Endoplasmic reticulum aminopeptidase 1 (ERAP1)
trims MHC class I-presented peptides in vivo
and plays an important role in immunodominance
Ian A. York*, Michael A. Brehm, Sophia Zendzian, Charles F. Towne, and Kenneth L. Rock
Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655
Communicated by Baruj Benacerraf, Dana–Farber Cancer Institute, Boston, MA, April 28, 2006 (received for review February 20, 2006)
CD8?T cells respond to short peptides bound to MHC class I
molecules. Although most antigenic proteins contain many se-
quences that could bind to MHC class I, few of these peptides
actually stimulate CD8?T cell responses. Moreover, the T cell
responses that are generated often follow a very reproducible
hierarchy to different peptides for reasons that are poorly under-
stood. We find that the loss of a single enzyme, endoplasmic
reticulum aminopeptidase 1 (ERAP1), in the antigen-processing
pathway results in a marked shift in the hierarchy of immunodomi-
same T cell receptor repertoire. In mice, ERAP1 is the major enzyme
that trims precursor peptides in the endoplasmic reticulum and, in
this process, can generate or destroy antigenic peptides. Conse-
quently, when ERAP1 is lost, the immune response to some viral
peptides is reduced, to others increased, and to yet others un-
changed. Therefore, many epitopes must be initially generated as
precursors that are normally trimmed by ERAP1 before binding to
MHC class I, whereas others are normally degraded by ERAP1 to
trimming and the resulting abundance of peptide–MHC complexes
are dominant factors in establishing immunodominance.
antigen presentation ? antigen processing ? peptidases
CD8?T cells recognize their targets through interactions between
their T cell receptor (TCR) and MHC class I on the surface of the
target cell. MHC class I is composed of a light chain (?2-
microglobulin), a highly polymorphic heavy chain, and a small
peptide that is produced by degradation of intracellular proteins.
MHC class I alleles bind to peptides with a particular set of
anchor residues that fit into pockets in the MHC class I
peptide-binding groove. Virus proteomes typically contain many
peptides with anchor residues suitable for particular MHC class
I alleles, yet immune responses are generally directed toward a
very limited number of epitopes. This phenomenon, in which
only a few epitopes are functionally immunogenic, is known as
immunodominance and is, as yet, incompletely understood
(reviewed in ref. 1). Although many explanations for immu-
nodominance have been proposed (1–9), their relative impor-
tance is not well established.
by proteasomes in the cytosol. Proteasomes can generate either the
mature epitope that is ultimately presented by MHC class I
the amino terminus. Such N-extended peptides are further pro-
cessed by aminopeptidases to generate the final presented epitope.
Although a number of peptidases have been proposed to play roles
in MHC class I antigen presentation, few have been shown to play
a major role in antigen presentation in intact cells. In cell lysates,
aminopeptidases, including leucine aminopeptidase (LAP) (10),
bleomycin hydrolase (BH) (11), and puromycin-sensitive amino-
peptidase (PSA) (11) can convert various N-extended peptides into
the mature epitopes. However, in intact cells and mice, LAP
ells infected with a virus or otherwise producing foreign
proteins can be recognized and eliminated by CD8?T cells.
appears dispensable for antigen presentation (12), and the impor-
tance of BH and PSA in antigen presentation remains to be
be important in destroying many peptides (13–15). It has recently
been proposed that most antigenic peptides are generated as very
long precursors (?15 residues) and that tripeptidyl peptidase II
(TPPII) is essential for trimming these precursors for antigen
presentation (16, 17). However, we find that, in vivo, proteasomes
generate relatively few very long peptides, and silencing of TPPII
has only a small effect on overall antigen presentation (I.A.Y., N.
Bhutani, S.Z., A. L. Goldberg, and K.L.R., unpublished data).
Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1; ER-
AAP) is an IFN ?-inducible ER-localized aminopeptidase ex-
pressed in many tissues, although at widely different levels (18–20).
