Download Induction of Protective Antitumor Immunity through Attenuation of ERAAP Function

Induction of Protective Antitumor Immunity
through Attenuation of ERAAP Function
Edward James, Ian Bailey, Gessa Sugiyarto and Tim Elliott
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of June 9, 2014.
J Immunol published online 22 April 2013
http://www.jimmunol.org/content/early/2013/04/23/jimmun
ol.1300220
http://www.jimmunol.org/content/suppl/2013/04/23/jimmunol.130022
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Supplementary
Material
Published April 24, 2013, doi:10.4049/jimmunol.1300220
The Journal of Immunology
Induction of Protective Antitumor Immunity through
Attenuation of ERAAP Function
Edward James,*,† Ian Bailey,*,1 Gessa Sugiyarto,*,† and Tim Elliott*,†
T
he repertoire of antigenic peptides derived from intracellular proteins consists of thousands of different 8–10-aa
peptides, many of which are generated as N-terminally
extended precursors following partial hydrolysis of substrates by
cytosolic proteases, including the proteasome (1, 2). These are
then transported into the endoplasmic reticulum by the TAP. A key
final step of the pathway involves aminopeptidases, which trim the
extra N-terminal residues to generate peptides (1) that are an
optimal length for loading onto MHC class I (MHC I) molecules.
This completes MHC I biogenesis and permits exit from the endoplasmic reticulum to the cell surface (3–6), where they present
the processed peptides to CTL. In humans, two aminopeptidases
perform this function, ERAP1 and ERAP2 (7–9); in mice, only
one aminopeptidase exists, ERAAP (10). ERAAP is critical for
the generation of many antigenic epitopes in vivo and can influence the generation of the antigenic peptide repertoire (11–14).
ERAAP can also destroy antigenic epitopes by trimming them to
lengths too small for MHC binding, thus its characterization as an
antigenic peptide editor (12).
In tumors, expression of ERAP1 and ERAP2 was shown to vary
considerably, with high/moderate levels in skin cancer and mel-
anoma and low levels in breast and lung cancer (15–17). Varying
expression of ERAP1/2 in tumors could indicate a mechanism by
which the tumors evade detection by CTL through alteration of
the peptide repertoire presented by MHC I, perhaps reducing the
generation of dominant tumor-derived peptide epitopes either by
reduced trimming activity or by destroying them by overtrimming.
To investigate the contribution of ERAAP to immunogenicity
and immunological control of tumor growth, we used the transplantable murine colon carcinoma model CT26 and modulated
ERAAP levels through small interfering RNA (siRNA). This model
allowed us to investigate the correlation among ERAAP expression, the generation of well-characterized tumor epitopes, and
protective and durable antitumor immunity. We found that reducing
ERAAP levels enhanced tumor immunogenicity, thereby opening
a window of opportunity for the potential use of small molecule
ERAAP inhibitors to enhance antitumor immunity. Accordingly,
treatment of CT26 with the ERAAP inhibitor leucinethiol (leuSH)
prior to injection or following tumor establishment in vivo led to
prolonged survival.
Materials and Methods
Cell lines and hybridomas
*Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, United Kingdom; and †Institute of Life Sciences, University of
Southampton, Southampton SO17 1BJ, United Kingdom
1
Current address: School of Life Sciences, University of Westminster, London, United
Kingdom.
All CT26 and P815 tumor cells, COS-7 cells, and the GSW11-specific
lacZ-inducible CD8+ T cell hybridoma CCD2Z were maintained as previously described (18). In all experiments, mice were injected s.c. with
105 CT26 tumor cells in PBS. All cell lines were tested and were mycoplasma free.
Received for publication January 22, 2013. Accepted for publication March 22, 2013.
This work was supported by Cancer Research UK Project Grant C25722/A8410 (to
E.J. and T.E.) and Cancer Research UK Programme Grant C7056/A11946 (to T.E.).
G.S. was awarded a Cancer Research UK Ph.D. studentship.
