinethiol dramatically increased cross-presentation of the
gp100 epitope by Mo-iDCs, whereas endogenous presentation
of the same gp100 epitope after electroporation of Mo-iDCs with
gp100 mRNA was almost completely blocked (Fig. 2F). Again, no
significant difference was observed concerning the expression of
costimulatory signals by cells after L-leucincethiol or vehicle
treatment (Supplemental Fig. 2). These results further supported
the essential role of suboptimally loaded HLA-A*0201 molecules
for cross-presentation of long peptides.
To confirm further the role of suboptimally loaded HLA-I
molecules, we used lentiviral constructs enabling the delivery of
peptides into the ER in a TAP-independent manner, through fusion
to the signal sequence of IL-2 (36). From the list of HLA-A*0201binding peptides eluted from TAP-deficient T2 cells (37), we
selected two short peptides with low/intermediate affinity for
HLA-A*0201 and one peptide with high affinity (Fig. 2G). As a
negative control, we also used a peptide known to bind to HLA-A1
but not to HLA-A2 (MAGE-A3168-176; EVDPIGHLY). We reasoned that after introduction of the constructs in T2 cells, the
high amounts of ER-delivered peptides should modulate crosspresentation efficiency, with the low–intermediate HLA-A2
binders (suboptimal peptides) favoring cross-presentation, as opposed to the high HLA-A2 binder and the nonbinder peptides.
This is exactly what we observed (Fig. 2G). As a control for the ER
delivery of the peptides, we observed the HLA-A1–restricted peptide was efficiently presented to specific CTL in a TAP-independent
manner (Supplemental Fig. 4). These results supported our model
that cross-presentation occurs by peptide exchange on suboptimally loaded HLA-I molecules.
Cross-presentation depends on nascent HLA-I molecules
The results obtained so far suggest that cross-presentation of the
long gp100 peptide occurs through peptide exchange on suboptimally loaded HLA-A*0201 molecules in a post-ER compartment containing the processed epitope derived from the
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We were intrigued by the consistent 2-fold increase of crosspresentation observed after TAP inhibition (Fig. 1E). We performed an extensive phenotyping of Mo-iDCs to verify that ICP47
transfection did not activate the production of IL-10 and IL-12,
nor did it modify the expression of costimulatory molecules
(Supplemental Fig. 2). Although it is well established that DCs
are the most efficient cross-presenting cells, other cells might
cross-present with a low efficiency. We reasoned that if the crosspresentation of long peptides was indeed favored by TAP blockade, it might be increased or revealed in other cells by the loss of
TAP. We therefore compared the cross-presentation of the long
gp100 peptide by TAP-deficient T2 cells and their parental TAPcompetent T-B lymphoblast hybrid cells T1 (29). We found that
T2 cells cross-presented long peptide gp100184–227 quite efficiently in a proteasome-independent manner, and much better than
its parental cells T1 (Fig. 2A, B). The cross-presentation ability of
T2 cells was not modified by ICP47 transfection, which excluded
off-target effects of ICP47 and further validated TAP inhibition as
responsible for the increased cross-presentation observed after
transfection of ICP47 in Mo-iDCs (Supplemental Fig. 3A). Because TAP blockade results in a shortage of peptide supply in the
ER, we considered two possible explanations for the increased
cross-presentation: either more empty HLA-A*0201 molecules
became available in the ER to load the cross-presented epitope,
provided it can reach the ER, or the ER contained more HLAA*0201 molecules loaded with suboptimal peptides derived from
signal peptides (30–33). Those suboptimal HLA-peptide complexes might then exit the ER and reach another compartment,
where the preloaded suboptimal peptides would be exchanged for
the cross-presented epitope.
If the increased cross-presentation after TAP inhibition was due
to more empty HLA-A*0201 molecules in the ER, then blocking
the release of signal peptides into the ER lumen by inhibiting
signal peptide peptidase (SPP) should result in even more empty
HLA-A*0201 molecules and a further increased cross-presentation.