ERAP1 was previously shown to influence the presentation of
several peptides in cultured cells. Human cells lacking ERAP1
increase in the amount of peptide associated with MHC class I but
also had markedly reduced presentation of specific peptides, such
as the immunodominant H-2Kb-binding peptide from chicken
ovalbumin, SIINFEKL (21, 22), whereas cells from mice lacking
?8 or 9 aa (20, 25), the optimal length for binding to MHC class I,
helping to explain why ERAP1 creates MHC class I epitopes in
some cases (by trimming N-extended precursors to the mature
epitope) and destroys them in others (by trimming mature epitopes
that are nine or more residues to a size that is too small to bind to
MHC class I).
To better understand the role and importance of ERAP1 in
normal immune responses, we generated and analyzed a mutant
mouse lacking ERAP1. We find that not only is ERAP1 important
in generating MHC class I epitopes but also that its absence
drastically alters immunodominance hierarchies, pointing to a
central role for antigen processing and MHC class I–peptide
complex abundance in establishing immunodominance.
Generation of ERAP1 Knockout (KO) Mice. C57?BL6 mice heterozy-
gous for floxed ERAP1 (in which loxP sites were present in the
introns flanking ERAP1 exons 5 and 6) were generated and
crossed with cre-deleter mice (26). The resulting loss of exons 5
and 6 causes a frame-shift mutation that would lead to early
termination of the exon4?exon7 fusion (see Fig. 6, which is pub-
because exon 5 of ERAP1 contains the enzyme’s active site, the
truncated protein (if expressed) would not be enzymatically active.
Conflict of interest statement: No conflicts declared.
Abbreviations: APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; ER, endoplasmic
reticulum; ERAP1, ER aminopeptidase 1; KO, knockout; LCMV, lymphocytic choriomenin-
gitis virus; MEF, mouse embryonic fibroblast; TCR, T cell receptor.
*To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
June 13, 2006 ?
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The resulting heterozygous ERAP1?/?mice were bred to homozy-
were fertile, grew normally, and showed no obvious signs of
pathology. Therefore, ERAP1 is not an essential gene for survival.
MHC Class I Levels in Fibroblasts from ERAP1 KO Mice Are Not Altered.
We prepared fibroblasts from day-14–17 embryos from
ERAP1?/?crosses and typed the cell lines to identify ERAP1?/?
and ERAP1?/?lines. Several independent mouse embryonic
fibroblast (MEF) cell lines were generated and analyzed for
surface MHC class I expression. ERAP1?/?and ERAP1?/?lines
had similar levels of H-2Kband H-2Db(Fig. 1A); however,
constitutive MHC class I expression on these fibroblasts was very
low, and, under these conditions, surface expression of MHC
class I is probably not limited by peptide supply.
Trimming of Peptide Precursors in the ER of Fibroblasts from ERAP1
KO Mice Is Reduced. We transfected three ERAP1?/?and three
ERAP1?/?MEF lines with various precursors of SIINFEKL (the
immunodominant H-2Kb-restricted epitope from chicken ovalbu-
min) using the mAb 25.D1.16 (27). First, we expressed SIINFEKL
as a ubiquitin fusion in the cytosol. Ubiquitin C-terminal hydrol-
yases efficiently cleave the peptide bond between the ubiquitin
moiety and SIINFEKL, thereby generating the mature epitope.
There was no statistically significant difference between ERAP1-
deficient and WT cells (Fig. 1B). This result is consistent with the
earlier findings that ERAP1 does not destroy the mature 8-residue
SIINFEKL peptide (20, 21, 25). These findings also demonstrate
that, with the exception of peptide trimming in the ER, the MHC
class I pathway is otherwise intact in ERAP1-deficient cells.