Address correspondence and reprint requests to Dr. Edd James, Cancer Sciences Unit,
Faculty of Medicine, University of Southampton, Southampton SO16 6YD, United
Kingdom. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: leuSH, leucinethiol; MHC I, MHC class I; shRNA,
short hairpin RNA; siRNA, small interfering RNA; TPPII, tripeptidyl peptidase II;
Treg, regulatory T cell.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300220
Mice, Abs, and in vivo depletion
BALB/c mice were bred locally under specific pathogen–free conditions
and were used at 6–8 wk of age. Mice were housed in conventional facilities during experimental procedures. All experiments were carried out
under license according to local ethical committee approval under United
Kingdom Home Office license guidelines. Hybridomas secreting CD8
(YTS 169.4.2.1, YTS 156.7.7, both rat IgG2b) and CD4 (YTS 191.1.2,
YTA 3.1.2, both rat IgG2b)-specific mAb were described previously (19)
and were grown in culture and purified by precipitation in saturated ammonium sulfate. For depletion, mice were injected with 100 mg each mAb
twice with a 1-d interval. For intratumoral administration of leuSH, mice
were challenged with CT26 tumor, and 100 mM leuSH in 0.1 M DTT in 50
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The endoplasmic reticulum aminopeptidase associated with Ag processing, ERAAP, plays an important role in the trimming of
antigenic peptides for presentation at the cell surface complexed with MHC class I molecules. Tumors express varying levels
of ERAAP, highlighting a possible mechanism of immune-evasion through alteration of the peptide repertoire. Using the CT26
tumor model, we investigated the effects of ERAAP modulation on peptide presentation and the use of ERAAP inhibition as an
antitumor therapy. We show that generation of the cross-protective tumor Ag GSW11 in the colorectal carcinoma CT26 is
increased when ERAAP expression is reduced. BALB/c mice with reduced ERAAP expression challenged with CT26 induced
protective immunity that was mediated by CD8+ T cells. This antitumor immunity also protected mice when rechallenged with
wild-type CT26 tumor; strong CD8+ T cell responses to GSW11 were observed, despite its presentation being considerably lower.
Furthermore, boosting the tumor immunogenicity through inhibition of ERAAP function with the small molecule inhibitor
leucinethiol in vitro, or in established tumors in vivo, abrogated tumor growth and prolonged survival. Thus, our results highlight
the promising possibility of using modulation of ERAAP to generate protective antitumor responses as a strategy for cancer
immunotherapy. The Journal of Immunology, 2013, 190: 000–000.
2
ml was injected into the tumor at days 7 and 10. Controls received DTTonly injections.
T cell–activation assays
The GSW11-specific lacZ-inducible T cell hybridoma CCD2Z was cocultured with CT26 (wild-type, ERAAP knockdown or inhibitor treated) or
APCs that were transfected with Ag cDNAs. The lacZ activity was measured as described previously (20). For inhibitor treatment of CT26, cells
were incubated with 10 mM MG132 (Calbiochem), 100 mM AAF-CMK
(Calbiochem), or 30 mM leuSH (Sigma) for 16 h. Following incubation,
the treated CT26 were assayed for stimulation of CCD2Z.
siRNA and short hairpin RNA knockdown
For stable ERAAP knockdown, ERAAP-specific siRNA (CT26-ERAAPmixed)
or scrambled siRNA (ERAAPhi CT26) was cloned into the short hairpin
RNA (shRNA) expression vector pSilencer 4.1 CMV/hygro (Life Technologies) and transfected into CT26 using FuGENE6 (Roche), according to
the manufacturer’s instructions. After 24 h, transfected cells were selected
by the addition of 0.5 mg hygromycin. CT26-ERAAPmixed cells were
subcloned and screened for ERAAP protein knockdown using Western blot
of CT26 cell lysates.
Total acid-soluble peptide pools from tumor cells CT26, CT26-ERAAPmixed,
ERAAPmed CT26, and ERAAPlo CT26 were extracted as described (12).
Briefly, 108 cells were washed with PBS and extracted with 400 ml
10% boiling formic acid in the presence of a 10 mM “martyr” peptide
ASNENMETM for 10 min. Cellular debris was removed by centrifugation
and fractionated by HPLC after filtration through a 10-kDa filter (Millipore,
Billerica, MA). Reverse-phase C18 column (Grace Vydac, Hesperia, CA;
2.1 3 250 mm, 5 mM, 300 Å) was run in 0.1% trifluoroacetic acid in
water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B).
