© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
In the absence of aminopeptidase ERAAP, MHC class I
molecules present many unstable and highly
immunogenic peptides
Gianna Elena Hammer, Federico Gonzalez, Edward James, Hector Nolla & Nilabh Shastri
Immunosurveillance by cytotoxic T cells requires that cells generate a diverse spectrum of peptides for presentation by
major histocompatibility complex (MHC) class I molecules. Those peptides are generated by proteolysis, which begins in
the cytoplasm and continues in the endoplasmic reticulum by the unique aminopeptidase ERAAP. The overall extent to which
trimming by ERAAP modifies the peptide pool and the immunological consequences of ERAAP deficiency are unknown.
Here we show that the peptide-MHC repertoire of ERAAP-deficient mice was missing many peptides. Furthermore, ERAAPdeficient cells presented many unstable and structurally unique peptide-MHC complexes, which elicited potent CD8+ T cell
and B cell responses. Thus, ERAAP is a ‘quintessential editor’ of the peptide-MHC repertoire and, paradoxically, its absence
enhances immunogenicity.
The peptide–major histocompatibility complex class I (pMHC I)
repertoire, containing thousands of different eight- to ten-residue
peptides, is generated by the antigen-processing pathway in almost all
vertebrate cells1,2. Because the pMHC I complexes serve as ligands for
the CD8+ T cell receptor, antigen processing is essential for immune
surveillance of intracellular viruses, bacteria or mutant proteins in
tumor cells. The peptides are generated by different proteases in two
distinct subcellular compartments3. Beginning in the cytoplasm, the
proteasome and possibly other proteases fragment intracellular proteins to cut them at the precise C terminus of the final peptide and
generate a mixture of N-terminally extended intermediates4–6. These
intermediates are then transported by TAP, the transporter associated
with antigen processing, into the endoplasmic reticulum7. There,
ERAAP (the endoplasmic reticulum aminopeptidase associated with
antigen processing) trims the extra N-terminal residues to generate
final peptides of the correct length, which are bound by MHC class I
molecules8,9. Those peptides are eventually presented as pMHC I
complexes on the cell surface.
Involvement of ERAAP in regulating pMHC I expression has been
demonstrated in cell lines8,9 and in ERAAP-deficient mice10,11. Those
studies have shown that although some peptides are unaffected by
ERAAP deficiency, the overall pMHC I repertoire is altered. Some
pMHC I complexes are not detected in ERAAP-deficient cells and
others are substantially upregulated. Furthermore, biochemical analysis of processed peptides has shown that those alterations in the
pMHC I repertoire are due to the inability to trim N-terminal residues
in the endoplasmic reticulum without ERAAP10. However, to what
extent ERAAP deficiency alters the total peptide repertoire and its
immunological consequences remain unknown.
Here we investigate the extent to which N-terminal trimming by
ERAAP shapes the overall pMHC I repertoire and the fate of
untrimmed precursors of the final peptides. We found that an absence
of ERAAP activity depleted the pMHC I repertoire of many peptides
and concomitantly generated a large volume of new pMHC I complexes on the cell surface. Those unique pMHC I complexes expressed
by ERAAP-deficient cells were structurally distinct from conventional
pMHC I complexes and elicited potent CD8+ T cell and B cell
responses in MHC-matched wild-type mice.
RESULTS
ERAAP-deficient CD8+ T cells respond to wild-type cells
In addition to having altered expression of endogenous peptides, cell
surface pMHC I is expressed less abundantly by ERAAP-deficient mice
than by wild-type mice10. Although those changes do not affect the
total number of CD8+ T cells, antigen-specific responses of female
ERAAP-deficient mice to pMHC I encoded by the Y chromosome
(HY) are lower. To further investigate why CD8+ T cell anti-HY
responses are low, we immunized female ERAAP-deficient or wildtype mice with cells from wild-type C57BL/6J mice. Two peptides
processed from the protein products of Uty (‘ubiquitously transcribed
tetratricopeptide repeat gene, Y chromosome’) and Jarid1d (‘jumonji,
AT-rich interactive domain 1D’; also called Smcy) were presented by
H-2Db MHC class I on cells from male C57BL/6J mice and, as
reported before, the average CD8+ T cell response of ERAAP-deficient
Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA. Correspondence should be addressed to
N.S. ([email protected]).
Received 5 September; accepted 13 October; published online 26 November 2006; doi:10.1038/ni1409
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a
Uty-H-2Db
CD8+IFN-γ + (%)
40
Jarid1d-H-2Db
11.0
5.5
0
0.0
WT
APC
8.7%
WT + APC
0.2%
101
100
WT ERAAP-KO
103
35.0
25.0%
102
ERAAP-KO
17.5
0.0
T cell:
APC:
CD8
CD8+IFN-γ + (%)
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
WT
103
102
20
WT ERAAP-KO
c
b
23.0%
101
100
10 –1 100 101 102 103 10 –1 100 101 102 103
WT ERAAP-KO
WT
WT
ERAAP-KO
IFN-γ
WT +
Figure 1 ERAAP-deficient mice elicit a CD8+ T cell response to autosomal antigens expressed on
wild-type cells. (a) Intracellular cytokine staining to detect the IFN-g responses of T cells from female
wild-type mice (WT) and ERAAP-deficient mice (ERAAP-KO). Female mice were immunized and
restimulated in vitro with cells from male wild-type mice; T cells were incubated with peptides derived
from Uty and Jarid1d. Each symbol represents one mouse; horizontal lines, average. Data are
representative of two independent experiments. (b) IFN-g production by CD8+ T cells cultured together
with APCs, assayed as described in a. Splenocyte samples from male wild-type mice (left), which
express HY antigens, or from female wild-type mice (right), were depleted of CD8 and were used as
APCs to restimulate the T cells from a. Numbers above boxed areas indicate percent CD8+IFN-g+ cells
among all CD8+ cells. Data are representative of responses from eight mice of either genotype.
(c) Responses of immunized mice to APCs. Each symbol represents one mouse; horizontal lines,
average. Numbers reported are above the background response of T cells alone. Data are representative
of two separate experiments with at least three mice each.
mice to those peptides was only 20% that of wild-type mice (Fig. 1a).
