Analysis of the Role of Tripeptidyl Peptidase II in MHC Class
I Antigen Presentation In Vivo1
Masahiro Kawahara,2* Ian A. York,†Arron Hearn,* Diego Farfan,* and Kenneth L. Rock3*
Previous experiments using enzyme inhibitors and RNA interference in cell lysates and cultured cells have suggested that trip-
eptidyl peptidase II (TPPII) plays a role in creating and destroying MHC class I-presented peptides. However, its precise con-
tribution to these processes has been controversial. To elucidate the importance of TPPII in MHC class I Ag presentation, we
analyzed TPPII-deficient gene-trapped mice and cell lines from these animals. In these mice, the expression level of TPPII was
reduced by >90% compared with wild-type mice. Thymocytes from TPPII gene-trapped mice displayed more MHC class I on the
cell surface, suggesting that TPPII normally limits Ag presentation by destroying peptides overall. TPPII gene-trapped mice
responded as well as did wild-type mice to four epitopes from lymphocytic choriomeningitis virus. The processing and presentation
of peptide precursors with long N-terminal extensions in TPPII gene-trapped embryonic fibroblasts was modestly reduced, but in
vivo immunization with recombinant lentiviral or vaccinia virus vectors revealed that such peptide precursors induced an equiv-
alent CD8 T cell response in wild-type and TPPII-deficient mice. These data indicate that while TPPII contributes to the trimming
of peptides with very long N-terminal extensions, TPPII is not essential for generating most MHC class I-presented peptides or
for stimulating CTL responses to several Ags in vivo. The Journal of Immunology, 2009, 183: 6069–6077.
ecules display peptides derived from the cell’s expressed genes. In
most situations, these presented peptides are from normal cellular
proteins and are ignored by CD8 T cells. However, if the abnormal
peptides are present, for example, ones containing mutations or
from viral proteins, then CTLs will recognize these complexes and
be stimulated to destroy the abnormal cell. This process protects
the host against tumors and viral infections.
Peptides that bind to MHC class I are produced from intracel-
lular proteins as a byproduct of normal protein catabolism (3–5).
The major protease responsible for the initial cleavage of cellular
proteins into oligopeptides is the proteasome, a large particle in the
cytosol and nucleus of cells. Most peptides produced by protea-
somes are very rapidly hydrolyzed into amino acids by the con-
certed action of aminopeptidases and endopeptidases in the cytosol
(6). However, a small fraction of peptides escape destruction and
are transported by TAP into the endoplasmic reticulum (ER),4
ytotoxic T lymphocytes survey the MHC class I mole-
cules on the surface of cells searching for ones that con-
tain immunogenic peptides (1, 2). The MHC class I mol-
where ones of the right size and sequence bind to newly assembled
class I molecules (7). MHC class I molecules bind peptides that are
of a precise size, which depending on the specific MHC class I
molecule are between 8 and 10 aa. Less than 5% of the peptides
produced by the proteasome are actually of the proper size to sta-
bly bind to any particular class I molecule (8). Proteasomes more
frequently generate peptides (?10–20%) that are too long to bind
to MHC class I molecules, but can serve as potential antigenic
precursors (8). These long precursors can be converted to MHC
class I-binding peptides by aminopeptidases, especially ER ami-
nopeptidase 1 (ERAP1; ERAAP) (9–14), or may be completely
degraded to amino acids by aminopeptidases and endopeptidases.
Where examined, most aminopeptidases preferentially degrade
relatively short peptides and in vitro have little or no activity on
peptides that are longer than ?16 aa (15, 16). An exception to this
rule is tripeptidyl peptidase II (TPPII). TPPII (EC 18.104.22.168) is an
abundant cytosolic aminopeptidase that sequentially removes trip-
eptides from the amino terminus of peptides, and it also has a
poorly understood endoproteolytic activity (17, 18). TPPII is ca-
pable of degrading quite long peptides (at least as long as 41 aa)
(17), and in vitro it is the major activity in cells that degrades
peptides longer than 15 aa (16). However, since only ?10% of
peptides produced by the proteasome are longer than 15 aa (8), the
importance of this activity is not clear.
Several groups have reported a role for TPPII in MHC class I
Ag presentation (16, 19–24). Most of these reports suggest a spe-
cialized role for TPPII in processing a limited number of presented
peptides; however, one group suggested that in intact cells, pro-
teasomes mainly generate very long peptides (in contrast to the
behavior of purified proteasomes in vitro), and that TPPII is es-
sential for processing these long peptides for Ag presentation (16).
We have previously tested the role of TPPII in MHC class I Ag
presentation in tissue culture, using small interfering RNA
(siRNA) to eliminate TPPII from human (HeLa) cells (21). We
*Department of Pathology, University of Massachusetts Medical School, Worcester,
MA 01655; and†Department of Microbiology and Molecular Genetics, Michigan
State University, East Lansing, MI 48824
Received for publication October 24, 2008. Accepted for publication September 4,
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the National Institutes of Health (to
K.L.R.). Core resources supported by the Diabetes Endocrinology Research Grant
DK42520 were also used. M.K. was supported by Japan Society for the Promotion of
Science Postdoctoral Fellowships for Research Abroad.
2Current address: Department of Chemistry and Biotechnology, School of Engineer-
ing, The University of Tokyo, Tokyo 113-8656, Japan.