The gradient was from 17 to 45% (solvent B) at a rate of 1%/min, with
fractions collected and dried in a vacuum centrifuge. Mock injections with
sample buffer alone were performed prior to each extracted sample, using
the same column and identical run conditions. The collected fractions were
assayed in the same experiment, using the same APCs and T cells, in parallel
with fractions from the cell extracts and synthetic peptide standards.
CD8+ T cell responses and MHC I expression
Spleen and draining lymph node cells from CT26-challenged mice (wildtype or ERAAP knockdown) were harvested between days 10 and 12. The
CD8+ T cell responses to GSW11 and AH1 Ags were measured by production of IFN-g by those cells in response to incubation with peptide.
CD8+ T cells, APCs, and peptides were cultured together for 4 h in the
presence of brefeldin A before being stained for surface CD8 and intracellular IFN-g (BD Biosciences). For assessment of cell surface MHC I
expression, H-2Dd–FITC (34-2-12) and H-2Ld-FITC (B22) Abs were incubated with CT26 for 30 min on ice and washed. Cells were then analyzed on a FACSCanto (BD Biosciences), and data were analyzed with
FlowJo software (TreeStar). CD8+ lymphocytes were gated, and the percentage of IFN-g–producing cells was calculated from total CD8+ T cells.
Statistical analysis
Statistical significance was assessed by the two-tailed paired Student t test
or one-way ANOVA, using a Bonferroni multiple-comparison posttest.
Survival data are presented as Kaplan–Meier plots.
Results
Destructive processing of tumor Ag GSW11 by ERAAP
In the absence of vaccination, protective immunity against the transplanted CT26 colon carcinoma requires the induction of a strong
CTL response against the GSW11 epitope (18). Anti-GSW11 CTLs
are preferentially suppressed by regulatory T cells (Tregs) and are
only allowed to expand when Tregs are depleted prior to tumor
challenge (18). Because GSW11-specific CD8+ T cell responses are
important for tumor rejection, we were interested to know how the
GSW11 epitope (GGPESFYCASW) was generated by proteases.
Because GSW11 is relatively “long” for a CTL epitope, does not
conform to the canonical H-2Dd binding motif (XGPXXXXX[I,L,
F]) (21), and has a very low affinity for MHC I (t1/2 = 20 min) (18),
we wondered whether it would be unusually susceptible to overtrimming by ERAAP, as observed for other CTL epitopes (12).
We incubated CT26 tumor cells with MG132 (a proteasome inhibitor), AAF-CMK (an inhibitor of tripeptidyl peptidase II
[TPPII]), and leuSH (an ERAAP inhibitor). We also transfected
COS-7 cells with the gp70 cDNA construct C8 (which contains the
GSW11 epitope) and/or a TAP inhibitor derived from human
herpesvirus 1, ICP47 (22, 23). The effect of the inhibitors on the
generation of GSW11 and its cell surface presentation by H-2Dd
were assessed using the GSW11-specific T cell hybridoma CCD2Z,
which is able to detect nanomolar concentrations of presented
peptide (Supplemental Fig. 1) (18). CCD2Z is a fusion between the
GSW11-specific CTL, CCD2, and BWZ.36 CD8a fusion partner
that contains an inducible reporter gene encoding b-galactosidase
(lacZ) (24). Activation of the CCD2Z T cell hybridoma is assayed
by measuring lacZ activity. MG132-treated CT26 and C8 and
ICP47 cotransfection resulted in complete abrogation of CCD2Z
stimulation (Fig. 1A, 1B). AAF-CMK treatment had no effect on
CCD2Z stimulation, indicating that TPPII has no role in the generation of GSW11 (Fig. 1A). In contrast, incubation of CT26 with
leuSH resulted in an increase in CCD2Z stimulation (Fig. 1A).
Therefore, we concluded that, although generation of GSW11
requires both the proteasome and TAP for presentation, it is overtrimmed and destroyed by ERAAP, resulting in a steady-state level
of presentation that is detectable but low.