To analyze peptide trimming in the ER, we expressed in MEFs
into the ER by a signal sequence (ss-ALEQLE-S8L). Presentation
of H-2Kb-SIINFEKL from this precursor was reduced to levels
barely above background staining (Fig. 1B). Therefore, in MEFs as
in human cells (21), ERAP1 is critical for trimming peptides in the
ER. Similarly, presentation of SIINFEKL from a cytosolic ubiq-
uitin fusion containing the 13-residue precursor LEQLE-
SIINFEKL was markedly reduced in ERAP1?/?MEFs. Therefore,
this peptide is transported into the ER by transporter associated
with antigen presentation (TAP) as an N-extended form, rather
than being processed by cytosolic aminopeptidases to SIINFEKL,
and trimming of this precursor to the mature epitope requires
In additional experiments, we analyzed the presentation of
SIINFEKL from full-length ovalbumin. In all experiments (n ?
6), presentation from this construct was highly variable between
individual MEF lines and, on average, did not differ significantly
between WT and ERAP1?/?cell lines. This result was surprising,
because it differed from earlier findings in HeLa cells, where the
presentation of SIINFEKL from the full-length protein was
markedly reduced in the absence of ERAP1 (21). However,
there may be effects of ERAP1 that are obscured by the large
variation between different MEF cell lines.
MHC Class I Levels Are Reduced on Splenocytes from ERAP1 KO Mice.
We stained spleen cells for the MHC class I molecules H-2Kband
H-2Dband, as a control, for the MHC class II I-Abmolecule. In
contrast to the findings with MEFs, H-2Kbwas reduced to ?60–
65% of control levels (P ? 0.05, Student t test) on B220?B cells,
CD4?and CD8?T cells, and on CD11c cells (predominantly
dendritic cells) from the spleen of mice lacking ERAP1 compared
with their WT littermates (Fig. 2). Expression of H-2Dbwas
moderately reduced on the same cell types, to ?75–80% of control
Student t test) only for CD11c?cells. In contrast, there was no
difference in the levels of I-Abbetween WT and ERAP1 on any of
selectively affects the surface expression of MHC class I molecules
on splenocytes. Similarly, thymocytes (all subsets) from ERAP1?/?
mice expressed significantly lower levels of both H-2Kband H-2Db
than did thymocytes from ERAP1?/?mice (data not shown).
CD8?T Cell Responses to Viral Epitopes Are Altered in ERAP1 KO Mice.
To determine whether, in the absence of ERAP1, responses to
MHC class I-restricted peptides were altered, we infected
ERAP1?/?, or age-matched WT C57BL?6 mice with recombinant
trimming in the ER. Fibroblast cell lines were prepared from ERAP1?/?(black
bars) and ERAP1?/?(gray bars) embryonic mice. (A) Cells were stained with
mAb directed against H-2Kb(mAb B8.24.3) or H-2Db(28.14.8S) and analyzed
by flow cytometry. (B) MEF cells were transfected with plasmids expressing
SIINFEKL as a cytosolic ubiquitin fusion (S8L), ovalbumin protein targeted to
the cytosol (Ova), LEQLE-SIINFEKL as a cytosolic ubiquitin fusion (N5-S8L), or
ALEQLE-SIINFEKL targeted to the ER by a signal sequence (ss.N5-S8L). After
48 h, the cells were stained with mAb 25.D1.16 (anti-H-2Kb-SIINFEKL) and
analyzed by flow cytometry. Data (representative of five experiments) show
the mean fluorescence intensity with background staining by an irrelevant
antibody subtracted (averages of three independent cell lines; error bars
Student t test) between ERAP1?/?and ERAP1?/?MEF cell lines.
ERAP1?/?mouse embryonic fibroblast lines are defective for peptide
I than those from ERAP1?/?mice. Splenocytes from ERAP1?/?mice (WT, black
bars) or ERAP1?/?mice (KO, gray bars) were stained with mAb to H-2Kbor
H-2Dbor, as a control, to I-Aband various antibodies to distinguish splenocyte
subsets (CD11c, predominantly dendritic cells; CD8?and CD4, predominantly
T lymphocyte subsets; B220, predominantly B cells) as indicated on the x axis.
Shown are mean fluorescence intensities with background staining by an
irrelevant antibody subtracted (averages of three mice; error bars represent 1
SD; representative of five experiments). Asterisks indicate statistically signif-
icant differences (P ? 0.05, Student t test) between ERAP1?/?and ERAP1?/?