We also measured interferon-g (IFN-g) produced by those
T cells when directly stimulated by antigen-presenting cells (APCs)
from male wild-type mice expressing HY antigen. When cultured
together with APCs from male wild-type mice, about 8% (average,
5%) of wild-type CD8+ T cells produced IFN-g, but did not respond
to self APCs from female wild-type mice (Fig. 1b,c). Notably, IFN-g
production induced by APCs from male wild-type mice was somewhat lower than the actual number of T cells that responded to
synthetic HY peptides, probably because of differences in ligand
density achieved with synthetic peptides compared with physiological
density. In contrast, not only was the fraction of ERAAP-deficient
CD8+ T cells producing IFN-g in response to cells from male wildtype mice 500% higher (average, 25% CD8+IFN-g+), but also a similar
number responded to cells from female wild-type mice (Fig. 1b,c).
That strong response to autosomal antigens probably ‘diluted out’ the
HY-specific response. We conclude that unlike female wild-type mice,
which responded only to Y chromosome–linked antigens of male cells,
the ERAAP-deficient mice elicited CD8+ T cell responses to a different
set of autosomal antigens.
ERAAP-deficient mice lack a large set of pMHC I complexes
Biochemical analysis of the pMHC I repertoire of ERAAP-deficient
mice has shown that a subset of peptides is absent from the cell surface
and is also not detected in cell extracts10. Because CD8+ T cells are
tolerant to self but respond with exquisite specificity to non–self
peptide–MHC class I complexes, we reasoned that if many peptides
were truly absent from ERAAP-deficient mice, the CD8+ T cell response
to wild-type cells described above could have been a consequence of
gross disruption of the pMHC I repertoire. To test that hypothesis, we
immunized female ERAAP-deficient mice with cells from male wild-
102
type mice, but after immunization, restimulated the T cells with spleen cells from female
wild-type mice. After 5 d in vitro, almost 38%
(average, 35%) of the CD8+ T cells from
immunized mice responded specifically to
the wild-type APCs (Fig. 2a,b), whereas their
response to ERAAP-deficient self APCs remained less than 1%. The same CD8+ T cells
did not respond to C57BL/6J mice lacking
H-2Kb and H-2Db MHC class I molecules
(called ‘H2-Kb–H-2Db–knockout’ here)12,
indicating that expression of their ligands
was dependent on classical MHC I molecules.
Furthermore, antibodies specific for the MHC
class I molecules H-2Kb and H-2Db but not
those to the MHC class II molecule H-2Ab
inhibited the IFN-g response of the CD8+
T cells by approximately 50% (Fig. 2c,d).
Thus, the ERAAP-deficient CD8+ T cells
responding to wild-type cells were specific
for peptides presented by either H-2Kb or
H-2Db MHC class I. The lack of tolerance of
ERAAP-deficient mice to pMHC I presented
on wild-type cells and the magnitude of the
CD8+ T cell response showed that the absence
of ERAAP caused substantial loss of peptides
normally present in the pMHC I repertoire.
We have called these ‘ERAAP-dependent peptides’ (Supplementary Fig. 1 online).
ERAAP-deficient cells are immunogenic in wild-type mice
If the only consequence of ERAAP deficiency was loss of ERAAPdependent peptides and other peptides expressed in the pMHC I
repertoire were unchanged or were increased relative to those in wildtype mice10,11, then wild-type mice should be tolerant to ERAAPdeficient cells, because wild-type cells express all those peptides as self
peptide–MHC class I complexes. Unexpectedly, when we immunized
wild-type mice with ERAAP-deficient splenocytes, they elicited a very
robust CD8+ T cell response (Fig. 3a,b). Over 43% (average, 35%)
of the wild-type CD8+ T cells produced IFN-g in response to
ERAAP-deficient APCs, compared with 1.5% producing IFN-g in
response to wild-type self APCs. The ligands recognized by the
CD8+ T cells required that the APCs be completely deficient in
ERAAP, as cells from mice heterozygous for the locus encoding
ERAAP (called ‘ERAAP-heterozygous’ here) failed to stimulate
IFN-g production. Notably, unlike the response to normal complexes
of ERAAP-dependent peptide and MHC class I (Fig. 2c,d), which was
inhibited by both antibody to H-2Kb (anti-H-2Kb) and anti-H-2Db,
neither those antibodies nor anti–H-2 Qa-1 (MHC class Ib) inhibited
the response to the ligands expressed by the ERAAP-deficient cells
(Fig. 3c,d and data not shown). ERAAP-deficient cells do express
pMHC I detectable with conventional MHC antibodies, at approximately 70% the expression of wild-type cells10,11. The wild-type
CD8+ T cells producing IFN-g were heterogeneous, as they expressed
several different T cell receptor variable region-b segments and were
similar to conventional CD8+ T cells in that they expressed CD8a,
CD8b and CD3 and lacked the natural killer cell marker NK1.1
(data not shown). Moreover, the magnitude of the wild-type
CD8+ T cell response to ERAAP-deficient cells was similar to the
‘alloresponse’ (CD8+ T cell response to MHC-mismatched cells) to
C57BL/6J.C-H2bm1 (bm1) cells (about 43% CD8+IFN-g+), which
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ERAAP-KO
0.2%
102
b
H-2Kb–H-2Db– KO
WT
0.3%
37.8%
c
WT APC + α-Ab
70
CD8+IFN-γ+ (%)
ER
AA
PKO
a
WT APC + α-Kb
19.8%
36.9%
102
WT APC + α-Db
15.2%
35
100 101 102 103
100 101 102 103
100
H
-2
Kb
–H
-2 WT
Db
–
KO
100 101 102 103
IFN-γ
CD8
0
100
100 101 102 103
IFN-γ
100 101 102 103
d
100 101 102 103
100
αAb
αKb
αDb
Figure 2 ERAAP-deficient mice lack peptides normally expressed in the pMHC I repertoire of wild-type cells. (a) Responses
50
of T cells to coculture with APCs. ERAAP-deficient mice were immunized with wild-type spleen cells, their spleen cells were
restimulated for 5 d in vitro with wild-type spleen cells, and then the restimulated T cells were cultured together with APCs
from ERAAP-knockout, wild-type or H2-Kb–H-2Db–knockout C57BL/6J mice and their responses were assessed as IFN-g
0
production. Rectangles enclose CD8+IFN-g+ cells; numbers above indicate percent of total CD8+ cells responding above the
background of T cell–only cultures. (b) Percent CD8+ IFN-g-producing cells detected in a from each immunized ERAAP-knockout
mouse in response to various APCs (horizontal axis). (c) T cell responses, analyzed as described in a, for cocultures of T cells and
+ WT APC
wild-type APCs incubated with blocking antibodies to MHC class II H-2Ab (a-Ab) or to MHC class I H-2Kb (a-Kb) or H-2Db (a-Db),
included to assess the MHC restriction of the responding CD8+ T cells. (d) Normalization of the CD8+ IFN-g responses of three mice analyzed in c to the
response to APCs in medium alone (arithmetic mean + s.d.). max, maximum. At least three immunized mice were used for each experiment and data are
representative of three separate experiments.