Conversely, if increased cross-presentation was due to more
HLA-A*0201 molecules loaded with suboptimal signal peptides,
inhibiting signal peptide release from the ER membrane should
reduce suboptimally loaded HLA-A*0201 molecules and reduce
cross-presentation in TAP-deficient cells. To inhibit SPP, we
treated Mo-iDCs with (Z-LL)2-ketone before electroporating
ICP47 or EGFP mRNA and incubating with long peptide
gp100184–227. SPP inhibition completely prevented the increase
of cross-presentation induced by TAP inhibition (Fig. 2C, left),
which was confirmed by the failure of the very same ICP47transfected DCs to present the HLA-A*0101–restricted epitope
MAGE-A3168–176 provided endogenously with a vaccinia vector
(22, 34) (Fig. 2C, right). The lack of effect of (Z-LL)2-ketone on
control cells transfected with EGFP instead of ICP47 indicated
LONG-PEPTIDE CROSS-PRESENTATION PATHWAY
The Journal of Immunology
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FIGURE 2. Long peptide cross-presentation is favored by suboptimally loaded HLA-A*0201 molecules in Mo-iDC. (A) T2 and T1 cells were pulsed
with peptide gp100184–227 for the cross-presentation assay. (B) T2 cells were treated with 0.5 mM epoxomicin or vehicle (DMSO) before and during
incubation with long peptide gp100184–227. After washing, they were tested for their ability to stimulate gp100-specific CTL to produce IFN-g. (C) Mo-iDCs
were treated overnight with (Z-LL)2-ketone (45 mM) or vehicle (DMSO) before transfection with mRNA encoding ICP47 or EGFP. Three hours after
transfection, some of the cells were pulsed with peptide gp100184–227 for the cross-presentation assay (left). Another portion of the same cells was infected
with a recombinant MAGE-A3–encoding vaccinia virus to verify efficient TAP blockade (right). (ZLL)2-ketone was maintained in the culture medium
during transfection and infection. Two hours postinfection, cells were washed and tested for their ability to stimulate CTL clone (Figure legend continues)
1716
Nascent HLA-I molecules used for cross-presentation follow a
non-classical secretion pathway
Low temperature incubation not only blocks recycling of surface
molecules; it also blocks the exit of secretory and surface proteins
from the trans-Golgi-network in the classical secretion pathway
(44, 45). We confirmed in our cellular system that incubation at
20˚C prevented classical secretion of HLA-I molecules (Fig. 4A)
and the presentation of endogenously derived class I–restricted
Ags (Fig. 4B). Because cross-presentation was not affected by low
temperature, we concluded that the nascent HLA-I molecules used
for cross-presentation followed a nonclassical secretion pathway.
The ER-Golgi Intermediate Compartment is an important early
secretion compartment involved in the sorting of ER-synthesized proteins. Cebrian et al. (12) reported a role for SEC22B in
escorting ER-resident molecules from the ER-Golgi Intermediate
Compartment to the phagosome, making it a good candidate to
escort HLA-I for long-peptide cross-presentation. However, we
observed that knocking down Sec22b with siRNA in Mo-iDCs did
not prevent cross-presentation (Fig. 4C). Recently, CD74 was
reported to escort MHC-I from the ER to the endolysosome
compartment to promote cross-presentation (13). However,
knocking down CD74 in Mo-iDC did not prevent crosspresentation of the long peptide either (Fig. 4D).
Taken together, the results reported above suggest that crosspresentation of the long gp100 peptide makes use of peptideloaded nascent HLA-I molecules that follow an alternative
secretory pathway to reach the cross-presenting vacuole, where preloaded peptides are exchanged for the cross-presented peptide before
transfer of the final peptide/HLA complexes to the cell surface.