York et al.
June 13, 2006 ?
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SIINFEKL from ovalbumin preceded by a signal sequence (Vac-
ES-Ova) and measured responses to two vaccinia-derived peptides
(B8R20–27and A47L138–146) (28) as well as SIINFEKL. The re-
sponse to B8R was significantly increased in ERAP1?/?mice
compared with WT mice, whereas the responses to A47L and
SIINFEKL were not significantly different whether mice were
infected with Vac-FLOVA or Vac-ES-OVA (Fig. 3). The response
to the subdominant H-2Kb-restricted peptide from ovalbumin,
KVVRFDKL (29), was not detectable in mice infected with
Vac-ES-OVA (which do not express this epitope) and was barely
above background in mice infected with Vac-FLOVA in the
presence or absence of ERAP1 (Fig. 3).
We also infected ERAP1?/?or WT mice with lymphocytic
choriomeningitis virus (LCMV). Eight days later, we measured the
frequency of CD8?T cells specific for particular LCMV peptides.
Of six LCMV H-2b-restricted epitopes tested, the frequencies of
somewhat increased (Fig. 4 A and B). Specifically, there were only
peptides GP34, GP118, NP205, and the H-2Db-restricted peptide
NP396, whereas those specific for the H-2Db-restricted epitope
GP33 were unchanged. T cells specific for the H-2Dbepitope
GP276 were modestly increased, up to 1.5-fold, although this
difference was statistically significant (P ? 0.049, Student t test) in
only one of three experiments.
Notably, all of the 8-mer epitopes (GP34, GP118, and NP205)
showed reduced immunogenicity, whereas the longest peptide, the
11-mer GP276 showed a trend toward increased immunogenicity.
This finding is consistent with the notion that ERAP1 is most
important for generating shorter peptides, whereas longer peptides
?50% of peptides down to 9 mer and the other 50% to 8 mer (25).
In addition to peptide size, internal sequences also affect the ability
of ERAP1 to trim peptides, but this specificity is not yet well
In WT mice, the magnitude of T cell responses to the different
LCMV epitopes follows a stereotypical hierarchy of immunodomi-
nance: NP396 ? GP34 ? GP276 ? GP33 ? NP205 ? GP118
(30–32) (Fig. 4 Inset). This pattern is reproducible between indi-
is, therefore, remarkable that the altered responses in the ERAP1-
reproducible hierarchy of immunodominance: GP276 ? NP396 ?
GP33 ? GP34 ? GP118 ? NP205 (Fig. 5 Inset).
ERAP1 Effects on CD8?T Cells Do Not Depend on Differences in the T
Cell Repertoire. The effects of ERAP1 on the magnitude of T cell
responses and the immunodominance hierarchy could be due to
changes in peptide processing in peripheral cells or in the thymus,
where different peptide presentation could lead to changes in TCR
repertoire because of effects on positive or negative thymic selec-
ERAP1?/?(black bars) and ERAP1?/?(gray bars) mice were infected with
recombinant vaccinia virus expressing full-length ovalbumin (Vac-FLOva) (A)
days later, splenocytes were stimulated with peptides corresponding to vac-
cinia epitopes (B8R and A47L) or ovalbumin epitopes (SIINFEKL and KVVR-
FDKL), permeabilized, stained with anti-IFN? antibody, and analyzed by flow
to each peptide (averages of three mice; error bars represent 1 SD; represen-
tative of at least three experiments). Asterisks indicate statistically significant
differences (P ? 0.05, Student t test) between ERAP1?/?and ERAP1?/?mice.