To confirm the role of ERAAP in destroying GSW11, CT26 tumor cells were transfected with ERAAP-specific siRNA. LaminB1specific siRNA knockdown was used as a control. ERAAP siRNA
knockdown resulted in an increase in CCD2Z stimulation (Fig. 1C),
consistent with the results following leuSH treatment (Fig. 1A).
As expected, laminB1 knockdown of CT26 did not affect CCD2Z
stimulation (Fig. 1C). The increase in GSW11 generation occurred
despite an overall decrease in MHC I levels; MHC I downregulation
is typically observed in ERAAP-knockout cells (Fig. 1D) (11–14).
This confirmed that the generation of GSW11 is ERAAP sensitive.
Level of anti-GSW11 CTL stimulation corresponds to the level
of ERAAP expression in CT26
To investigate the effect of GSW11 presentation on antitumor
immunity, we created stable ERAAP knockdown lines of CT26
using an ERAAP-specific shRNA vector. A polyclonal line was
established (CT26-ERAAPmixed) from which two clones were
generated that showed greater stimulation of CCD2Z than controltransfected CT26 (Fig. 2A). Analysis of ERAAP expression revealed
that these showed an ∼80% (ERAAPlo CT26) and 65% (ERAAPmed
CT26) reduction in protein levels (Fig. 2B), thus establishing a
correlation between the level of ERAAP expression and the level
of CCD2Z stimulation.
ERAAP expression is directly correlated with the quantity of
GSW11 presented
To confirm that the increase in CCD2Z stimulation observed with
reduced ERAAP protein was due to an increase in the generation
of GSW11, we generated peptide extracts from tumor cells with
different ERAAP expression levels. These were fractionated by
reverse-phase HPLC, and the amount of GSW11 was quantified in
the fractionated mixture using CCD2Z as a biological detector. The
profile obtained from CT26 peptide extracts revealed one significant peak that coeluted with synthetic GSW11 (Fig. 3A). Analysis
of the polyclonal line CT26-ERAAPmixed revealed a 3-fold increase
in GSW11 compared with CT26 (Fig. 3B). Greater increases in
GSW11 were observed for ERAAPmed and ERAAPlo CT26 (8- and
30-fold increases, respectively; Fig. 3C, 3D). Importantly, the amount
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Extraction and HPLC analysis of endogenously processed
peptides
ERAAP MODULATION OF ANTITUMOR RESPONSES
The Journal of Immunology
3
of GSW11 generated correlated with the amount of ERAAP
protein present within the cells. The presence of an additional
peak in ERAAP-knockdown CT26 extracts eluting ahead of
GSW11 is also seen in CT26 cells when developed for a longer
period of time; this was shown to be an oxidized adduct (or dimer)
that probably forms during the extraction process (18). Therefore,
we concluded that the increased response of CCD2Z observed after
ERAAP knockdown is due to an increase in the amount of GSW11
generated in these cells.
Silencing ERAAP causes tumor rejection
Having shown that ERAAP-knockdown CT26 generated and presented significantly more GSW11 compared with CT26, we wanted
to determine whether the increase in GSW11 presentation was able
to overcome Treg suppression and induce GSW11-specific T cells
in vivo. We inoculated BALB/c mice with ERAAPlo, ERAAPmed,
or untreated CT26 and assessed tumor growth and survival. Mice
inoculated with CT26 had detectable tumors from day 8 that
continued to grow until reaching the humane end point, with a
median survival time of 20 d (Fig. 4A, 4C). All mice challenged
with ERAAPlo and ERAAPmed CT26 had tumor growth at days 8–
12 that was subsequently cleared in the majority of cases (8/12),
indicating a role for an effective adaptive antitumor response in
these mice. Mice challenged with ERAAPlo CT26 showed a significant increase in survival time compared with CT26, with .90%
of mice rejecting the tumor (p , 0.001; Fig. 4A, 4C). Inoculation
with ERAAPmed CT26 induced tumor protection in 50% of mice
(Fig. 4A), with a median survival time of 64.5 d compared with
20 d for CT26 (p , 0.005; Fig. 4A, 4C). These results showed
that the increased generation of GSW11 resulting from ERAAP
knockdown produced a protective antitumor response whose
intensity correlated negatively with the level of ERAAP expression.