Cross-presentation of other long peptides
We used another long peptide, derived from melanocytic protein
Melan-A to determine whether other long peptides followed
the same cross-presenting pathway as gp100184–227. Long peptide
Melan-A15-40A27L (15KGHGHSYTTAE26ELAGIGILTV35ILGVL40),
which comprises the heteroclitic antigenic peptide Melan-A26-35A27L
with an alanine-to-leucine substitution in position 27 to increase
binding to HLA-A*0201 (46, 47), was efficiently cross-presented
to Melan-A26–35-specific CTL by Mo-iDCs in a proteasome- and
TAP-independent manner (Fig. 5A, B). Although ICP47 efficiently blocked TAP, as indicated by the reduced HLA-I surface
expression (Supplemental Fig. 3B), it did not increase crosspresentation of the long Melan-A peptide as it did for the long
gp100 peptide. Consistently, there was no effect of L-leucinethiol
on the cross-presentation either (Fig. 5C). This might result from
the higher affinity of Melan-A26-35A27L for HLA-A*0201 as
compared with gp100209–217 (Supplemental Fig. 3C). The higher
the affinity of the cross-presented epitope, the easier peptide
A10, which recognizes peptide MAGE-A3168–176 presented by HLA-A*0101 (22). (D) T2 cells were treated with (ZLL)2-ketone (45 mM) or vehicle for 2 or
16 h at 37˚C, then labeled with Alexa-633-conjugated anti-HLA-I murine mAb W6/32 at 4˚C. HLA-class I expression level was represented by geometric
mean fluorescence intensity (GMFI). (E) T2 and T1 cells were treated with (Z-LL)2-ketone (45 mM) or vehicle overnight before being pulsed with peptide
gp100184–227 for the cross-presentation assay. (F) Mo-iDCs were treated with L-leucinethiol (30 mM) or vehicle (Tris(2-carboxyethyl)phosphine [TCEP]) for 3 h
before pulsing with peptide gp100184–227 for cross-presentation (left) or electroporation with gp100 mRNA to test endogenous presentation (right). After 3 h of
additional incubation in the presence of L-leucinethiol or vehicle, cells were tested for their ability to stimulate gp100-specific CTL. (G) Peptides of various affinities
for HLA-A2 were targeted into the ER of T2 cells with lentiviral constructs. The constructs used IL-2 signal sequence (IL-2ss) as a targeting motif and IRESmCherry as the selection marker. Forty-eight hours after transduction, mCherry-positive cells were sorted, cultured for 48 h, and used for a cross-presentation assay.
The proper expression of the constructs was monitored by FACS for mCherry fluorescence, as indicated in the column at the right (MFI). Control non–HLA-A2–
binding peptide EVDPIGHLY corresponds to peptide MAGE-A3168–176, which binds HLA-A1. The affinity of the peptides for HLA-A*0201 as predicted by
algorithms BIMAS and SYFPEITHI are indicated. HLA-A2 binding affinity was also estimated from the peptide concentration (EC50) needed to achieve 50%
of maximal stabilization of HLA-A2 molecules on T2 cells after acid strip, and from the maximal level of stabilization relative to peptide gp100209–217 (relative
efficacy). EC50 shown are the mean of three experiments. Each graph is representative of at least three independent experiments. For all graphs, assays were
performed in triplicate or more and repeated at least three times. MFI, mean fluorescence intensity; ND, not detected.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
engulfed long peptide. Because the vacuolar pathway of crosspresentation has been considered to make use of recycling MHC-I
molecules that are derived from the cell surface (15–18), we asked
whether those suboptimally loaded HLA-I were recycled or were
newly synthesized. To avoid unspecific effects of inhibitors like
primaquine on vesicle trafficking, we chose to block HLA-I
recycling by incubating cells at low temperature, which is
reported to block recycling of surface molecules (38). We observed that cross-presentation was not reduced at 20˚C (Fig. 3A).