CD8?T cell responses to vaccinia virus are altered in ERAP1?/?mice.
infected with LCMV. Eight days later, splenocytes were stim-
ulated with peptides corresponding to LCMV epitopes, per-
with tetramers consisting of H-2Kbor H-2Dbcomplexed with
the indicated LCMV epitopes and analyzed by flow cytometry
peptide (averages of three mice; error bars represent 1 SD;
representative of at least three experiments). Asterisks indi-
cate statistically significant differences (P ? 0.05, Student t
as in A and B, arranged to show the normal and altered
www.pnas.org?cgi?doi?10.1073?pnas.0603095103York et al.
tion. The number and frequency of T cell subsets were not altered
in the thymus (see Table 1, which is published as supporting
information on the PNAS web site) or spleen (see Table 2, which
is published as supporting information on the PNAS web site) of
not grossly affected by the absence of ERAP1. Nevertheless, it
that led to the observed changes. To test this issue, we used an
adoptive transfer approach to normalize the repertoire of respond-
ing T cells in the WT and ERAP1-deficient mice.
LCMV-immune splenocytes from WT B6?SJL mice (congenic
for CD45.1) that had been infected with LCMV 5 months previ-
ously were transferred into ERAP1?/?or ERAP1?/?mice, and the
recipients were infected with LCMV to stimulate a recall response.
After 7 days, donor CD45.1 splenocytes were analyzed by intracel-
lular cytokine staining. In WT mice, the transferred memory cells
showed the typical WT pattern of immunodominance. In contrast,
the responses of the transferred memory cells in ERAP1?/?mice
were very different from those in ERAP1?/?mice (Fig. 5). These
differences followed the same altered pattern of immunodomi-
nance that was observed in the primary response, except that, if
ERAP?/?mice was generally greater, all the more remarkable
because the repertoire of the transferred memory cells was initially
biased toward the WT pattern of immunodominance. Therefore,
these results demonstrate that a major factor in establishing the
pattern of immunodominance in the LCMV system is antigen
presentation by host antigen-presenting cells (APCs).
In studies using cultured cells, ERAP1 either enhanced MHC class
I antigen presentation by trimming peptides to the proper size for
MHC class I binding or reduced presentation by trimming peptides
22). Recent studies of cells from ERAP1 (ERAAP) KO mice
showed similar effects, although, in most cases, antigen presenta-
tion was reduced in the absence of ERAP1 (23, 24). Here, we
confirm and extend these findings and show that, in vivo, ERAP1
plays an important role in immune response to viruses, either
enhancing or reducing cytotoxic T lymphocyte (CTL) responses to
particular viral epitopes and, thereby, helping establish immu-
nodominance hierarchies. In addition to supporting a role for
aminopeptidases in antigen presentation, these findings indicate
that, in LCMV infections, immunodominance is, to a very large
extent, determined by antigen processing and epitope density on
Consistent with previous studies (21, 22), we found that MEF
cell lines from ERAP1-deficient mice presented SIINFEKL on
H-2Kbmuch less efficiently than did WT MEFs, when trans-
fected with precursors of SIINFEKL with 5- or 6-residue N-
terminal extensions (Fig. 1B). Mature SIINFEKL was presented
at similar levels in the WT and ERAP1?/?MEFs (Fig. 1B),
demonstrating that all steps in the antigen-presentation pathway
other than ER trimming were intact in the ERAP1?/?cells.
These findings confirmed that ERAP1 is the major trimming
enzyme in the ER lumen. Interestingly, ER-targeted SIINFEKL
with a relatively short N-terminal extension (ALL-SIINFEKL, a
3-residue extension) was presented relatively well in ERAP1?/?
cells, being reduced by ?50% (but still easily detectable) com-
pared with WT cells (data not shown), raising the possibility that
another as-yet-unidentified ER aminopeptidase may contribute
weakly to trimming of some ER peptides. In humans, another
aminopeptidase that is closely related to ERAP1, termed L-
RAP (31) or ERAP2 (32), is also expressed in some tissues,
on peptides in the ER, may also modify the activity of ERAP1,
because the two enzymes form a complex (32). However,
ERAP2 is not present in mice.