3Address correspondence and reprint requests to Dr. Kenneth L. Rock, University
of Massachusetts Medical School, Department of Pathology, Room S2-109, 55
Lake Avenue North, Worcester, MA 01655. E-mail address: Kenneth.Rock@
4Abbreviations used in this paper: ER, endoplasmic reticulum; DC, dendritic cell;
KO, knockout; LCMV, lymphocytic choriomeningitis virus; MEF, mouse embryonic
fibroblast; siRNA, small interfering RNA; TPPII, tripeptidyl peptidase II; WT, wild
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
found that under these conditions, overall MHC class I Ag pre-
sentation was only slightly affected, even though presentation of
peptides derived from very long model peptides (14–17 residues)
was markedly reduced in the absence of TPPII. We concluded that
(as with purified proteasomes in vitro) proteasomes in intact cells
mainly generate relatively short peptides that can be degraded by
many intracellular peptidases, and that TPPII is not essential for
MHC class I Ag presentation in tissue culture. In this study, we
investigated whether TPPII is also dispensable for Ag presentation
in primary and cultured mouse cells and intact mice.
Materials and Methods
Mice and PCR
Mice containing a “gene-trap” cassette that abrogates TPPII expression
were purchased from Lexicon Pharmaceutical. These mice were produced
by insertional mutagenesis of embryonic stem cells using a retroviral cas-
sette containing a splice acceptor and a poly(A) signal. In these cells, the
retroviral cassette was inserted between exons 2 and 3, so that a spliced
TPPII containing exons 1 and 2 (98 out of 1262 aa) is expressed instead of
full-length TPPII (see Fig. 1A). The presence of the gene-trap cassette was
tested by genomic PCR using primers specific for a wild-type (mTPPIIg-
F1, 5?-AGAATAGCCCATGTGCCAAC-3?; mTPPIIg-R1, 5?-CAACG
AAACTTGCCTTCACA-3?) and a gene-trapped allele (mTPPIIg-F1, 5?-
CTTGCAGTTGCATC-3?), respectively. Real-time PCR of TPPII was
performed on spleens, kidneys, and embryonic fibroblasts to quantify the
knockdown level of full-length TPPII. We first used a TaqMan system
(Applied Biosystems), in which the amplicon sequence was not provided
by the manufacturer. Therefore, we also designed a set of defined primers
that could be applied for a quantitative PCR SYBR Green assay. The prim-
ers used were TPPII ex11 forward (5?-GTGCCTAACTGGACATTGAG-
3?) and TPPII ex12 reverse (5?-CAACATTATTTGCTTTCAGCCCTG-3?)
for amplification around exons 11 and 12. The real-time PCR was per-
formed on a MyiQ machine (Bio-Rad). We used the mice backcrossed with
C57BL/6 mice (The Jackson Laboratory) for six generations in all exper-
iments except the one using mouse embryonic fibroblasts (MEFs), in which
we used the mice backcrossed for two generations, using MEFs derived
from littermates as controls. In all experiments, we used age- and sex-
matched mice for analysis. All mice were housed under specific pathogen-
free conditions in the animal facility in the University of Massachusetts
Medical School. Handling of the mice was performed according to insti-
tutional guidelines in the University of Massachusetts Medical School.
MEFs were produced from crosses of gene-trapped heterozygous mice as
previously described (25). The genomic DNA extracted from the cells was
analyzed by PCR to genotype each cell line. MHC class I Ag presentation
was analyzed in homozygous gene-trapped, homozygous wild-type (WT),
and heterozygous MEFs. MEFs were cultured in DMEM supplemented
with 20% FBS in a 37°C/10% CO2incubator.
Mouse bone marrow-derived dendritic cells (DCs) were generated using
standard protocols (26). Briefly, bone marrow cells were cultured in HCM
media (RPMI 1640 (Invitrogen) supplemented with 10% FBS, 2 mM L-
glutamine, 10 mM HEPES, 50 ?M 2-ME, penicillin/streptomycin, and
nonessential amino acids) in a 37°C/5% CO2incubator. On day 1, the cells
were plated in the presence of 10 ng/ml mouse GM-CSF and 5 ng/ml
mouse IL-4. On day 4, an additional 10 ng/ml mouse GM-CSF and 5 ng/ml
mouse IL-4 were added to the culture media.
293T cells were cultured in DMEM (Invitrogen) supplemented with
10% FBS and 2 mM L-glutamine.
Construction of plasmids and recombinant lentiviral vectors
We tested presentation of a model peptide SIINFEKL (S8L), the H-2Kb-
restricted immunodominant epitope from chicken OVA. Construction of
plasmids expressing N-extended S8L peptides has been previously de-
scribed (21). Briefly, S8L preceded by N-terminal extensions of various
lengths was inserted downstream of ubiquitin to generate a ubiquitin-pep-
tide fusion protein. When expressed in cells, the N-terminal ubiquitin is
rapidly cleaved by ubiquitin C-terminal hydrolases, yielding peptides with
defined N-terminal residues (27). GFP was expressed from the same tran-
script as the ubiquitin-peptide using an internal ribosome entry site.
Recombinant lentiviral vectors expressing full-length OVA, ubiquitin,
ubiquitin fused with S8L, and ubiquitin fused with 9-aa N-extended S8L
were constructed by inserting each gene and an IRES-GFP cassette at an
EcoRI-BamHI site of FUGW (provided by Dr. E. Latz, University of Mas-
sachusetts Medical School, Worcester, MA) (28).