To investigate the cells responsible for this tumor protection, we
depleted mice of either CD4 or CD8 T cells prior to challenge
with ERAAPlo CT26. Depletion of CD8 T cells completely abrogated the ability to clear tumor (Fig. 4B). Depletion of CD4
T cells also resulted in a small decrease in tumor rejection, although the majority of mice survived tumor challenge (Fig. 4B),
confirming that the rejection of ERAAPlo CT26 tumor was primarily mediated by CD8+ T cells.
FIGURE 2. Generation of stable ERAAP knockdown CT26. (A) CT26 were stably transfected with
ERAAP (CT26-ERAAPmixed) or scrambled shRNA
(ERAAPhi CT26) and assessed for stimulation of CCD2Z.
Two clones established from the polyclonal CT26ERAAPmixed cell line, ERAAPmed CT26 and ERAAPlo
CT26, were also assessed for CCD2Z stimulation. (B)
ERAAP expression in all CT26 cell lines was assessed
by Western blot. Data shown are representative of three
experiments.
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FIGURE 1. GSW11 is overtrimmed by ERAAP.
(A) CT26 tumor cells were incubated with leuSH
(ERAAP inhibitor), AAF-CMK (TPPII inhibitor), or
MG-132 (proteasome inhibitor). Generation of GSW11
was assessed using the lacZ-inducible GSW11-specific
T cell hybridoma CCD2Z. (B) COS-7 cells were
transfected with H-2Dd and the GSW11-encoding
C8 or vector only cDNA and/or ICP47 (TAP inhibitor). GSW11 generation was assessed by activation
of CCD2Z. (C) CT26 cells were transfected with
ERAAP or laminB1-specific siRNA or were mock
transfected, and GSW11 generation was assessed
by activation of CCD2Z. (D) The effect of ERAAP
siRNA knockdown on cell surface MHC class I
expression was assessed by flow cytometry. Data
shown are representative of four experiments.
4
ERAAP MODULATION OF ANTITUMOR RESPONSES
FIGURE 3. Knockdown of ERAAP expression increases the amount of GSW11 generated. Peptides
were extracted from CT26 (A), CT26-ERAAPmixed
(B), ERAAPmed CT26 (C), or ERAAPlo CT26 (D) cells
and fractionated on a reverse-phase C18 column by
HPLC. Each fraction was dried, resuspended in PBS,
and tested for its ability to stimulate lacZ activity in
CCD2Z T hybridoma with P815 cells as APCs. The
arrow indicates the peak activity of synthetic GSW11
peptide run under identical conditions. Data shown
are representative of four experiments.
To investigate the specificity of the anti-ERAAPlo CT26 response
in more detail, we inoculated mice with either ERAAPlo CT26 or
CT26 and assessed the anti-AH1 and anti-GSW11 CD8+ T cell
responses in spleens and draining lymph nodes using intracellular
cytokine staining. As shown previously (18), anti-AH1 CD8+
T cell responses were observed following inoculation with CT26
(∼0.4%), with no GSW11-specific responses detected above that
seen in unstimulated controls (Fig. 5A). Interestingly, despite detectable anti-AH1 responses in tumor and draining lymph nodes,
these mice succumb to tumor by day 25. In contrast, all mice
challenged with ERAAPlo CT26 had GSW11-specific CD8+ T cell
responses that were much greater than those observed in CT26-
challenged mice (∼0.75%, p , 0.001; Fig. 5A), similar to those
observed in mice depleted of Tregs (18). The anti-GSW11 response dominated over AH1-specific responses (∼0.75% versus
∼0.6%; p , 0.05) in these mice, even though the latter were also
increased compared with CT26 challenge (∼0.6% versus ∼0.4%;
Fig. 5A). Because .90% of mice challenged with ERAAPlo CT26
reject the tumor, the correlation of protection and the appearance
of GSW11-specific CD8+ T cells suggests the importance of these
cells in clearance of the tumor. Although AH1-specific CD8+ T cells
were also increased, the modest difference suggests that these
responses are unlikely to play a major role in tumor clearance.