We found no significant difference in cell-surface MHC class I
between ERAP1?/?and WT MEFs. However, it is likely that, in
primary fibroblasts (which express much lower levels of MHC class
I and other components of the MHC class I antigen-presentation
pathway than do the transformed cells used in previous studies),
peptide is not always limiting for surface expression of MHC class
I. Surface levels of MHC class I were significantly reduced on
ERAP1?/?B cells, T cells, and thymocytes. T cell and thymocyte
subset ratios were not altered in KO vs. WT mice, suggesting that
or negative thymic selection. Importantly, MHC class I surface
are critical for immune surveillance and for activating T cell
responses. T and B cells and DC express mainly immunoprotea-
somes, and, in HeLa-Kb cells in which immunoproteasomes were
induced by chronic treatment with IFN?, ERAP1 knockdown also
the observation that ERAP1 acts only very slowly on peptides of
eight or fewer residues but rapidly hydrolyzes ?50% of nonamers
to octamers (20, 25). Because H-2Kbmainly binds peptides that are
8 aa in length, ERAP1 will primarily function to generate these
peptides, whereas, in contrast, ERAP1 can both generate and
destroy many of the 9-mer peptides that bind to H-2Db(33). In any
case, these data show an important role for ERAP1 in MHC class
I antigen presentation in mouse primary cells.
One of the most important reasons to develop an ERAP1 KO
of immune responses in vivo. We infected ERAP1?/?or ERAP1?/?
tissue (recombinant vaccinia viruses) and measured the frequency
of CD8?T cells specific for various epitopes from these viruses.
ERAP1 proved to be very important in establishing immune
responses to viral epitopes: Six of the nine epitopes that gave a
detectable response showed significantly different responses in the
response, whereas, in four, the response was markedly reduced in
the absence of ERAP1. These observations show that, in vivo, as in
vitro, ERAP1 plays an important role in modifying antigenic
It is noteworthy that the effects of ERAP1 are, to some extent,
predictable based on its biochemical characterization (20, 25).
T cell repertoire. Splenocytes from LCMV-immune B6?SJL mice were adoptively
transfer, recipient mice were infected with LCMV, and the recall CD8?T cell
Shown is the percentage of CD8?CD45.1?T cells producing IFN? in response to
Student t test) between ERAP1?/?and ERAP1?/?mice.
Altered CD8?T cell responses in ERAP1?/?mice are not due to altered
York et al.
June 13, 2006 ?
vol. 103 ?
no. 24 ?
Three of the peptides with altered responses are octamers that are
presented by H-2Kb(LCMV GP34, NP205, and GP118); these
of nonamers slowly and the other half rapidly. One of the two
nonamers analyzed in the LCMV response (NP396, presented by
H-2Db), showed a reduced response in the absence of ERAP1,
whereas the other nonamer (GP33, presented on H-2Db) was not
affected by the lack of ERAP1. ERAP1 rapidly trims peptides
between 10 and 16 residues long; the CD8?response to LCMV
GP276, which is 11 residues long, was significantly enhanced in
secondary responses and showed a similar trend in primary re-
sponses in the absence of ERAP1. Therefore, presentation of the
shortest peptides, which are most likely to depend on ERAP1 for
trimming, is the most reduced by its loss; whereas presentation of
was enhanced in its absence.
An exception to this trend occurred with the vaccinia virus
In the absence of ERAP1, B8R induced about twice as many CTL
as in its presence. This difference between LCMV and vaccinia
peptides may be related to the primary sequence of the peptides or
of CD8?T cells responding to the B8R epitope changes when
vaccinia is administered to different tissues (28), and it is tempting
to speculate that this change may be related to different levels of
ERAP1 in different tissues. In addition, consistent with observa-
tions on MEFs, the immune response to ovalbumin (SIINFEKL)
was not affected when mice were infected with recombinant
vaccinia virus expressing full-length ovalbumin or SIINFEKL tar-
geted to the ER by a signal sequence (Fig. 3 A and B). The main
point, however, is that ERAP1 clearly plays an important role in
vivo in processing peptides for MHC class I antigen presentation.