We then confirmed that low temperature efficiently blocked HLA-I
recycling in our Mo-iDCs cellular system using a two-step approach. We first showed that internalization of HLA-I, which
conditions recycling, was almost completely blocked at 20˚C
(Fig. 3B, left), in line with the observation that endocytosis of
MHC-I depends on membrane rafts, which are highly temperature sensitive (39), as opposed to the clathrin-dependent endocytosis of mannose receptor CD206 (38, 40, 41) (Fig. 3B,
right). We then evaluated the recycling step at different temperatures (Fig. 3C). Surface HLA-I molecules of Mo-iDCs were first
labeled with conformation-dependent anti–HLA-I murine mAb
W6/32. Next, HLA-I/Ab complexes were allowed to internalize
for 12 min at 37˚C or 20˚C in the presence of primaquine
(220 mM) to prevent recycling. After stripping non-internalized
HLA-I/Ab complexes and washing away primaquine, we followed the reappearance of HLA-I/Ab complexes at the cell
surface at 20˚C or 37˚C by FACS using labeled anti-murine IgG
Ab. When both internalization and recycling were performed at
37˚C, we observed a fast kinetics of recycling, which was
consistent with the observation that HLA-I are recycled
through the RAB-35–dependent fast recycling pathway (42,
43). When recycling was performed at 20˚C, we observed a
striking reduction in the number of HLA-I recycled to the cell
surface. When both internalization and recycling were performed at 20˚C, HLA-I recycling to the cell surface was completely blocked. Because cross-presentation was not blocked
at 20˚C but recycling of HLA-I was, we concluded that crosspresentation of long peptides did not use recycled HLA-I for
peptide exchange. To confirm further that cell surface HLA-I
molecules are dispensable for long-peptide cross-presentation,
we acid-stripped T2 cells and reconstituted cell surface HLA-A2
with short peptides of different HLA-A2 binding affinities before
the cross-presentation assay. Although the extent of surface HLA-A2
reconstitution varied according to the peptide affinity for HLA-A2,
the cross-presentation ability of the cells remained unaffected
(Fig. 3D). If cross-presentation does not use recycled HLA-I
molecules, it must use nascent HLA-I. This was confirmed by the
inhibitory effect of protein synthesis inhibitor cycloheximide on
cross-presentation of the long gp100 peptide (Fig. 3E).
LONG-PEPTIDE CROSS-PRESENTATION PATHWAY
The Journal of Immunology
1717
exchange can take place, so that the limited pool of suboptimally
loaded HLA-I molecules available in TAP- or ERAP-competent
cells might become sufficient for optimal cross-presentation.
This is supported by the fact that L-leucinethiol strongly increased cross-presentation of the wild-type Melan-A long peptide (Melan-A15–40) (Fig. 5C), whose epitope (Melan-A26–35) has
a much lower affinity for HLA-A2 (Supplemental Fig. 3C) and
is otherwise not cross-presented efficiently. As with the gp100
peptide, low temperature (20˚C) did not block cross-presentation
of the long Melan-A peptide (Fig. 5D), further supporting the
involvement of an alternative secretion pathway of the HLA-I
molecules used for cross-presentation. These results indicate
that the cross-presentation of other long peptides follow the
same pathway as the gp100 long peptide, involving vacuolar
peptide exchange on nascent suboptimally loaded HLA-I
molecules, which follow a nonclassical secretion pathway.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 3. Cross-presentation of the long gp100 peptide makes use of nascent HLA-I molecules. (A) Mo-iDCs were preincubated for 1 h at 20˚C and
then assayed for cross-presentation (2 h) of the long gp100 peptide at the indicated temperature. Degranulation assay performed at 20˚C was used as readout, which is quicker and less temperature-sensitive than the cytokine production assay. Values are means 6 SD of triplicates. (B) Surface HLA-I (left) or
CD206 (right) molecules of Mo-iDC were labeled with Alexa-633-conjugated W6/32 or Alexa-488-conjugated anti-CD206. After washing, cells were
incubated at 37˚C in the presence of 220 mM primaquine to prevent recycling. Internalization was stopped by transferring cells to 4˚C at different time
points. The remaining surface Ag/Ab complexes were stripped off by citric acid (pH 3.0) treatment. Internalized HLA-I or CD206 were then quantified by
FACS analysis measuring geometric mean fluorescence intensity (GMFI) increase with time and shown as a ratio to the GMFI of Ab-labeled cells without
acid-strip (100%). GMFI of cells labeled with isotype control Ab was considered as 0%. (C) HLA-I recycling was evaluated on Mo-iDCs at different
temperatures. Anti–HLA-I murine mAb W6/32 labeled Mo-iDC were incubated for 12 min at the indicated temperature in the presence of primaquine
(220 mM). After acid stripping with 50 mM citric acid (pH 3.0) and washing away of primaquine, the reappearance of HLA-I/Ab complexes at the cell
surface was followed at different temperatures and analyzed by FACS using labeled anti-murine IgG Ab. Geometric mean fluorescent intensity (GMFI) of
non–acid-stripped cells was considered as 100%. Values are means 6 SD of duplicates. (D) Surface HLA-A2 of T2 cells were acid stripped and
reconstituted at 20˚C overnight in the presence of b2-microglobulin and short peptides with different HLA-A2 binding affinities (Fig. 2G). Cells were
pulsed with long peptide gp100184–227 for the cross-presentation assay. In parallel, surface HLA-A2 expression was detected with conformation-dependent
anti–HLA-A2 Ab BB7.2 and analyzed by FACS. The expression level is shown as mean fluorescence intensity. (E) Mo-iDCs were treated with cycloheximide (1 mg/ml) and tested for cross-presentation of the long gp100 peptide. Values are means 6 SD of triplicates. Each representative experiment
shown was repeated at least three times.