express a mutant H-2Kb MHC class I (Supplementary Fig. 2 online)
and have been shown to activate 1–10% of all naive CD8+ T cells in
wild-type mice13. We called this population of CD8+ T cells ‘BEko
T cells’ because they were elicited in C57BL/6J mice by ERAAPknockout cells (Supplementary Fig. 1).
BEko T cell ligands require TAP and H-2b
We were puzzled by the inability of antibodies to MHC to inhibit
BEko T cell responses and considered the possibility that unlike the
ERAAP-deficient CD8+ T cell response to wild-type cells, the BEko
T cells might instead be specific for other potential ligands, such as
nonclassical MHC class I molecules14. We therefore characterized the
BEko T cell ligands on ERAAP-deficient cells by assaying T cell
responses to APCs from mice of different genetic backgrounds. In
contrast to the robust IFN-g response to ERAAP-deficient cells, BEko
T cells did not respond to APCs doubly deficient in both ERAAP
and TAP1 or singly deficient in TAP1 (Fig. 4a). Likewise, BEko T cells
were not activated by several other APCs, including those lacking
classical MHC I (H2-Kb–H-2Db–knockout), ERAAP-heterozygous
APCs or APCs lacking ERAAP but expressing different H-2d MHC
molecules (Fig. 4b).
b
102
1.5%
ERAAP-KO
CD8+IFN-γ+ (%)
WT
43.8%
101
100
To further confirm that the generation of BEko T cell ligands was
a direct consequence of ERAAP deficiency, we used a fibroblast cell
line derived from ERAAP-deficient C57BL/6J mice10. We first
treated ERAAP-deficient fibroblasts stably transfected with cDNA
encoding ERAAP or vector control with IFN-g to upregulate MHC
class I molecules and then used those as APCs for BEko T cells.
The ERAAP-deficient fibroblasts activated 37% of BEko T cells to
produce IFN-g, which is approximately one half the response to
splenic APCs (Fig. 4a), indicating that ligand expression was not
restricted to spleen cells alone (Fig. 4c). Most notably, when we stably
transfected the cDNA encoding ERAAP into the fibroblasts, those cells
failed to activate the BEko T cells, directly demonstrating an inverse
relationship between ligand expression and the presence of functional
ERAAP. Our results thus showed that the BEko T cell ligands
expressed by ERAAP-deficient cells required both TAP and H-2b
MHC. Thus, despite their inability to engage antibodies to MHC
class I, the BEko T cell ligands shared similarities to conventional
pMHC I. The ligands contained peptides of cytoplasmic origin,
which were transported by TAP and were presented by the H-2b
MHC I molecules on the cell surface, but only in the complete absence
of ERAAP.
c
50
ERAAP-KO APC
+ α-Ab
102
25
+ α-Kb
48.6%
+ α-Db
49.6%
45.9%
101
CD8
a
CD8
100
0
100 101 102 103
IFN-γ
100 101 102 103
WT
ERAAP-KO
ERAAP-het
100 101 102 103 10–1 100 101 102 103 10–1 100 101 102 103
IFN-γ
Figure 3 C57BL/6J wild-type mice elicit a robust CD8+ T cell response to ERAAP-deficient cells. (a) Responses of CD8+
T cells to coculture with APCs. Wild-type mice were immunized with ERAAP-deficient spleen cells, their spleen cells were
restimulated for 5 d in vitro with ERAAP-deficient spleen cells, and then the restimulated T cells were cultured together
with wild-type, ERAAP-deficient or ERAAP-heterozygous (ERAAP-het) APCs and their responses were detected by assay of
IFN-g production. (b) Percent CD8+ IFN-g-producing cells detected in a from each immunized wild-type mouse in response to
various APCs (horizontal axis). Each symbol represents one mouse; horizontal lines, arithmetic mean. (c) T cell responses,
analyzed as described in a, for cocultures of T cells and wild-type APCs incubated with blocking antibodies to MHC class II
H-2Ab or to MHC class I H-2Kb or H-2Db. (d) Normalization of the CD8+ IFN-g responses of three mice analyzed in c to the
response to APCs in medium alone (arithmetic mean + s.d.). Data are representative of three separate experiments with at
least three mice each.
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d
100
Response (% of max)
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
101
Response (% of max)
CD8
101
50
0
α-Ab α-Kb α-Db
103
ARTICLES
ERAAP-het
103
ERAAP-KO
0.1%
ERAAP-TAP-DKO
TAP-KO
1.2%
1.6%
72.0%
102
CD8
101
100
b
100 101 102 103
100 101 102 103
c
70
60
50
40
20
CD8+IFN-γ+ (%)
100 101 102 103
103
+ vector
+ hERAAP cDNA
37.6%
0.0%
102
101
CD8
10
100
E
W
-2 RA
T
K b AP
–H
-h
e
-2
Db t
–K
ER
AA TA O
P- PTA KO
PD
KO
H
-2 d H-2 d
ER W
AA T
PKO
100 101 102 103
100 101 102 103
ERAAP-KO fibroblasts
IFN-γ
H
ER
AA
P-
KO
0
BEko T cell ligands are relatively unstable
ERAAP deficiency is expected to result in a higher abundance of
N-terminally extended peptides in the endoplasmic reticulum, which
are normally trimmed in that compartment. We reasoned that if such
peptides bound MHC class I molecules and the complexes actually
made it to the cell surface, they would be unique to ERAAP-deficient
cells and could conceivably serve as ligands for BEko T cells. Those
MHC class I molecules assembled with N-terminally extended peptides would also be expected to be less stable than conventional pMHC
I, as MHC class I molecules bind best to peptides of the appropriate
length and consensus motifs15,16. To test the stability of BEko T cell
ligands, we assessed their expression on cells treated with brefeldin
A17. Brefeldin A inhibits egress from the endoplasmic reticulum and
thus prevents newly assembled pMHC I from reaching the cell surface.