Having shown that challenge with ERAAPlo CT26 induces robust
protective antitumor immunity that is CD8 dependent, we went on
to show that tumor challenge also induced immunological memory.
FIGURE 4. ERAAPlo CT26 induces CD8+
T cells capable of mediating tumor rejection.
BALB/c mice were challenged with wild-type (n =
6), ERAAPmed (n = 6), or ERAAPlo (n = 7) CT26,
and tumor survival (A) and tumor growth (C) were
assessed. **p , 0.01, ***p , 0.001, versus CT26.
(B) Untreated CD4+ or CD8+ T cell–depleted
ERAAPlo CT26–challenged mice were assessed for
tumor survival. Control mice were challenged with
wild-type CT26 (n = 5/group). *p , 0.05, versus
ERAAPlo CT26. Data represent two independent
experiments with at least five mice/group.
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ERAAPlo CT26 induces CD8+ T cell responses to CT26 Ags
GSW11 and AH1
The Journal of Immunology
5
We rechallenged ERAAPlo CT26 immune mice with ERAAPlo
CT26 50 d after the primary challenge. All mice that rejected
the first tumor challenge also rejected the second (Fig. 5C).
Examination of CD8+ T cell responses in mice 10 d after rechallenge revealed increased anti-GSW11 and anti-AH1 T cell
responses compared with those observed in the primary challenge with dominant anti-GSW11 responses (∼2.25% versus ∼1%,
p , 0.01; Fig. 5D).
To consider ERAAP silencing as a potential antitumor therapy,
the induced immunity following primary challenge with ERAAPlo
CT26 would also need to be effective against tumor cells with
normal levels of ERAAP (ERAAP silencing may not be effective
in all tumor cells or those at additional sites in the body). The
presence of anti-AH1 and anti-GSW11 CD8+ T cell responses in
ERAAPlo CT26–challenged mice suggests that ERAAPlo CT26
immune mice would be able to clear wild-type CT26, despite a
lower level of epitope production. To assess this, ERAAPlo CT26
immune mice were immunized with CT26 50 d after primary tumor
challenge. All ERAAPlo CT26–immune mice were protected from
CT26 tumor challenge, whereas control naive mice succumbed to
the tumor by day 25 (Fig. 5C). Assessment of CD8+ T cell responses
10 d following CT26 challenge revealed strong anti-GSW11 and
anti-AH1 responses greater than those observed after primary
ERAAPlo CT26 challenge (2 and 1.25% versus 0.8 and 0.6%;
Fig. 5A, 5E). This suggests that the dominant protective CD8+ T cell
responses in these mice were directed toward GSW11 and AH1
peptide epitopes. Therefore, these memory CTL responses to epitopes shared between ERAAPlo CT26 and CT26 (GSW11 and AH1)
allow rejection of CT26 tumor following rechallenge, even though
the presentation of these epitopes is considerably lower in the latter.
The results also highlight the validity of attenuating ERAAP function as a strategy for the induction of protective antitumor immunity.
Chemical inhibition of ERAAP prolongs tumor survival in vivo
We investigated whether attenuation of ERAAP activity in CT26
with leuSH treatment either prior to injection or in situ would
trigger tumor rejection. Treatment of CT26 with LeuSH in vitro
results in a transient inhibition of ERAAP that persists for 24–
48 h (Supplemental Fig. 2). Mice challenged with CT26 treated
in vitro with leuSH showed prolonged survival in four of seven
recipients (Fig. 6A; p , 0.05), with two mice surviving beyond
day 50. Tumor was present in these two mice but was stable (Fig.
6C), indicating that a protective anti-CT26 response was induced
that favored tumoristasis over rejection.