1718
LONG-PEPTIDE CROSS-PRESENTATION PATHWAY
Discussion
The first major conclusion from our work is that long-peptide crosspresentation follows a vacuolar pathway. This conclusion contrasts
with a number of previous reports supporting a cytosolic pathway
(8–11). Our proposed model implying a key role of suboptimally
loaded HLA-I molecules provides a potential explanation for
those discrepancies. Indeed, the support for a cytosolic pathway
usually comes from the observation that cross-presentation is
impaired by TAP inhibition. This is interpreted to indicate that
the cross-presented epitope needs to be transported across
membranes, in line with the cytosolic pathway (1–3). However,
if, as we propose, cross-presentation critically depends on suboptimally loaded MHC molecules, TAP inhibition may block
cross-presentation indirectly by reducing the availability of suboptimally loaded MHC-I. In fact, HLA-A2 is an exception among
MHC-I in the fact that it can load signal peptides in the absence of
TAP and therefore increase its pool of suboptimal peptides. Other
MHC class I molecules need TAP to load suboptimal peptides.
Therefore, in non–HLA-A2–restricted cross-presenting model
systems, TAP inhibition may indirectly—and paradoxically—
block long-peptide cross-presentation even though it follows a
vacuolar pathway.
This alternative interpretation may apply to the study reported by
Rosalia et al. (11) who studied cross-presentation of long peptides
by mouse and human DC. They studied the role of the proteasome
and TAP in murine bone marrow-derived DCs, and observed reduced cross-presentation using epoxomicin (1 mM) or using cells
derived from TAP-KO mice. Although it is difficult to compare
results obtained in different species using different peptides and DC
types, the latter observation might be explained by an indirect effect
of TAP inhibition in a vacuolar pathway, as discussed above.
Other reports studied cross-presentation of HLA-A2–restricted
peptides similar or identical to the ones we analyzed (8–11). Faure
et al. (10) used proteasome inhibitor lactacystin (40 mM) to
evaluate cross-presentation of Melan-A and gp100 long peptides
by human Mo-DCs. In line with our results, they observed no
inhibition of the cross-presentation of the gp100 peptide. For
Melan-A, they observed partial inhibition, with high donor-todonor variation. They did not evaluate the effect of TAP inhibition. In Segura et al. (9),the same group used lactacystin (2.5 mM)
to evaluate cross-presentation of a long Melan-A peptide by various DC, using an IFN-g production assay. They observed some
inhibition, again with donor-to-donor variation, but the IFN-g
signal was weak (∼100 pg/ml), probably at the limit of sensitivity
of the IFN-g ELISA. Again, they did not evaluate the effect of
TAP inhibition. If the effects reported by Segura et al. (9) are
confirmed, the difference with our data might result from the fact
that different DC types were used.
Contrary to these studies, Ménager et al. (8) did evaluate the
effect of TAP inhibition on the cross-presentation of a long HLAA2–restricted Melan-A peptide. They did so using a synthetic
peptide corresponding to the N-terminal 35-amino acid residues
of TAP inhibitor ICP47. They reported inhibition of crosspresentation using this long ICP47 peptide at 50 or 100 mM.