The persistence of previously generated pMHC I on the surface of cells
treated with brefeldin A therefore reflects their stability. When we
treated wild-type cells with brefeldin A for 2 or 4 h, there was no
decrease in the number of ERAAP-deficient CD8+ T cells producing
IFN-g in response to the conventional ERAAP-dependent peptide–
MHC I ligands recognized by those cells (Fig. 5a,b). In contrast, the
b
103
0.0%
102
101
100
IFN-γ
+ Medium
66.0%
+ BfA 2 h
79.1%
BEko T cell response dropped precipitously (by 80%) after only 2 h of
culture of the ERAAP-deficient APCs in brefeldin A, indicating that
the ligands were rapidly lost from the cell surface (Fig. 5c,d). The
disparate stability of the T cell ligands did not correlate with the
overall expression of conventional pMHC I, which, as assayed by flow
cytometry, decreased by approximately 50% on both wild-type and
ERAAP-deficient cells after 4 h of treatment with brefeldin A10. Thus,
the ERAAP-dependent peptide–MHC I ligands recognized by ERAAPdeficient T cells and those recognized by BEko T cells could be
distinguished not only by different antibody inhibition but also by
their instability on the cell surface.
BEko T cell ligands arise in ERAAP-inhibited wild-type cells
ERAAP functions in the endoplasmic reticulum and is not expected to
influence the cytoplasmic peptide pool before TAP transport. The
precursor peptides that enter the endoplasmic reticulum should
therefore be identical in both wild-type and ERAAP-deficient cells.
Only the lack of N-terminal trimming by ERAAP should therefore
account for the ligands expressed on the surface of ERAAP-deficient
cells recognized by BEko T cells. To independently establish that the
c
125
ERAAP-KO APCs
103
100
75
102
50
101
CD8
WT APCs
ERAAP-KO APC
Response (% of max)
a
CD8
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
100 101 102 103
IFN-γ
Figure 4 Wild-type CD8+ T cells responding to
ERAAP-deficient cells (BEko T cells) are specific
for ligands expressed on ERAAP-deficient cells
in a TAP- and H-2b-dependent way. (a) IFN-g
production of CD8+ T cells from wild-type C57BL/
6J mice, immunized and restimulated twice
in vitro as described in Figure 3a, in response to
APCs from ERAAP-heterozygous mice,
ERAAP-deficient mice, mice doubly deficient in
ERAAP and TAP (ERAAP-TAP-DKO) or mice
singly deficient in TAP (TAP-KO). (b) Percent
CD8+ IFN-g+ cells detected after stimulation of
BEko T cells with APCs from C57BL/6J mice
(genotype, horizontal axis). (c) Elimination of
BEko ligands from ERAAP-deficient fibroblasts by
expression of ERAAP-encoding cDNA. Fibroblasts
derived from ERAAP-deficient mice stably
expressing either empty vector (+ vector) or cDNA
encoding human ERAAP (+ hERAAP cDNA) were
treated with IFN-g and were used as APCs for
BEko T cells. Numbers above boxed areas
(a,c) indicate percent CD8+IFN-g+ cells among all
CD8+ cells. Data are representative of two
separate experiments with two immunized mice.
25
WT APC
+ Medium
0.0%
7.1%
100
0
Med.
2h
4h
+ BfA
100 101 102 103
IFN-γ
100 101 102 103
d
Figure 5 Ligands for BEko T cells but not those for wild-type-specific ERAAP-deficient CD8+ T cells are unstable.
(a) IFN-g production of T cells from ERAAP-deficient mice, immunized and restimulated as described in Figure 2a,
in response to culture together with APCs (genotype, above plots) incubated in medium alone or with 8 mg/ml of
brefeldin A (BfA) for 2 h before coculture. Numbers above boxed areas indicate percent CD8+IFN-g+ cells among all
CD8+ cells. (b) Responses of ERAAP-deficient CD8+IFN-g+ T cells (mean percent and s.d.) to brefeldin A–treated APCs
relative to the response to control APCs treated with medium only (Med). (c,d) Response of BEko T cells to culture with
APCs, analyzed as described in a,b. Data are representative of two separate experiments with three mice each.
104
+ BfA 2 h
50.0%
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100
Response (% of max)
a
75
50
25
0
Med.
2h
4h
+ BfA
NATURE IMMUNOLOGY
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Medium
103
+ DTT
57.8%
Figure 6 BEko T cells are specific for precursors of self peptides that are
N-terminally extended and presented by H-2Kb or H-2Db MHC class I
molecules. BMDCs were cultured for 5 h in medium alone, with the
reducing agent dithiothreitol (+ DTT) or with dithiothreitol and leucinethiol
(+ DTT + LeuSH), then were used as APCs for BEko T cells, whose IFN-g
responses were then analyzed as described in Figure 3a. Numbers above
boxed areas indicate percent CD8+IFN-g+ cells among all CD8+ cells. Data
are representative of three separate experiments.
+ DTT + LeuSH
57.1%
63.5%
1.0%
60.5%
ERAAP-KO
BMDCs
102
101
100
103
BMDCs lacking both H-2Kb and H-2Db MHC class I molecules had
no expression of BEko ligands, as those APCs stimulated only 3% of
the BEko T cells. That result independently confirmed the idea that
BEko T cells were specific for peptides presented by H-2Kb or H-2Db
MHC class I and showed that the unique ‘non–self peptide–MHC
class I complex’ expressed on ERAAP-deficient cells could also have
arisen from the normal intracellular pool of ‘self’ peptides in wild-type
APCs when N-terminal trimming by ERAAP was inhibited. We have
therefore called those peptides ‘ERAAP-unedited, novel’ peptides
(Supplementary Fig. 1).