Peptides used in these experiments are summarized in Table I.
Transfection and transduction in vitro
MEFs were transiently transfected using FuGENE6 (Roche) as described
previously (12). In some cases, MEFs were treated with 50 U/ml murine
IFN-? (BD Biosciences) 5 h after transfection.
For production of lentivirus, 293T cells were transiently transfected with
each recombinant lentiviral vector together with packaging construct delta-
8.91 and a VSV-G expression plasmid (gifts from Dr. E. Latz, University
of Massachusetts Medical School, Worcester, MA) using TransIT-293
transfection reagent (Mirus Bio). Supernatant containing recombinant len-
tivirus was collected after 2 and 3 days of transfection and stored at ?80°C
until just before use. For in vivo injection, the supernatant was further
concentrated by ultracentrifugation (29). Lentiviral titers (transducing
units) were determined using NIH3T3 cells based on the GFP-positive cell
ratio on day 2.
Bone marrow cells were transduced with recombinant lentivirus in
the presence of 4 ?g/ml polybrene (Sigma-Aldrich) on day 2. Half of the
media was replaced on day 3 to reduce a toxic effect of polybrene on the
cells. On day 7, cells were stained and analyzed by flow cytometry.
Handling of viral vectors was performed according to the guidelines of
biosafety level 2?laboratories established by the Recombinant DNA Com-
mittee of the University of Massachusetts Medical School.
Poly(I:C) treatment of mice
Mice were injected i.p. with 200 ?g of poly(I:C) (GE Healthcare) in a total
volume of 100 ?l of PBS. Spleens from the mice were harvested after 24 h
and then stained for flow cytometric analysis.
Viral infection in vivo and peptide stimulation in vitro
Mice were injected i.p. with 5 ? 104PFU per mouse of lymphocytic
choriomeningitis virus (LCMV) Armstrong (a gift from Dr. R. Welsh, Uni-
versity of Massachusetts Medical School, Worcester, MA) or with 1.25 ?
106PFU per mouse of recombinant vaccinia virus expressing full-length
OVA, ubiquitin, ubiquitin fused with S8L, or ubiquitin fused with 9-aa
N-extended S8L. Mice were injected in the footpad with 3.3 ? 105trans-
ducing units per mouse of recombinant lentivirus or with 4.8 ? 105PFU
per mouse of recombinant vaccinia virus. Eight (LCMV and recombinant
lentivirus) or seven (recombinant vaccinia virus) days later, splenocytes
(LCMV and recombinant vaccinia virus) or lymphocytes from a draining
lymph node (recombinant lentivirus) were harvested and incubated for 5 h
with 5 ?M of the appropriate peptide, or with 0.5 ?g/ml anti-CD3? (BD
Biosciences), in the presence of GolgiPlug (BD Biosciences). Peptides that
were used to stimulate IFN-? production after LCMV infection were gp33
(KAVYNFATC), NP205 (YTVKYPNL), gp276 (SGVENPGGYCL), and
NP396 (FQPQNGQFI). For recombinant vaccinia and lentivirus, S8L pep-
tide was used. All peptides were synthesized by Anaspec. Cells were
stained for CD8, CD44 (BD Biosciences), and intracellular IFN-? (eBio-
science) using commercial Abs, and analyzed by flow cytometry.
Abs and flow cytometry
The mAb 25.D1.16 (which recognizes S8L in combination with H-2Kb)
(30), Y3 (anti-H-2Kb) (31), 28.14.8S (anti-H-2Db) (32), and H36.4.5 (anti-
influenza hemagglutinin; for isotype control) (a gift of W. Gerhard, The
Wistar Institute, University of Pennsylvania) (33) were used as primary
Abs in staining MEFs and lentivirus-transduced DCs for flow cytometry.
After treatment with donkey anti-mouse F(ab?)2fragments conjugated to
Cy5 (Jackson ImmunoResearch Laboratories), flow cytometry was per-
formed on a FACSCalibur apparatus (BD Biosciences), followed by anal-
ysis with FlowJo software (Tree Star). Transfected cells were identified by
Table I. Peptides expressed by plasmids
Name of Peptide Peptide SequenceLength
6070ROLE OF TRIPEPTIDYL PEPTIDASE II IN MHC-I ANTIGEN PRESENTATION
gating on GFP fluorescence. In the case of staining of DCs, PE-conjugated
anti-CD11c Ab (eBioscience) was also used as a secondary Ab.
For staining cells isolated from spleens, bone marrows, and draining
lymph nodes, AF6-88.5 (anti-H-2Kb), KH95 (anti-H-2Db), and AF6-120.1
(anti-I-Ab), BB7.2 (anti-HLA-A2; for isotype control), and anti-Gr1 Abs
conjugated to a fluorophore were used according to the manufacturer’s
directions (BD Biosciences). Other fluorophore-conjugated Abs against
cell surface markers (CD4, B220, CD11c, CD86) were purchased from
eBioscience. The cells were then analyzed by flow cytometry.