However, this experiment was lacking a control with an irrelevant
peptide. We have observed competition effects between long
peptides for cross-presentation. Because the TAP inhibitor used by
Ménager et al. (8) was, in essence, a long peptide, it is expected to
compete with the long peptide for cross-presentation. Therefore, it
is likely that the inhibition observed was due to competition rather
than TAP inhibition. Ménager et al. (8) also studied the effect of
proteasome inhibitor epoxomicin at high doses (1–5 mM), and
observed significant inhibition only at 5 mM. We used epoxomicin
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FIGURE 4. HLA-I molecules used for cross-presentation traffic to the cross-presenting vesicle through a nonclassical secretion pathway. (A) Low
temperature blocks conventional secretion of HLA-I. After acid stripping, reappearance of HLA-I at the cell surface of Mo-iDCs was followed by FACS at
the indicated temperature with Alexa-633–conjugated anti–HLA-I mAb W6/32. Values are means 6 SD of three independent experiments. (B) Low
temperature blocks endogenous Ag presentation by HLA-I molecules. HLA-B51–positive or –negative Mo-iDCs were acid-stripped and incubated at the
indicated temperature for 2 h and assayed for their ability to stimulate RU134–42/HLA-B51–specific CTL clone CTL381/84 in a degranulation assay
performed at 20˚C. (C and D) Mo-iDCs were transfected with (C) Sec22b- or (D) CD74-targeting siRNA by electroporation on day 3 (CD74) or day 4
(Sec22b) after differentiation and assayed for their ability to cross-present the long gp100 peptide to gp100209–217-specific CTL after 72 h (for CD74) or
48 h (Sec22b) of transfection. Efficiency of knocking down was analyzed by Western blot with cells collected at the same time as the cross-presentation
assay. The black lines indicate where parts of the image were joined. Values shown in (C) and (D) are means 6 SD of triplicates. The experiment in (C) was
repeated three times, and the experiment in (D) was repeated twice.
The Journal of Immunology
1719
at 0.5 mM, and provided controls demonstrating complete inhibition of endogenous presentation of gp100 at this dose (Fig. 1). It
is known that epoxomicin at high doses induces a number of other
effects in the cell, including ER stress. Therefore, the effect observed by Ménager et al. (8) might be unspecific because of the
high dose used. In line with this conclusion is the fact that the
authors did not observe any modulation of cross-presentation
when they changed the proteasomal subunit composition of
DCs, whereas the Melan-A antigenic peptide is known to be
processed differently by the standard proteasome and the immunoproteasome (21, 48–50).
To our knowledge, our study on long-peptide cross-presentation
is the first to evaluate both the effect of TAP inhibition and the
effect of proteasome inhibition in carefully controlled conditions. Our results clearly indicate a vacuolar pathway of crosspresentation of these long peptides by human monocyte-derived
DCs.
The second major conclusion of our work is that long-peptide
cross-presentation depends on newly synthesized HLA-I molecules that are loaded with suboptimal peptides. In our view, suboptimal peptides mean peptides that are able to bind HLA-I, but
with a low affinity so that they can be readily exchanged. The
binding of a low-affinity peptide may be needed to stabilize the
MHC-I and allow it to exit to ER and reach the post-ER compartment, where the low-affinity peptide would be exchanged
for the cross-presented peptide. The exact nature of the crosspresenting compartment is unclear at this stage and is under
study in our laboratory. Candidates include the late endosomal
MHC class II compartment, the phagosome and the IRAPcontaining vesicles. Another highly relevant question we are
currently addressing is to delineate the exact secretion pathway
followed by the nascent MHC-I molecules used for crosspresentation. Answering such questions will bring new insights
into intracellular trafficking pathways and may influence the development of vaccines based on long peptides to trigger CD8 T cell
responses in patients with cancer.