We next investigated whether inhibition of ERAAP by topical
delivery of leuSH could prolong survival in mice where tumor was
established. CT26-challenged mice with a palpable tumor (∼0.5 cm2)
were injected intratumorally with leuSH at day 7, with a repeat dose
given at day 10. Intratumoral administration of leuSH resulted
in the attenuation of tumor growth, leading to prolonged survival
in five of seven mice (p , 0.05; Fig. 6B, 6D). Tumor growth was
arrested in three mice, with two showing a dramatic reduction in
tumor size (Fig. 6D). In all instances of tumor growth arrest, the
tumor size remained stable throughout the remainder of the experiment, again indicating the presence of a tumoristatic T cell
response. Control mice injected with DTT alone showed no retardation of tumor growth (Fig. 6D). Attenuation of tumor growth
in most mice after inhibition of ERAAP function provides proof of
principle that small molecule inhibitors of ERAAP might have
therapeutic value, particularly for tumors in which high expression
correlates with poorer prognosis.
Discussion
We used ERAAP silencing to induce a robust protective antitumor
immunity to CT26 and show that this immunity is CD8+ T cell
mediated, which also protects against challenge with wild-type
CT26. Although it is not known whether modulation of ERAAP
expression or activity leads to immune-evasion by human tumors,
it is interesting to note that ERAP1/2 expression in human cancers
is variable; one study (15) showed that, unlike ovarian, breast, and
lung cancers, which tended to downregulate ERAAP expression,
colorectal adenocarcinoma showed enhanced expression relative
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FIGURE 5. ERAAPlo CT26 challenge induces responses to GSW11-specific T cells. (A)
Responses to AH1 and GSW11 Ags were assessed in mice challenged with either CT26 or
ERAAPlo CT26. (B) Representative dot plots
of AH1- and GSW11-specific CD8+ T cell responses in CT26- or ERAAPlo CT26–challenged
mice. Percentages adjacent to outlined areas
indicate CD8+ IFN-g–producing cells. (C)
ERAAPlo CT26–immune mice were rechallenged 50 d later with either ERAAPlo CT26
or CT26, and tumor survival was assessed.
Control naive mice were challenged with CT26
(n = 5/group). Data are representative of two
independent experiments. Anti-AH1 and antiGSW11 CD8+ T cell responses from ERAAPlo
CT26–rechallenged (D) or CT26-rechallenged
(E) ERAAPlo CT26–immune mice were assessed.
*p , 0.05, **p , 0.01, ***p , 0.001.
6
ERAAP MODULATION OF ANTITUMOR RESPONSES
to normal gut tissue. This observation is consistent with the model
that we investigated in which a potent tumor epitope is overprocessed by ERAAP, rendering it cryptic. Downregulation of
ERAAP in CT26 led to the increased generation of protective
epitopes and, in particular, the cryptic epitope GSW11, leading to
strong antitumor CTL and tumor rejection. Importantly, ERAAPlo
CT26–immune mice were also able to reject CT26 when challenged 50 d after rejection of the primary tumor. We also showed
that transient ERAAP inhibition with leuSH attenuated tumor
growth in established tumors, demonstrating the potential of this
strategy as a therapy.
ERAAP-deficient cells were shown to present peptides with Nterminal extensions, as well as novel peptides uncovered in the
absence of ERAAP (21, 25, 26). Interestingly, some peptide epitopes were overtrimmed by ERAAP, highlighting its role as a
peptide editor (12). The increased T cell responses, together
with the 30-fold increase in peptide generation when ERAAP is
downregulated/inhibited, strongly indicate that the potent antitumor epitope GSW11 is overtrimmed. The precise mechanism for
peptide trimming by ERAAP is not known; however, from other
examples of aminopeptidases, it is likely that it trims a single
amino acid at a time in a bind, trim, release mechanism. This
suggests that ERAAP must compete for peptide binding with other
molecules, such as MHC I, and this competition would ultimately
determine whether a peptide is overtrimmed; GSW11 has a poor
affinity for H-2Dd (its half-life for dissociation is only 20 min),
which might bias competition toward ERAAP binding over MHC
I, thereby providing more opportunity for ERAAP to bind and
overtrim GSW11.