Tissue extracts were prepared in homogenization buffer (50 mM Tris (pH
7.4), 0.25 M sucrose, 5 mM DTT, 5 mM MgCl2, 2 mM ATP, 10% glyc-
erol) with complete protease inhibitor cocktail minitablets (Roche) using a
dounce homogenizer on ice. The homogenate was spun at 325 ? g for 10
min, and the supernatant was further spun at 16,000 ? g for 20 min. The
total amount of protein in the supernatant was quantified by BCA assay
(Pierce). The supernatant was mixed with SDS/DTT sample buffer (New
England Biolabs) and was heated to 100°C for 5 min. The resulting lysate
was resolved by SDS-PAGE and was transferred to a nitrocellulose mem-
brane. After the membrane was blocked with 5% milk in PBS containing
0.1% Tween 20, the blot was probed with chicken anti-TPPII Ab (Cedar-
lane Laboratories) or mouse anti-?-tubulin Ab (Abcam), followed by HRP-
conjugated anti-chicken IgY (Promega) or HRP-conjugated anti-mouse
IgG (Jackson ImmunoResearch Laboratories), and detection was per-
formed using an ECL system (Pierce Biotechnology).
Radiolabeling and immunoprecipitation
Thymocytes (25 ? 106) were incubated in 700 ?l of RMPI 1640 without
methionine (Sigma-Aldrich) at 37°C and 5% CO2for 2.5 h, and then 500
?Ci of L-[35S]-methionine L-[35S]-cysteine mix (EasyTag Express protein
labeling mix; PerkinElmer) was added and the incubation continued for
4 h. Cells were spun down, washed with PBS, and lysed on ice by addition
of 700 ?l of ice-cold lysis buffer (Tris-buffered saline (pH 8.0), 1% (w/v)
deoxycholic acid, 0.5% (v/v) Nonidet P-40) with protease inhibitors (com-
plete protease inhibitor cocktail tablets; Roche). The resulting lysates were
cleared by centrifugation through a 0.22-?m-pore cellulose acetate mem-
brane (SpinX; Corning).
For immunoprecipitation, protein A-conjugated magnetic beads (15 ?l/
sample Dynabeads protein A; Invitrogen) were washed with lysis buffer
(Tris-buffered saline (pH 8.0), 1% (w/v) deoxycholic acid, 0.5% (v/v) Non-
idet P-40) and incubated 2 h at 4°C with rabbit anti-H-2Kbexon 8 (2
?l/sample; a gift from J. Yewdell, National Institutes of Health, Bethesda,
MD) or rabbit anti-?2-microglobulin (1 ?l/sample; Dako) and then added
to radiolabeled cell lysates. A third of each cell lysate volume was incu-
bated with 15 ?l of Ab beads overnight at 4°C with shaking. The beads
were then washed three times with lysis buffer using a magnet (Dyna-
Mag-2; Invitrogen), resuspended in 50 ?l/sample of sample buffer and
reducing agent (XT sample buffer and reducing agent; Bio-Rad), and
heated to 90°C for 10 min. Thirty microliters per sample was run in a 12%
30/0.8 acrylamide/bisacrylamide (Duracryl; Proteomic Solutions), 360 mM
bis-Tris (Calbiochem/EMD Chemicals) gel. The running buffer was 50
mM MOPS, 50 mM Tris, 1 mM EDTA, 0.1% (w/v) SDS, and 5 mM
sodium bisulfite. The gel was washed with distilled water, incubated for 1 h
in scintillation phosphor solution (Autofluor, National Diagnostics), dried
under vacuum, and autoradiography film was exposed to it at ?80°C for
1–5 days. The films were developed and scanned, and the band intensities
were quantified using ImageJ software (National Institutes of Health).
TPPII gene-trapped mice are viable
Mice with a gene trap disrupting TPPII were obtained from Lex-
icon Pharmaceuticals. In the gene-trapped allele, the gene-trap cas-
sette (containing a splice acceptor and a poly(A) signal) was in-
serted between exons 2 and 3, leading to expression of a truncated
TPPII (98 out of 1262 aa) and impairment of expression of full-
length TPPII (Fig. 1A). To evaluate the reduction in TPPII, TPPII
mRNA levels were analyzed in tissues and cells by real-time quan-
titative PCR. TPPII mRNA levels derived in spleens, kidneys and
MEFs from gene-trapped mice were reduced down to 3.2, 7.5, and
2.9% of WT mice, respectively. We also analyzed levels of TPPII
mRNA by amplifying the active site region in exon 11 and found
that mRNA levels in the gene-trapped MEFs were 4.9% of those
in WT MEFs. These results indicate that the TPPII mRNA level is
severely reduced (?95% reduction) in the gene-trapped mice; the
residual mRNA level is possibly due to alternative splicing. To
evaluate the effects of the reduction in mRNA levels on TPPII
protein levels, we analyzed spleen, liver, heart, and kidney tissue
from WT and gene-trapped mice by semiquantitative Western
blotting. TPPII protein was present in WT mice but was undetect-
able in the gene-trapped animals, demonstrating that TPPII protein
levels are reduced by at least 87.5% (Fig. 1B). These analyses
indicated that the TPPII gene-trapped mice are markedly deficient
Mice with a gene trap disrupting TPPII (Fig. 1A) were viable
and fertile, and they were normal in appearance and behavior. In a
previous description of TPPII knockout (KO) mice, splenomegaly
due to extramedullary hematopoiesis and evidence of chronic in-
flammation were observed (34). However, in our TPPII-deficient
gene-trapped mice, we did not observe any evidence of extramed-
ullary hematopoiesis or inflammation, including splenomegaly, in-
creased splenic myeloid cells (GR-1?) cells (Fig. 2), or abnormal
histology (data not shown) or evidence of systemic cytokine effects
Surface MHC class I levels are increased on a subset of
In order for MHC class I molecules to be transported to the cell
surface, they must first bind a peptide in the ER. Therefore, surface
expression of MHC class I molecules is an indirect measure of overall
peptide supply. Elimination of TPPII might either reduce the peptide
supply to MHC class I if TPPII contributes to the degradation of
MHC class I-binding peptides, or it might increase peptide supply
if TPPII helps produce such peptides. We therefore measured
MHC class I surface expression on splenic B and T cells, CD11c?