0.9%
0.5%
3.2%
102
101
100
ERAAP-deficient cells express structurally unique pMHC I
Because the ERAAP-unedited, novel peptides are not trimmed by
ERAAP in the endoplasmic reticulum, we reasoned that they would
retain their N-terminal extensions and would therefore be presented
by MHC class I molecules on the cell surface. Accommodating longer
peptides in the peptide-binding groove of MHC class I would
probably cause structural alterations that could be used to distinguish
complexes of ERAAP-unedited, novel peptide and MHC class I from
conventional pMHC I complexes. We therefore investigated whether
ERAAP-deficient cells would elicit antibody responses in wild-type
mice. We immunized wild-type mice with ERAAP-deficient spleen
cells and assayed their serum for antibodies by flow cytometry. Serum
from each of the two immunized mice contained antibodies that
specifically stained ERAAP-deficient cells with a mean fluorescence
intensity more than twice the staining of wild-type cells (Fig. 7a). To
further characterize the specificity of the antibody response of wildtype mice to ERAAP-deficient cells, we generated antibody-secreting
hybridomas using spleen cells from the immunized mice. One of the
monoclonal antibodies, which we called ‘WEko.70’ (for ‘wild-type
anti-ERAAP-knockout’), specifically stained ERAAP-deficient cells
with a mean fluorescence intensity about ten times greater than that
102
103
100
101
102
103
dearth of ERAAP enzymatic activity was responsible for the generation
of the unique BEko T cell ligands, we treated bone marrow–derived
dendritic cells (BMDCs) with leucinethiol, a potent inhibitor of
aminopeptidases, including ERAAP18. The BEko T cells produced
IFN-g in response to untreated ERAAP-deficient BMDCs or to
ERAAP-deficient BMDCs treated with dithiothreitol (required to
keep the leucinethiol inhibitor reduced and active) but not to similarly
treated BMDCs from wild-type C57BL/6J or H2-Kb–H-2Db–knockout
mice (Fig. 6). Notably, after 5 h of treatment of BMDCs with
leucinethiol, the wild-type BMDCs expressed BEko T cell ligands
and stimulated IFN-g responses similar to those of ERAAP-deficient
BMDCs (Fig. 6). Leucinethiol treatment induced only a marginal
change in the response to ERAAP-knockout BMDCs, indicating that
induced expression of BEko ligands on wild-type cells was due mainly
to inhibition of ERAAP and that other leucinethiol-sensitive aminopeptidases had at best a minor function. Notably, leucinethiol-treated
a
Antiserum
80
2°
WT
ERAAP-KO
60
b
Anti-Kb
WEko.70
100
ERAAP-KO
ERAAP-KO
40
WT
WT
b
H-2Kb–H-2Db– KO
b
H-2K –H-2D – KO
20
0
10–1
100
101
102
103 10–1
100
101
102
103
10–1
100
101
Fluorescence intensity
102
103
c
30
20
10
8
Relative cell number
101
4
0
Anti-Kb
–
100
+
103
+/
102
–/
–
101
+/
100
IFN-γ
–/
–
+/
–
+/
+
CD8
H-2Kb–H-2Db–KO
BMDCs
103
Mean fluorescence intensity
WT
BMDCs
101
100
Relative cell number
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
0.6%
102
WEko.70
100
WEko.70-biotin
80
ERAAP-KO
60
ERAAP-TAP-DKO
40
TAP-KO
20
0
10–1 100
101
102
103
Fluorescence intensity
Figure 7 ERAAP-deficient cells express a large volume of structurally unique pMHC I complexes. (a) Fluorescent intensity of wild-type (dashed black lines)
or ERAAP-deficient (solid red lines) spleen cells (samples depleted of B cells) stained with antibodies and/or serum obtained from wild-type mice immunized
and boosted five times with ERAAP-deficient spleen cells; relative cell number is presented as a function of fluorescent intensity. Spleen cells from
H2-Kb–H-2Db–knockout mice serve as a negative control (middle, right). Left, staining with fluorescent secondary antibody alone (21; shaded histogram)
or with antiserum plus secondary antibody. Middle, staining with WEko.70. Right, conventional H-2Kb MHC class I expression (detected by staining with
monoclonal antibody 28.13.3S; Anti-Kb). Data are representative of three experiments. (b) Staining of ERAAP-deficient cells (–/–), ERAAP-heterozygous cells
(+/–) and wild-type cells (+/+) with anti-H-2Kb and WEko.70 s (mean fluorescent intensity and s.d.). Cells from at least three different mice of each genotype
were used for staining; data are representative of three separate experiments. (c) Staining of whole spleen cell suspensions (nondepleted) from ERAAPdeficient mice (solid red line), mice singly deficient in TAP (solid black line) or mice doubly deficient in ERAAP and TAP (dashed red line) with biotinylated
WEko.70 and fluorescent strepavidin as the secondary antibody (shaded histogram). Data are representative of six mice for each genotype.
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of wild-type or ERAAP-heterozygous spleen cells, showing that
complete ERAAP deficiency was essential for epitope expression
(Fig. 7a,b). Furthermore, the intensity with which WEko.70 stained
ERAAP-deficient cells was approximately 50% the intensity with
which the isotype-matched monoclonal antibody 28.13.3S stained
conventional H-2Kb MHC class I on the same cells, showing that
the epitope for WEko.70 was abundantly expressed.
TAP is essential for the expression of pMHC I on the cell surface7,19.
To determine whether expression of the epitope for WEko.70 was
indeed the consequence of lack of ERAAP function in the MHC class I
antigen-processing pathway, we assessed expression of the epitope for
WEko.70 on cells from mice doubly deficient in both ERAAP and TAP.
WEko.70 staining was readily detectable on all spleen cells from
ERAAP-deficient mice, but the mean fluorescence intensity of
ERAAP-deficient cells also deficient in TAP was 90% lower and was
similar to that of control ERAAP-sufficient, TAP-deficient cells
(Fig. 7c). Furthermore, WEko.70 did not stain cells from H2-Kb–H2Db–knockout mice (Fig. 7a), demonstrating that like the BEko T cell
ligands, expression of the epitope for WEko.70 required TAP as well as
the H-2Kb or H-2Db MHC class I molecules. We concluded that
WEko.70 recognizes abundant and structurally distinct pMHC I
molecules unique to ERAAP-deficient cells.
DISCUSSION
Our analysis of the pMHC I repertoire in ERAAP-deficient mice has
established that N-terminal trimming is a key step in the generation of
many endogenous peptides. Failure to carry out that step results in the
loss of many conventional pMHC I complexes and the concomitant
emergence of a set of unstable but highly immunogenic pMHC
I complexes. Both the loss and gain of pMHC I complexes in
ERAAP-deficient mice demonstrate that ERAAP is a ‘quintessential
editor’ of the pMHC repertoire and that its absence has salient
immunological consequences.