The induction of anti-GSW11 CD8+ T cell responses is fundamental to the clearance of CT26 tumor. In naive mice challenged
with CT26, responses to GSW11 are absent, but we showed that
GSW11-specific T cell responses are observed following Treg
depletion prior to challenge, revealing that Tregs suppress these
T cells (18). Downregulation of ERAAP in CT26 (ERAAPlo CT26)
led to an increased abundance of GSW11 and the subsequent induction of GSW11-specific T cell responses similar to those observed in CT26-challenged Treg-depleted mice. This suggests that
increasing the peptide abundance in CT26 tumors facilitates
effective priming and activation of GSW11-specific T cells, thus
overcoming Treg suppression. There are many mechanisms for
Treg suppression, with strategies to overcome the effects of Tregs
on protective T cell responses based on the depletion/blocking
of important cells or interactions, such as Treg depletion (19),
CTLA-4 (27), PD-L1 (28). These approaches, although showing
promise, rely on the modulation of all immune responses, not just
those restricted to the tumor, and were shown to cause undesirable
autoimmune responses. Therefore, the use of ERAAP downregulation may provide a new approach by which Treg suppression of
CTL responses can be overcome.
Alteration of ERAAP activity was shown to induce immune
responses to tumors (mediated by NK cells) (29) and ERAAP-
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FIGURE 6. ERAAP inhibition attenuates tumor
growth in vivo. (A) Survival of BALB/c mice challenged
with in vitro leuSH-treated CT26 or CT26 (n = 7/
group). (B) CT26-challenged BALB/c mice were
injected with leuSH and DTT or DTT only intratumorally
on days 7 and 10 (n = 7/group). Tumor-growth kinetics
of BALB/c mice challenged with in vitro (C) or in vivo
(D) leuSH-treated CT26. Arrows indicate times of intratumor injections. *p , 0.05.
The Journal of Immunology
Acknowledgments
We thank Nasia Kontouli for technical expertise and Denise Boulanger for
helpful discussions.
Disclosures
The authors have no financial conflicts of interest.
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deficient cells (mediated by CD8+ T cells restricted by MHC Ia
and Ib) (25, 26), but a link between ERAAP expression/function
and immune control of human tumors has yet to be made. There is
growing evidence for the importance of tumor-infiltrating lymphocytes
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there is a negative correlation between tumor survival and tumorinfiltrating Tregs, which adds further significance to our observation that ERAAP downregulation allows anti-GSW11 CTLs to
overcome epitope-specific immunosuppression by Tregs (32). The
same is likely to be true for other cryptic epitopes that arise as
a result of ERAAP silencing, which, for other Ags, were shown to
be highly immunogenic (21, 25). Therefore, it is probable that these
Ags play a role in the rejection of ERAAP-silenced tumor cells
(gene knockdown or leuSH treated). However, these Ags, like
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for CT26 clearance in ERAAPlo CT26–immune mice; however,
this may be indicative of other similar epitopes because anti-CT26
CD8+ T cells of other specificities (albeit subdominant) were
identified (18).
We found that molecular inhibition of ERAAP in CT26 by
preincubation with leuSH, which induces a transient loss of
ERAAP activity lasting ,48 h (Supplemental Fig. 2), also induced
antitumor immunity. This indicated the possibility of using topical
ERAAP inhibitors in vivo in a therapeutic setting, and we found
that intratumoral injection of established and palpable tumors with
leuSH attenuated their growth in most instances. LeuSH is not an
attractive therapeutic candidate for many reasons, including its lack
of specificity, redox number (requiring coadministration of a reducing agent), and high Kd (∼50 mM), which do not justify the
further pharmacokinetic studies and dosing trials that would be
needed to optimize these therapy experiments. Nevertheless, its
clear effect on immune control of tumor growth in the majority of
mice and a corresponding increase in survival indicate that more
specific, potent inhibitors of ERAAP may have significant therapeutic potential. Furthermore, even in cases in which in vivo leuSH
treatment did not lead to long-term survival (5/7 cases), such
treatment may prolong the “equilibrium phase” of tumor growth
(33) during which progression of an established tumor is kept in
check via natural immunity. In turn, this may widen a window of
opportunity for adjunct CTL-targeted therapies (e.g., vaccination) or immunomodulation (e.g., anti–CTLA-4, Treg ablation).
In this way, small molecule inhibitors of ERAAP might be delivered with biological immunotherapeutics in the future, taking
their place among a growing armory of anticancer combination
therapies.
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