A, Gene trap diagram. The TPPII gene-trapped mice were produced by
insertional mutagenesis of embryonic stem cells using a retroviral cassette
containing a splice acceptor and a poly(A) signal. In these cells, the ret-
roviral cassette was inserted between exons 2 and 3, so that a spliced TPPII
containing exons 1 and 2 (98 out of 1262 aa) is expressed instead of full-
length TPPII. B, Undetectable expression level of the TPPII protein in
TPPII gene-trapped mice. Tissue extracts were subjected to Western blot-
ting either with anti-TPPII Ab or with anti-?-tubulin Ab.
Construction and assessment of TPPII gene-trapped mice.
6071The Journal of Immunology
splenic DCs and thymocytes, as well as on bone marrow-derived
DCs. The most striking finding in this analysis was an increase in
MHC class I levels on thymocytes. H-2Kband H-2Dbon all thy-
mic subsets from TPPII gene-trapped mice were 20–30% higher
than those on thymocytes from WT C57BL/6 mice (Fig. 3, A and
B). There was also a trend for increased levels of MHC class I
molecules on DCs (Fig. 3, C and D) and splenic lymphocytes (Fig.
4, A, B, D, and E) from TPPII-deficient mice, but in most cases this
was not statistically significant. When mice were treated with the
type I IFN inducer poly(I:C), MHC class I levels increased on
peripheral lymphocytes from TPPII-deficient vs WT mice, but
were not significantly different between the strains. I-Abwas sim-
ilarly expressed on DCs and splenic B cells from TPPII gene-
trapped mice compared with WT animals (Figs. 3, E and F, and
4, C and F).
TPPII gene-trapped mice respond normally to viral infection
To test the contribution of TPPII to immune responses to a viral
infection, we examined the CTL responses to LCMV. C57BL/6
and TPPII gene-trapped mice were infected with LCMV. At the
peak of the response on day 8 after infection, splenic lymphocytes
were isolated, stimulated in vitro with individual LCMV epitopes,
and stained for intracellular IFN-? levels. The frequency of CTL
producing IFN-? in response to four LCMV epitopes in TPPII
gene-trapped mice was not significantly different from those in
C57BL/6 mice, suggesting that any contribution of TPPII to cre-
ating or destroying these presented peptides is not sufficient to
affect immune responses in vivo (Fig. 5).
TPPII knockdown affects the trimming of long peptide
precursors in some cell types
In our previous study using HeLa-Kb cells, we demonstrated that
siRNA-mediated silencing of TPPII inhibited by ?50% the trim-
ming of long peptide precursors (from 14 to 17 aa long) to generate
MHC class I-presented peptides (21). To examine whether TPPII
matopoiesis. The bars represent averages with SDs (n ? 3). A, Spleens of
5- to 6-mo-old BL6 and TPPII gene-trapped mice. B, Splenocytes were
stained with anti-Gr1 Ab to identify splenic granulocytes. C, Comparison
of the percentage of spleen weights of whole body weights. D, Comparison
of number of splenocytes.
TPPII gene-trapped mice do not show extramedullary he-
show differences in MHC class I, but not in MHC class
II, levels. The mice were injected i.p. with PBS. After
24 h, thymocytes were stained with appropriate Abs and
analyzed by FACS. Bone marrow cells were also har-
vested and cultured for 6 days, followed by addition of
PBS or poly(I:C) (pI:C) and staining with appropriate
Abs on day 7 and analysis by FACS. A–D, Expression
levels of H-2Kb(A and C) and H-2Db(B and D) ex-
pressed as geometric mean fluorescence intensity. The
Student t test was used to determine statistical signifi-
cance (?, 0.01? p ? 0.05; ??, p ? 0.01). Error bars
represent SDs (n ? 3). Data are representative of three
independent experiments. E and F, Flow cytometry
CD11chighcells) stained for I-Abafter 6 days of culture,
followed by 24 h of treatment with (E) PBS or (F)
poly(I:C). Bone marrow-derived DCs from WT mice
are indicated by dashed light traces. Bone marrow-de-
rived DCs from TPPII gene-trapped mice are indicated
by solid dark traces.