Several independent studies have demonstrated that ERAAP is
important for the normal presentation of several different pMHC I
complexes derived from endogenous or viral sources10,11,20. Here we
have used the ability of CD8+ T cells to detect differences between self
and foreign peptides in the overall pMHC I repertoire. ERAAPdeficient mice responded vigorously to cells from their wild-type
counterparts, indicating that differences were readily detectable in the
pMHC I repertoire expressed by those otherwise genetically identical
mice. ERAAP-deficient mice thus lack a large set of peptides that are
normally expressed by wild-type mice and cause self-tolerance in the
wild-type CD8+ T cell repertoire. The loss of those complexes of
ERAAP-dependent peptide and MHC class I is consistent with other
analyses of post-proteasomal antigen-processing steps in the cytoplasm and the endoplasmic reticulum4–6,21. Those studies suggest
that complexes of ERAAP-dependent peptide and MHC class I are
derived from N-terminally extended precursors that arrive from the
cytoplasm into the endoplasmic reticulum, where they are trimmed
by ERAAP. The inability to trim the ERAAP-dependent peptides
depletes the pMHC I repertoire of peptides presented by H-2Kb as
well as H-2Db MHC class I molecules and allows ERAAP-deficient
mice, which are not tolerant to those pMHC I complexes, to respond
to those now ‘foreign’ peptides expressed by wild-type cells. The loss
of tolerance to many pMHC I complexes shows that the precursors to
all of those peptides depend on ERAAP to convert them into their
final forms.
The reciprocal immunization of wild-type mice with cells from
ERAAP-deficient mice has also demonstrated the existence of novel,
highly immunogenic pMHC I complexes. Wild-type mice responded
106
vigorously to ERAAP-deficient cells by eliciting CD8+ T cell responses
(BEko T cells), which were specific for the unconventional complexes
of ERAAP-unedited, novel peptide and MHC class I. In a plausible
scenario, complexes of ERAAP-unedited, novel peptide and MHC
class I could arise because of the failure to trim the precursors of
ERAAP-dependent peptides by ERAAP. Our results suggest that those
ERAAP-dependent peptide precursors do not leave the endoplasmic
reticulum22 but instead emerge in their intact unedited form as
complexes of ERAAP-unedited, novel peptide and MHC class I on
the cell surface. Because such extended peptides are unique in ERAAPdeficient cells, they serve as ligands for both BEko CD8+ T cells and
WEko antibodies. Our data did not allow us to determine exactly how
many complexes of ERAAP-unedited, novel peptide and MHC class I
were generated in the absence of ERAAP. Nevertheless, their ability to
elicit vigorous CD8+ T cell and B cell responses as well as the
magnitude of surface staining on ERAAP-deficient cells suggest that
they constitute a substantial fraction of the total pMHC I. Some
unique peptides expressed exclusively by tapasin- or TAP-deficient
cells have also been reported23,24. However, to our knowledge, cells
deficient in either TAP or tapasin are not immunogenic in wild-type
animals25. The immunogenicity of complexes of ERAAP-unedited,
novel peptide and MHC class I is therefore a likely consequence of
their large number and diversity.
The existence of these unstable, but immunologically unique
complexes of ERAAP-unedited, novel peptide and MHC class I also
provides an insight into the longstanding observation that normally
MHC class I molecules present only those peptides that conform to
their consensus motifs16,26. It has been argued that those sequence
motifs are a consequence of structural constraints imposed by the
peptide-binding grooves of MHC I molecules. Structurally distinct
complexes of ERAAP-unedited, novel peptide and MHC class I were
presented on the surface of ERAAP-deficient cells, which indicated
instead that exclusive presentation of consensus motif peptides is a
result of the enzymatic activity of ERAAP, which generates precise N
termini by trimming longer precursors. Thus, the length restriction of
the precise peptides that define the pMHC I repertoire is a function of
MHC class I molecules as well as of ERAAP. The idea that MHC I
molecules are capable of presenting noncanonical longer peptides is
also supported by occasional reports of long peptides bound by MHC
class I and recognized by CD8+ T cells27,28. Structural analyses of such
complexes have shown that the longer peptides are present in the
MHC class I peptide-binding groove with a central bulge or with the C
terminus extending outside the groove29–32. The structural features of
the N-terminally extended, ERAAP-unedited, novel peptides and how
they are accommodated in the MHC class I peptide-binding groove
remain to be determined.
Structural differences in specific peptides presented by MHC class I,
such as those derived from minor histocompatibility antigens, are well
known for their ability to elicit CD8+ T cell responses33. However, the
ability of ERAAP-deficient cells to generate an antibody response in
MHC-matched wild-type mice was unexpected. Generally, antibodies
specific for pMHC I complexes are elicited only in allogeneic MHCmismatched mice, including those specific for abundantly expressed
single peptide–MHC class I complexes34. The precise epitope bound
by WEko.70 is not yet known, but its expression required TAP and
MHC class I molecules as well as a total absence of ERAAP. Expression
of the complexes of ERAAP-unedited, novel peptide and MHC class I
was also dependent on the same factors, which suggested that
WEko.70 recognizes a structural feature shared by many of those
complexes. Furthermore, WEko.70 was generated in MHC-matched
wild-type mice, which showed that such structural features of
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complexes of ERAAP-unedited, novel peptide and MHC class I must
be unique and abundant to account for their immunogenicity. Thus,
the antibody response of wild-type mice to ERAAP-deficient cells is
another immunological consequence of ERAAP deficiency.
Finally, we note the paradox that inhibiting a key component of the
antigen-processing pathway actually enhanced the immunogenicity of
cells similar to that of allogeneic cells with mismatched MHC.
Notably, the enhancement of immunogenicity as a consequence of
ERAAP inhibition did not require mismatched MHC, polymorphic
differences in minor histocompatibility genes or mutations in genes
encoding normal proteins. Thus, it may be possible to enhance the
immunogenicity of tumors by inhibiting ERAAP or, conversely, loss of
ERAAP function in self tissues could result in autoimmunity.