One striking result of ERAP1’s broad effect on antigen presen-
tation was its impact on immunodominance. The CTL response to
LCMV, as with virtually all immune responses, is focused on a
limited number of peptides, and, in mice with the same genetic
background, the response to different peptides follows a strict and
stable hierarchy, with (in C57BL?6 mice) the peptide NP396
inducing the most CTL and GP118 the fewest (31). LCMV is a
maintaining its immunodominance hierarchy in the absence of
CD28 costimulation (34) and in the absence of immunoprotea-
somes (35) and showing only slight changes in immunodominance
the immunodominance hierarchy was disrupted: The response to
several peptides was reduced by 1?2 to 2?3, whereas the response
to other peptides was unchanged or even increased. Remarkably,
even when responding cells were all from the WT mice that had
previously been exposed to LCMV (and were, therefore, already
skewed toward the WT pattern of immunodominance), the pattern
of altered immunodominance emerged in ERAP1?/?mice.
These findings have important implications for the mechanisms
responsible for immunodominance. The causes of immunodomi-
nance are as yet incompletely understood. Although high-affinity
binding to MHC class I is important for responses and immu-
nodominance (37–40), many peptides with high affinity for MHC
class I are not immunodominant, and, conversely, several immu-
lymphocytes themselves have been proposed to play an important
role in establishing immunodominance through several different
mechanisms. CD8?T cells responsive to immunodominant
epitopes may be the most abundant ones in the T cell repertoire,
either naturally (3, 44, 45) or as a result of cross-reactive immunity
others for APCs (6, 7) by suppressing responses to subdominant
epitopes (8, 9) or by more efficiently generating IFN? (46). Other
explanations for immunodominance include the induction of tol-
(9), kinetics of protein expression (47), and agonist?antagonist
peptide interactions with TCR (48). However, there is little defin-
itive data demonstrating the relative importance of these explana-
surface [which could be influenced by many factors, including the
amount of peptide generation and destruction by proteasomes (49)
and peptidases] may be another factor (50, 51), but quantitation of
cell-surface MHC class I–peptide complexes suggests that peptide
density may not be a major cause of immunodominance (52, 53)
(reviewed in ref. 1); however, accurate quantitation of cell-surface
peptide–MHC complexes is notoriously difficult. These various
mechanisms are not mutually exclusive and may all participate to
varying extents in different situations.
LMP2-deficient mice (54), in which immunodominance of a few of
the many influenza-derived epitopes is affected. However, in this
model, altered T cell repertoire was also important for some,
although not all, of the alterations in immunodominance. In the
ERAP1-deficient mouse model, we can rule out T cell-intrinsic
factors, such as a biased T cell repertoire, because the same effects
were seen even when the responding T cells from the same
LCMV-immune WT host were transferred into ERAP1?/?versus
ERAP1?/?mice. In addition, the results from these adoptive
transfer experiments indicate that T cell immunodomination or
competition at the level of the APC does not underlie the major
mechanism of immundominance for LCMV, and, because the
source of the epitopes (i.e., protein expressed from the virus) is the
same in WT and ERAP1?/?mice, antigen-intrinsic differences can
be ruled out. The CTL transfer experiments map the effects of
its effects through alterations in the generation and destruction of
the individual viral epitopes.
These findings are particularly remarkable because LCMV stim-
ulates an extraordinarily strong immune response. Similarly, we
observed changes in CD8?T cell responses to vaccinia virus,
another strongly immunogenic virus. Thus, the changes in the
under very limiting conditions. Together, these findings demon-
strate that the major factor in establishing the immunodominance
hierarchy to LCMV epitopes is almost certainly the abundance of
peptide epitopes at the cell surface. By altering antigen processing
and, therefore, affecting epitope abundance, the absence of
ERAP1 reprogrammed the immunodominance hierarchy in the
immune response to LCMV.