TPPII gene-trapped and WT thymocytes
6072ROLE OF TRIPEPTIDYL PEPTIDASE II IN MHC-I ANTIGEN PRESENTATION
similarly contributes to trimming of long precursors in murine
cells, we isolated multiple independent MEF lines from the prog-
eny of TPPII?/?mouse crosses, thus generating homozygous
gene-trapped, heterozygous, and WT MEFs. The homozygous
TPPII gene-trapped and WT lines were indistinguishable in terms
of their morphology and growth characteristics for at least 25
Since TPPII is a cytosolic peptidase, we examined the ability of
the gene-trapped MEFs to generate presented peptides from pre-
cursors expressed in the cytosol. For this experiment, several in-
dependent MEF lines were transfected with plasmids expressing
SIINFEKL (S8L) as ubiquitin fusion proteins, either as the mature
epitope (SIINFEKL) or with N-terminal extensions of varying
lengths as summarized in Table I. Cleavage by ubiquitin C-termi-
nal hydrolases, which reside in the cytosol, releases the peptide of
interest without an initiating methionine, thus generating peptides
similar to those generated by the proteasome. Subsequent trim-
ming of the N-terminal residues releases SIINFEKL (S8L), which,
if presented on H-2Kb, can be detected by staining with the mAb
MEFs were transfected with various constructs and analyzed by
flow cytometry by gating on cell populations expressing compa-
rable amounts of GFP. Because absolute levels of surface H-2Kb
were variable between different independent MEF lines, peptide
presentation was normalized to the H-2Kblevel of each cell. Pre-
sentations of mature S8L and of S8L with a 2-aa N-terminal ex-
tension were not statistically different between WT, heterozygous,
and KO MEFs. In contrast, presentation of S8L with a 4- to 10-aa
N-terminal extension was significantly reduced in the KO MEFs,
compared with WT and heterozygous MEFs (Fig. 6A). These re-
sults are similar to (although less marked than) those previously
observed in human HeLa cells (21) and indicate that TPPII con-
tributes to the trimming of very long N-terminal extensions in
some primary murine cells.
To further test the role of TPPII in trimming N-extended pep-
tides in another primary cell type, we transduced bone marrow-
derived DCs with lentiviruses expressing various S8L precursors.
Lentivirus transduction does not affect DC maturation or Ag pre-
sentation (35). We constructed recombinant lentiviral vectors en-
coding S8L as a ubiquitin fusion protein, S8L with a 9-residue
N-terminal extension as a ubiquitin fusion protein (N9-S8L), ubiq-
uitin alone (as a control), or full-length OVA. Bone marrow-de-
rived DC progenitors from WT or KO mice were transduced with
each lentivirus on day 2 and stained with H-2Kb-S8L-specific Ab
25.D1.16 on day 7 by gating on GFP?CD11c?cells. These cells
presented the full-length OVA and S8L similarly. However,
LCMV infection in vivo. Splenocytes from TPPII?/?and BL6 mice in-
fected with LCMV were harvested on day 8, and stimulated in vitro with
peptides corresponding to LCMV epitopes for 5 h. Cells were then stained
for intracellular IFN and analyzed by flow cytometry by gating on
CD8?CD44?cells. The graph represents average percentages of IFN-??
cells with SDs as error bars (n ? 6). There was no significant difference
between TPPII?/?and BL6 mice in their response to any of the four
TPPII gene-trapped and WT mice respond similarly to
show differences in MHC class I, but not in MHC class
II, levels. The mice were injected i.p. with PBS or
poly(I:C) (pI:C). After 24 h, splenocytes were stained
with appropriate Abs and analyzed by FACS. Each
graph shows geometric mean fluorescence intensity
(GMFI), which represents the expression levels of
H-2Kb(A and D), H-2Db(B and E), or I-Ab(C and F).
The Student t test was used to determine statistical sig-
nificance (?, 0.01? p ? 0.05). Error bars represent SDs
(n ? 3). Data are representative of three independent
TPPII gene-trapped and WT splenocytes
6073 The Journal of Immunology
TPPII-deficient DCs also showed reduced presentation compared
with WT cells of the N9-S8L construct when it was expressed at
high levels (GFPhighcells) (Fig. 6B). Interestingly, the difference
in N9-S8L presentation by WT and KO cells was not seen at more
limiting Ag concentrations (GFPlowcells) (Fig. 6C), presumably
because the alternate trimming mechanisms for long peptides only
become limiting at the higher Ag dose.
Analysis of the role of TPPII in trimming long precursors
Since TPPII has some effect on the trimming of long precursor
peptides in some cell types, we sought to examine its role in this
process in animals. He et al. reported that s.c. injection of lentivi-
rus resulted in direct transduction of skin-derived DCs and potent
and prolonged Ag presentation (36). We injected recombinant len-
tiviruses into footpads of TPPII gene-trapped mice and C57BL/6
mice as described (36, 37). We used recombinant lentivirus har-
boring genes for the various S8L-derived peptides described above
and for full-length OVA. In this system the magnitude of the CD8
T cell response was dependent on the amount of virus injected, and
therefore we used limiting doses of viruses so as to be on a sen-
sitive portion of the dose-response curve. On day 8 after infection,
lymphocytes from the draining popliteal lymph node were stimu-
lated in vitro with the S8L peptide, followed by staining for intra-
cellular IFN-? levels. The frequency of IFN-??CTLs to S8L and
to the N-extended S8L precursor was not different between WT
and TPPII-deficient mice (Fig. 7A), even at limiting doses of the
virus (where responses were not maximal).
We next immunized mice with recombinant vaccinia viral vec-
tors encoding the same set of genes as recombinant lentiviral vec-
tors described above. We infected WT and gene-trapped mice ei-
ther by footpad injection or i.p. injection with the various
recombinant vaccinia viruses. On day 7, splenocytes were simi-
larly stimulated in vitro with the S8L peptide, followed by staining
for intracellular IFN-? levels. No consistent, statistically signifi-
cant difference in response to any of the constructs was seen, al-
though a statistically significant decrease of IFN-??CTLs to
N9S8L was observed in one of two i.p. injection experiments (Fig.