Acknowledgments
We thank the Centre d’hématologie of the Cliniques universitaires SaintLuc (Brussels) for providing blood samples from hemochromatosis patients; Aline Depasse for technical help; Aude Bonehill and Carlo Heirman
(Vrije Universiteit Brussel, Brussels) for help with mRNA preparation for
electroporation; Zhaojun Sun, Didier Colau, and Florence Depontieu for
help at various stages of the project; Julie Klein and Mandy Macharis
for editorial assistance; and Pierre Coulie for critical reading of the
manuscript.
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FIGURE 5. Cross-presentation of the long Melan-A peptide. (A) Mo-iDCs were treated with 0.5 mM epoxomicin or with vehicle (DMSO) before
and during incubation with peptide Melan-A15–40 or the heteroclitic peptide Melan-A15–40A27L. Melan-A26–35–specific CTL clone 549/18 was
added, and cytokine production was measured after 18 h. (B) Mo-iDCs were transfected with mRNA encoding ICP47 or EGFP. Three hours after
transfection, they were incubated with peptide Melan-A15–40A27L for 2 h. Melan-A26–35–specific CTL was added and cytokine production was
measured. (C) Mo-iDCs were pulsed with peptide Melan-A15–40 or Melan-A15–40A27L in the presence of L-leucinethiol (30 mM) or vehicle (Tris[2carboxyethyl]phosphine) after pretreatment with the inhibitor for 3 h. Melan-A26–35–specific CTL was added, and cytokine production was
measured. (D) Cross-presentation of the long peptide Melan-A15–40A27L was performed at the indicated temperature and evaluated in a degranulation assay performed at 20˚C. For all experiments, values are means 6 SD of triplicates from a representative experiment repeated at least three
times. ND, not detected.
1720
Disclosures
The authors have no financial conflicts of interest.
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LONG-PEPTIDE CROSS-PRESENTATION PATHWAY
Suppl. Fig. 1
A
short peptide
long peptide
short peptide
1.4
1.4
0.5
0.5
0.4
0.3
0.4
0.3
0.2
0.2
0.1
0.1
0
0
5.5
16.6
50
no pep
long peptide
1.8
IFN-γ (ng/ml)
IFN-γ (ng/ml)
1.8
B
MG132
DMSO
Conc. of cytochalasin B (µg/ml)
Supplementary Figure 1. Effect of cytochalasin B (A) and MG132 (B) on cross-presentation of
the long gp100 peptide.
(A) Mo-iDCs were treated with cytochalasin B at indicated concentrations and tested for cross-presentation of long peptide gp100184-227. As positive control, cells were pulsed with short peptide gp100209-217
and tested similarly. Values are means +/-SD of triplicates. One representative experiment of three
repetitions is shown here.
(B) Mo-iDCs were pretreated with MG132 (20µM) for 30 min, and tested for cross-presentation of long
peptide gp100184-227 in the presence of MG132. As positive control, cells were pulsed with short peptide
gp100209-217 and tested similarly. Values are means +/-SD of triplicates.
Suppl. Fig. 2
A
7
Vehicle (TCEP)
ICP47 mRNA
Leucinethiol
B
Cytokine (pg/ml)
IL-10
IL-12
5
Vehicle (DMSO) EGFP
4.46
0.00
4
Vehicle (DMSO) ICP47
4.52
0.00
3
(Z-LL)2-keton EGFP
9.12
0.00
Z-LL)2-keton ICP47
2.30
0.00
Vehicle (TCEP)
0.21
0.00
L-leucinethiol
0.07
0.00
6
IFN-γ (ng/ml)
EGFP mRNA
2
1
0
Vehicle
DMSO
(Z-LL)2Keton
Vehicle
DMSO
gp100184-227
(Z-LL)2Keton
gp100184-227 no peptide
no peptide
C
Surface
marker
CD80
CD86
CD83
HLA-II
HLA-I
CD11C
CD14
No antibody
Vehicle (DMSO) EGFP
Vehicle (DMSO) ICP47
(Z-LL)2-keton EGFP
(Z-LL)2-keton ICP47
Vehicle (TCEP)
Leucinethiol
MFI
(log)
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
5
Supplementary Figure 2. Effects of ICP47 mRNA electroporation, (Z-ll)2-keton and L-leucinethiol treatments on crosspresentation of the long gp100 peptide and expression of costimulatory signals by Mo-iDCs.