Materials and Methods
ERAP1 Mutant Mice. Heterozygous mice with a conditional KO of
ERAP1 were generated under contract by OzGene (Bentley,
Australia). Briefly, LoxP sequences were inserted between exons
4 and 5 and between exons 6 and 7 (Fig. 6). A phosphoglycerine
kinase (PGK) Neo cassette flanked by FLP recombinase target
(FRT) sequences, inserted between exon 6 and the second loxP
site, was used to confer resistance to C57BL?6 ES cells that had
successfully integrated the targeting vector and was removed by
treatment with Flippase (FLP) recombinase. This procedure
produced ES cells with exons 5 and 6 of ERAP1 flanked by LoxP
sites (Fig. 6). ES cells were microinjected, and chimeric mice
were bred to generate heterozygous F1 mice. These floxed mice
were crossed with Cre-deleter C57BL?6 mice (26), leading to the
removal of exons 5 and 6 on one chromosome. Mice were bred
Plasmids. Plasmids used in these experiments were pTracerSR?
(pTracerSV40; Invitrogen) in which the SV40 promoter has been
replaced by the SR? promoter (55) pTracer-SR?-Cyto-OVA (ex-
the signal sequence removed, yielding a cytosolic protein that is
www.pnas.org?cgi?doi?10.1073?pnas.0603095103York et al.
rapidly degraded by proteasomes) (56), pUG1 (a control vector
GFP, under the control of the CMV promoter), pUG-S8L (same
as pUG1 but with SIINFEKL fused to the C terminus of ubiquitin,
so that C-terminal ubiquitin hydrolases rapidly release SIINFEKL
from ubiquitin) (57), pUG-N5-S8L (pUG expressing LEQLE-
SIINFEKL fused to the C terminus of ubiquitin), and pUG-ss-
AN5-S8L (pUG with the Ad-E3gp19K signal sequence preceding
Cells and Flow Cytometry. Six 9-week-old homozygous KO mice or
their WT littermates were killed, and splenocytes or thymocytes
were stained with anti-CD8, anti-CD4, anti-CD11c, anti-H-2Kb,
anti-H-2Db, anti-B220, and anti-I-Abin various combinations and
Dickinson and eBioscience, San Diego).
MEFs were prepared as described (12) from heterozygous
females crossed to heterozygous males. After culture, DNA from
each cell line was analyzed by genomic PCR, and homozygous WT
and KO lines (three each) were stained with B8.24.3 (anti-H-2Kb)
(58) or 28.14.8S (anti-H-2Db) (58) and analyzed by flow cytometry.
FuGene6 (Roche) according to the manufacturer’s protocol, with
plasmids expressing various SIINFEKL precursors stained with
mAb 25.D1.16 (specific for the combination of SIINFEKL and
identified by GFP expression. Transfection efficiency, based on
GFP expression, ranged from ?5% to 25%, depending on the
Viral Infections and Detection of Epitope-Specific CD8?T Cells. Ho-
mozygous KO mice, age-matched WT littermates, or C57BL?6
mice (The Jackson Laboratory), were infected i.p. with 5 ? 104
plaque-forming unit (PFU) of LCMV (strain Armstrong) or 5 ?
106PFU of vaccinia recombinants expressing full-length ovalbu-
min (Vac-FLOVA) (50) or A-SIINFEKL preceded by a signal
sequence (Vac-ES-Ova) (50) provided by Jon Yewdell (National
Institutes of Health, Bethesda). Seven or 8 days later, epitope-
specific CD8?T cells were detected by using intracellular
cytokine staining or tetramers as described in refs. 4, 28, and 59.
Adoptive Transfer of LCMV-Immune Splenocytes. To generate
LCMV-immune mice, WT B6?SJL mice (CD45.1 from Taconic
Farms) were infected with 5 ? 104PFU of LCMV and were used
at 5–6 months after infection. Splenocytes from LCMV-immune
B6?SJL mice were adoptively transferred into ERAP1?/?or
ERAP1?/?mice. One day after transfer, recipient mice were
infected with 5 ? 104PFU of LCMV, and the recall CD8?T cell
response generated by the donor cells was analyzed by intracellular
cytokine staining as described in ref. 4. Donor CD8?T cells were
identified by staining with a mAb specific for the congenic marker
CD45.1 (A20; eBioscience).
We thank Keith Daniels for constructing MHC class I tetramers and
Sharlene Hubbard for expert assistance with mice. This work was
supported by National Institutes of Health grants (to K.L.R.).
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