7, B and C).
cultured DCs, respond differently to N-extended epitope precursors. A,
TPPII?/?, TPPII?/?, or TPPII?/?MEFs were transfected with plasmids
expressing, in the cytosol, the OVA H-2Kb-restricted epitope SIINFEKL
(S8L) extended at the N-terminus by 0, 2, 4, 6, 8, or 10 aa as ubiquitin
fusion constructs, or full-length OVA, and coexpressing GFP on the same
mRNA. After 24 h the MEFs were stained with 25.D1.16 Ab. Cells were
gated on similar levels of GFP expression. Data are normalized to expres-
sion level of H-2Kbin each MEF line. The Student t test was used to
determine statistical significance (?, 0.01? p ? 0.05; ??, p ? 0.01). Error
bars represent SDs (n ? 9 for TPPII?/?, n ? 7 for TPPII?/?, n ? 4 for
TPPII?/?). B and C, TPPII?/?or BL6 bone marrow-derived DCs were
transduced with recombinant lentiviral vectors encoding S8L or N9S8L as
ubiquitin fusion constructs, or full-length OVA, and then stained with
25.D1.16 Ab at 5 days posttransduction. Cells were gated on (B) high or
(C) low levels of GFP expression. Data are normalized by expression level
of H-2Kbin each DC line. The Student t test was used to determine sta-
tistical significance. Error bars represent SDs (n ? 6).
TPPII gene-trapped and WT embryonic fibroblasts, but not
extended epitope precursors in vivo. A, Mice were injected in the footpad
with recombinant lentivirus encoding full-length OVA, ubiquitin alone
(?), ubiquitin fused with SIINFEKL (S8L), or ubiquitin fused with S8L
extended by 9 aa at the N terminus (N9S8L). B, Mice were injected in the
footpad with recombinant vaccinia virus encoding the same constructs. C,
Mice were injected i.p. with 1.25 ? 106PFU per mouse of recombinant
vaccinia virus encoding the same constructs. Eight days later (A) or seven
days later (B and C), lymphocytes from a draining lymph node (A) or
splenocytes (B and C) were harvested and stimulated for 5 h with 5 ?M of
the appropriate epitope. Cells were then stained for intracellular IFN-? and
analyzed by flow cytometry by gating on CD8?CD44?cells. The graphs
represent average percentages of IFN-??cells with SDs as error bars (n ?
5). Data are representative of two independent experiments. The Student t
test was used to determine statistical significance (?, 0.01? p ? 0.05). The
difference in response to i.p. vaccinia-N9S8L seen between TPPII KO and
WT mice in C (p ? 0.03) was not seen in the repeat experiment (p ? 0.59).
TPPII gene-trapped and WT mice respond similarly to N-
6074 ROLE OF TRIPEPTIDYL PEPTIDASE II IN MHC-I ANTIGEN PRESENTATION
Immune surveillance for virally infected cells and tumors is de-
pendent on cells processing their proteins into fragments and dis-
playing a fraction of these peptides on MHC class I molecules on
the cell surface. The magnitude of the CTL response to a particular
peptide and the immunodominance hierarchy of responses are at
least partly dependent on the numbers of MHC class I-peptide
complexes at the cell surface. This in turn is influenced by several
factors, such as the affinity of binding between the peptide and
MHC class I allele, the number of protein precursors, and the fre-
quency with which the peptide is generated or destroyed by the
Ag-processing pathway. To understand and predict the specificity
of responses, therefore, it is important to identify proteases that
generate or destroy peptides that bind, or could bind, MHC class I
Most intracellular proteins are degraded by proteasomes, and
proteasomes are essential for generating most MHC class I-pre-
sented epitopes (38). Experiments with model peptides and pro-
teasome inhibitors have shown that the proteasome generally pro-
vides the only activity in cells that can make the proper cleavage
to generate the C terminus of an MHC class I epitope (39–41). In
contrast, peptides that have the correct C-terminal residue for bind-
ing MHC class I, but that are too long at the N terminus, can be
trimmed by aminopeptidases into mature epitopes. Epitopes can
also be destroyed if aminopeptidases or endopeptidases trim them
below the minimum size needed for MHC class I binding (6).
Therefore, several groups have pursued the question of whether
trimming by aminopeptidases is normally important in Ag presen-
tation, and if so which aminopeptidases contribute to this process
A number of aminopeptidases have been proposed to play a role
in MHC class I Ag presentation, based on biochemical observa-
tions, experiments in tissue culture, or KO mice. ER aminopepti-
dase 1 (ERAP1) has been shown to play an important role in trim-
ming peptides in the ER for Ag presentation and in the generation
of CD8 T cell responses in mice in ways that increase or decrease
responses to certain peptides (9–14). There is also strong evidence
that peptide trimming by aminopeptidases in the cytosol contrib-
utes to Ag presentation (10, 39, 42, 43, 45). Although biochemical
experiments with various aminopeptidases (including leucine ami-
nopeptidase, bleomycin hydrolase, and puromycin-sensitive ami-
nopeptidase) suggested they may have important functions in Ag
processing (42, 43), mice lacking these aminopeptidases have
shown at most very limited effects on Ag presentation and immune
responses (25, 46, 47). To fully understand the contribution of
various peptidases to Ag presentation, it is therefore important to
generate and analyze peptide-deficient mice.