Mo-iDCs were either pretreated overnight with (Z-LL)2-ketone (45 μM) or vehicle (DMSO) before transfection with mRNA encoding
ICP47 or EGFP (as in figure 2C), or treated with L-leucinethiol (30 µM) or vehicle (Tris(2-carboxyethyl)phosphine [TCEP]) on the day
of the test (as in figure 2F). Three hours after mRNA transfection (as in figure 1E and 2C) or L-leucinethiol treatment (as in figure 2F),
part of the cells were pulsed with peptide gp100184-227 for the cross-presentation assay with maintained inhibitors (A), culture cell
supernatant was collected and tested for IL-10 and IL-12 presence by Bio-Plex (BIO-RAD; Confidence threshold: 10.37-2153.35
pg/ml for IL-10 and 9.31-2379.54 pg/ml for IL-12) (B), and part of the cells were analyzed for expression of costimulatory molecules
and cell surface markers by FACS (C).
Suppl. Fig. 3
A
Mo-iDCs
5
8
IFN-γ (ng/ml)
4
IFN-γ (ng/ml)
T2 cells
10
3
2
1
6
4
2
0
EGFP
0
ICP47-EGFP
long peptide gp100184-227
EGFP
ICP47-EGFP
short peptide gp100209-217
no peptide
C
B
Mo-iDCs
T2 cells
EGFP
80
12
ICP47
HLA class I level
(GMFI x 1,000)
% of initial expression
of HLA class I
100
60
40
20
0
ELAGIGILTV
EAAGIGILTV
8
RRYQNSTEL
ITDQVPFSV
4
0
0
50
100
150
200
Time (min)
250
300
0
3
6
9
12
15
Peptide concentration (μM)
Supplementary Figure 3. Controls for TAP inhibition by ICP47 (A, B), and evaluation of the affinity of
short peptides for HLA-A2 (C)
(A) Increase of long-peptide cross-presentation upon ICP47 transfection in Mo-iDCs but not in TAP-deficient
T2 cells. Mo-iDCs and T2 cells were transduced with lentiviral constructs encoding ICP47-ires-EGFP or
EGFP. Then total population of Mo-iDCs (>90 % EGFP+ cells) or sorted EGFP+ T2 cells were incubated with
long peptide gp100184-227 and tested for their ability to stimulate CTL-811/327.4.
(B) (related to Figure 5B) ICP47 mRNA electroporation successfully blocked HLA-I expression on Mo-iDC.
After acid stripping, reappearance of HLA class I molecules at the cell surface of EGFP- or ICP47-mRNA
transfected DCs was followed by FACS using Alexa-633-conjugated W6/32. The GMFI of labeled cells
without stripping was considered as 100%. One representative experiment out of at least three is shown.
(C) To measure the affinity of short peptides for HLA-A2, T2 cells were incubated with the indicated serially
diluted peptides for 3 hours at 37°C, stained with Alexa-633-conjugated HLA-class I specific antibody W6/32
and analyzed by FACS.
Suppl. Fig. 4
B
HLA-A1 Ladder ß-actin
1
2
1: EGFP+ clone
2: EGFP- clone
1
2
450
IFN-γ (pg/ml)
A
300
150
0
mcherry
+
-
Supplementary Figure 4. Presentation by T2-HLA-A1 cells of
ER-targeted peptide MAGE-A3168-176
(A) T2 cells were transduced with a lentivirus encoding
HLA-A1-IRES-EGFP, and EGFP-positive cells were cloned (named
T2-A1). HLA-A1 expression was confirmed by RT-PCR.
(B) T2-A1 cells were transduced with a lentivirus expressing
IL-2ss-MAGE-A3168–176 - IRES-mcherry encoding ER-targeted
MAGE-A3 peptide EVDPIGHLY as in Figure 2G. Then mcherry-positive and negative cells were sorted and tested for their ability to stimulate IFN-γ production by MAGE-A3168–176 -HLA-A1-specific CTL.