METHODS
Mice, cell lines, immunization and CD8+ T cell responses. Use of all mice was
with the approval of the Animal Care and Use Committee of the University of
California at Berkeley. ERAAP-deficient mice and fibroblast cell lines derived
from those mice have been described10. ERAAP-deficient mice were backcrossed nine times to the C57BL/6J (H-2b) background, and ERAAPheterozygous mice were their littermates. Mice deficient in both ERAAP
and TAP, mice expressing H-2d MHC, and H2-Kb–H-2Db–knockout
mice have been described10,12. Wild-type C57BL/6J, TAP-deficient, wild-type
B10.D2 (H-2d) and C57BL/6J.C-H2bm1 mice were purchased from the
Jackson Laboratory.
Female ERAAP-deficient and wild-type mice were immunized intraperitoneally with 20 106 spleen cells from male wild-type or ERAAP-deficient mice,
respectively. Then, 10 d after immunization, whole spleens were restimulated
for 5 d in vitro in cultures containing 20 U/ml of interleukin 2 (BD Biosciences)
and irradiated spleen cells from female mice of the same genotype used for
immunization. Subsequent restimulations were done similarly with 50 U/ml of
interleukin 2 every 7 d, after removal of dead cells by Ficoll fractionation (GE
Healthcare). CD8+ T cell HY peptide responses were assayed as described10.
The CD8+ T cell responses of the immunized mice were measured by assay for
inducible IFN-g production by those cells in response to APCs from female
mice. Unless otherwise indicated, APCs were splenocytes from female mice and
were incubated overnight in 200 ng/ml of lipopolysaccharide (Sigma) and
samples were depleted of CD8+ cells by Dynabeads conjugated to anti-CD8
(Dynal Biotech). Restimulated CD8+ T cells and APCs were cultured together
for 4 h in the presence of brefeldin A before being stained for surface CD8 and
intracellular IFN-g (BD Biosciences). Cells were then analyzed on a FACScan
(Coulter) and data were analyzed with FlowJo Software (Treestar). Numbers are
reported as above the background response of T cells alone.
Antibody blocking and treatment of APCs with brefeldin A and IFN-c.
For inhibition of presentation by MHC class I molecules, APCs were incubated
for 20 min at 4 1C with supernatant from hybridoma 5F1 (anti-H-2Kb),
B22.249 (anti-H-2Db) or M5/1114 (anti-H-2Ab) before being cultured together
with CD8+ T cells as described above. The coculture medium also contained
50% of the same hybridoma supernatant used during the 4 1C incubation. For
analysis of the cell surface stability of the MHC class I ligands, APCs were
preincubated for 2 or 4 h at 37 1C in medium containing 8 mg/ml of brefeldin A
(Sigma). CD8+ T cells producing IFN-g were counted as described above.
ERAAP-deficient fibroblasts expressing either vector or cDNA encoding human
ERAAP were generated as described10 and were treated for 2 d with 500 U/ml
of recombinant mouse IFN-g (Biosource) before being used as APCs for
BEko T cells.
BMDCs and leucinethiol treatment. Bone marrow was extracted from female
ERAAP-deficient, wild-type and H2-Kb–H-2Db–knockout mice and cells were
cultured for 5–7 d in 20 ng/ml of granulocyte-macrophage colony-stimulating
factor to enrich the growth of DCs. Nonadherent cells were collected and were
replated in medium only or in medium containing 0.5 mM dithiothreitol alone
or dithiothreitol plus 30 mM leucinethiol (Sigma) as described18. After 5 h,
nonadherent cells were used as APCs for BEko T cells.
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Antibody responses of wild-type mice to ERAAP-deficient cells and generation of WEko.70. Female wild-type C57BL/6J mice were immunized intraperitoneally with 20 106 spleen cells from male ERAAP-deficient mice.
Mice were boosted every 3 weeks and antiserum and spleens were collected
after five boosts. Spleens were fused with the P3X63-Ag8.653 myeloma fusion
partner according to the standard protocol for generating antibody-secreting
hybridomas. For screening for positive cells and for analysis of antibody
specificity, supernatants from terminal cultures or antiserum were used to
stain ERAAP-deficient, wild-type and H2-Kb–H-2Db–knockout spleen cell
samples depleted of B cells with Dynabeads conjugated to anti–mouse
immunoglobulin G. The specificity of the antibody for ERAAP-deficient cells
was demonstrated by comparison of the mean fluorescence intensity of the
staining of ERAAP-deficient cells to that of cells doubly deficient for ERAAP
and TAP, ERAAP-heterozygous cells or wild-type cells. Ascites fluid from mice
injected with 28.13.3S (immunoglobulin M) was used to stain conventional
H-2Kb MHC class I. The secondary antibody was fluorescein isothiocyanate–
conjugated goat anti–mouse immunoglobulin G (Cappel). Supernatant from
the WEko.70 hybridoma was purified with a protein L column (Pierce) and was
biotinylated with EZ-Link NHS-PEO solid-phase biotinylation reagents
(Pierce). Biotinylated WEko.70 was used to stain whole spleen cell suspensions,
and phycoerythrin-conjugated strepavidin (Caltag) was used as the secondary
staining reagent.
Statistical methods. Mean values and standard deviations were calculated with
GraphPad Prism (GraphPad Software).
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS
Supported by the US National Institutes of Health (N.S.).
AUTHOR CONTRIBUTIONS
G.E.H. contributed to the experimental design, immunized mice, did
experiments, screened hybridomas, analyzed data and cowrote the manuscript;
F.G. generated and backcrossed ERAAP-deficient mice and generated the WEko
hybridomas and the biotinylated derivative of WEko.70; E.J. contributed to the
experimental design and did skin grafts; H.N. sorted cells; and N.S. established
the initial scientific questions, provided guidance and cowrote the manuscript.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/natureimmunology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
1. Shastri, N., Schwab, S. & Serwold, T. Producing nature’s gene-chips. The generation of
peptides for display by MHC class I molecules. Annu. Rev. Immunol. 20, 463–493
(2002).
2. Cresswell, P., Ackerman, A.L., Giodini, A., Peaper, D.R. & Wearsch, P.A. Mechanisms of
MHC class I-restricted antigen processing and cross-presentation. Immunol. Rev. 207,
145–157 (2005).
3. Shastri, N., Cardinaud, S., Schwab, S.R., Serwold, T. & Kunisawa, J. All the peptides
that fit: the beginning, the middle, and the end of the MHC class I antigen-processing
pathway. Immunol. Rev. 207, 31–41 (2005).
4. Kunisawa, J. & Shastri, N. The group II chaperonin TRiC protects proteolytic intermediates from degradation in the MHC class I antigen processing pathway. Mol. Cell
12, 565–576 (2003).
5. Kunisawa, J. & Shastri, N. Hsp90a chaperones large proteolytic intermediates in the
MHC class I antigen processing pathway. Immunity 24, 523–534 (2006).
6. Rock, K.L., York, I.A. & Goldberg, A.L. Post-proteasomal antigen processing for
major histocompatibility complex class I presentation. Nat. Immunol. 5, 670–677
(2004).
7. Van Kaer, L., Ashton-Rickardt, P.G., Ploegh, H.L. & Tonegawa, S. TAP1 mutant mice are
deficient in antigen presentation, surface class I molecules, and CD4–8+ T cells. Cell
71, 1205–1214 (1992).
8. Serwold, T., Gonzalez, F., Kim, J., Jacob, R. & Shastri, N. ERAAP customizes peptides
for MHC class I molecules in the endoplasmic reticulum. Nature 419, 480–483
(2002).
9. York, I.A. et al. The ER aminopeptidase ERAP1 enhances or limits antigen presentation
by trimming epitopes to 8–9 residues. Nat. Immunol. 3, 1177–1184 (2002).
10. Hammer, G.E., Gonzalez, F., Champsaur, M., Cado, D. & Shastri, N. The aminopeptidase ERAAP shapes the peptide repertoire displayed by major histocompatibility
complex class I molecules. Nat. Immunol. 7, 103–112 (2006).
107
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
11. Yan, J. et al. In vivo role of ER-associated peptidase activity in tailoring peptides for
presentation by MHC class Ia and class Ib molecules. J. Exp. Med. 203, 647–659
(2006).
12. Perarnau, B. et al. Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral
CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses.
Eur. J. Immunol. 29, 1243–1252 (1999).
13. Sherman, L.A. & Chattopadhyay, S. The molecular basis of allorecognition. Annu. Rev.
Immunol. 11, 385–402 (1993).
14. Loveland, B., Wang, C-R., Yonekawa, H., Hermel, E. & Lindahl, K.F. Maternally
transmitted histocompatibility antigen of mice: A hydrophobic peptide of a mitochondrially encoded protein. Cell 60, 971–980 (1990).
15. Madden, D.R., Gorga, J.C., Strominger, J.L. & Wiley, D.C. The structure of HLA-B27
reveals nonamer self-peptides bound in an extended conformation. Nature 353, 321–
325 (1991).
16. Rammensee, H.G., Bachmann, J. & Stevanovic, S. in MHC Ligands and Peptide Motifs
1–457 (Landes Bioscience, Austin, Texas, 1997).
17. Yewdell, J.W. & Bennink, J.R. Brefeldin A specifically inhibits presentation of protein
antigens to cytotoxic T lymphocytes. Science 244, 1072–1075 (1989).
18. Serwold, T., Gaw, S. & Shastri, N. ER aminopeptidases generate a unique pool of
peptides for MHC class I molecules. Nat. Immunol. 2, 644–651 (2001).
19. Cresswell, P., Bangia, N., Dick, T. & Diedrich, G. The nature of the MHC class I peptide
loading complex. Immunol. Rev. 172, 21–28 (1999).
20. York, I.A., Brehm, M.A., Zendzian, S., Towne, C.F. & Rock, K.L. Endoplasmic reticulum
aminopeptidase 1 (ERAP1) trims MHC class I-presented peptides in vivo and plays an
important role in immunodominance. Proc. Natl. Acad. Sci. USA 103, 9202–9207
(2006).
21. Saveanu, L., Fruci, D. & van Endert, P. Beyond the proteasome: trimming, degradation
and generation of MHC class I ligands by auxiliary proteases. Mol. Immunol. 39, 203–
215 (2002).
22. Koopmann, J-O. et al. Export of antigenic peptides from the endoplasmic reticulum
intersects with retrograde protein translocation through the Sec61p channel. Immunity
13, 117–127 (2000).
108
23. van Hall, T. et al. Selective cytotoxic T-lymphocyte targeting of tumor immune escape
variants. Nat. Med. 12, 417–424 (2006).
24. Purcell, A.W. et al. Quantitative and qualitative influences of tapasin on the class I
peptide repertoire. J. Immunol. 166, 1016–1027 (2001).
25. Wolpert, E.Z. et al. Generation of CD8+ T cells specific for transporter associated with
antigen processing deficient cells. Proc. Natl. Acad. Sci. USA 94, 11496–11501
(1997).
26. Falk, K., Rotzschke, O., Stevanovic, S., Jung, G. & Rammensee, H-G. Allele-specific
motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature
351, 290–296 (1991).
27. Probst-Kepper, M. et al. An alternative open reading frame of the human macrophage
colony-stimulating factor gene is independently translated and codes for an antigenic
peptide of 14 amino acids recognized by tumor-infiltrating CD8 T lymphocytes. J. Exp.
Med. 193, 1189–1198 (2001).
28. Green, K.J. et al. Potent T cell response to a class I-binding 13-mer viral epitope and
the influence of HLA micropolymorphism in controlling epitope length. Eur. J.
Immunol. 34, 2510–2519 (2004).
29. Guo, H-C. et al. Different length peptides bind to HLA-Aw68 similarly at their ends but
bulge out in the middle. Nature 360, 364–366 (1992).
30. Probst-Kepper, M. et al. Conformational restraints and flexibility of 14-meric peptides
in complex with HLA-B*3501. J. Immunol. 173, 5610–5616 (2004).
31. Tynan, F.E. et al. T cell receptor recognition of a ‘super-bulged’ major histocompatibility complex class I-bound peptide. Nat. Immunol. 6, 1114–1122
(2005).
32. Collins, E.J., Garboczi, D.N. & Wiley, D.C. Three-dimensional structure of a
peptide extending from one end of a class I MHC binding site. Nature 371,
626–629 (1994).
33. Roopenian, D., Choi, E.Y. & Brown, A. The immunogenomics of minor histocompatibility antigens. Immunol. Rev. 190, 86–94 (2002).
34. Porgador, A., Yewdell, J.W., Deng, Y., Bennink, J.R. & Germain, R.N. Localization,
quantitation, and in situ detection of specific peptide-MHC class I complexes using a
monoclonal antibody. Immunity 6, 715–726 (1997).
VOLUME 8
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