TPPII is an abundant intracellular peptidase that cleaves triplets
of amino acids from the N terminus of peptides and has been
reported to also act as an endoprotease (19). The physiological
functions of TPPII have been unclear. Early studies suggested that
TPPII might be able to compensate for the loss of proteasomes
(48), although subsequent experiments did not support this possi-
bility (49). In cell extracts, TPPII has been shown to help generate
a few MHC class I-presented peptides, and studies with inhibitors
have suggested a similar role in vivo (16, 19, 48).
A recent paper suggested that in fact TPPII may be essential for
almost all MHC class I Ag presentation, and the authors proposed
a model in which proteasomes in intact cells normally produce
only long peptides (more than 16 aa long) and TPPII was needed
to trim these long precursors; in this model, TPPII was an essential
bridge between proteasome-generated peptides and degradation to
amino acids by conventional peptidases (16). We have previously
tested this model in cultured human cells by using siRNA to knock
down TPPII levels (21). We found that, while TPPII was indeed
important (although not essential) for converting long peptides to
shorter forms, TPPII was not required for MHC class I Ag pre-
sentation in cultured cells: presentation of peptides from full-
length proteins required proteasomes, but TPPII knockdown did
not reduce (and if anything slightly increased) MHC class I peptide
production. We tentatively concluded that relatively few very long
precursor peptides are normally generated in vivo and therefore
TPPII is not required for most Ag presentation. However, the pos-
sibility remained that TPPII is required for a natural immune re-
sponse in intact animals (e.g., if the HeLa cells we tested are not
representative of normal cells, or if the initiation of an immune
response has different requirements for Ag presentation).
Mice containing a gene trap between exons 2 and 3 have a
?90% reduction in the expression of TPPII mRNA and at least a
?87.5% reduction in the expression of protein. They are viable,
fertile, and are grossly normal in appearance and behavior; in par-
ticular, and in contrast to observations in TPPII KO mice (34), the
gene-trapped TPPII-deficient mice did not show extramedullary
hematopoiesis or splenitis. These differences may be because of
differences in housing or in mouse background, or may be because
of very low (although undetectable in our assays) levels of func-
tional TPPII in our mice. The lack of chronic inflammation in these
gene-trapped mice offers the ability to compare Ag presentation in
the presence and near-absence of TPPII, without the confounding
effects of inflammation on MHC class I Ag presentation.
Some cells (particularly thymocytes) from TPPII gene-trapped
mice express a higher level of surface MHC class I than do those
from C57BL/6 mice (Fig. 2). In contrast, the synthesis of heavy
chains (measured by immunoprecipitation of metabolically labeled
H-2Kb) and light chains (?2-microglobulin) was not significantly
different in thymocytes from WT vs TPPII-deficient mice (data not
shown). Since MHC class I can only reach the cell surface when
it is bound to an appropriate peptide, this suggests that the overall
supply of presented peptides may be reduced by TPPII. In other
words, the net effect of TPPII may be to destroy more peptides
than it helps produce. It is possible that residual TPPII in the gene
trap mice is contributing some function, and a more pronounced
phenotype would be observed in the complete absence of this pep-
tidase. However, our findings are similar to observations with
TPPII KO mice (24).
In previous studies, the most important contribution of TPPII to
Ag presentation was its ability to trim very long precursor peptides
(16, 21). However, this activity has only been examined in cultured
human tumor cells and not in primary cells or cells of murine
origin. Therefore, we made minigene constructs to test the trim-
ming of full-length protein, short and long antigenic precursors,
and mature epitope in primary cells from gene-trapped mice. For
these studies, we produced embryonic fibroblast cell lines (MEFs),
transfected these cells with plasmids expressing various precursors
of the H-2Kb-binding peptide SIINFEKL, and quantified presen-
tation of H-2Kb-SIINFEKL as a measure of Ag processing. Pre-
sentation of SIINFEKL from short precursors tested was similar in
WT, heterozygous, and homozygous gene-trapped cell lines, while
presentation of SIINFEKL from precursors with 4- to 10-aa N-
terminal extensions was significantly reduced by TPPII deficiency.
A similar defect in processing a long precursor (N9-S8L) was ob-
served in TPPII-deficient bone marrow-derived DCs under condi-
tions of high Ag expression. These results are consistent with our
previous findings with HeLa-Kb cells (21), although the extent of
reduction in the MEFs was less marked than in the previous ex-
periments, perhaps because of the variability between independent
MEF cell lines, different species (human vs mouse), expression
6075 The Journal of Immunology
level of TPPII, and/or presence of other aminopeptidases in the
different cell types.
We also tested the effect of TPPII knockdown on the generation
of an authentic antiviral immune response to viral proteins and
antigenic precursors. We infected TPPII gene-trapped mice and
C57BL/6 mice with LCMV, and after 8 days we analyzed the
frequency of CD8?T cell responses to four LCMV MHC class
I-restricted epitopes. Consistent with the recent findings (23, 24),
we found no difference in the responses to any of the epitopes (Fig.
5). Additionally, we specifically tested the importance of TPPII on
generation of MHC class I epitopes from precursors with long
N-terminal extensions, using recombinant lentiviruses and vac-
cinia viruses expressing ubiquitin-fused minigenes. TPPII gene-
trapped mice generated similar levels of CTL to these precursors
as did WT mice, demonstrating that TPPII is not essential for
generating immune responses to long N-extended precursors in
vivo. We presume that this is because the reduction in presentation
we observe in TPPII-deficient DCs is too small to affect responses
in vivo. Alternatively, since we only see a defect in presentation at
high Ag concentrations it is possible that such high amounts of Ag
expression are not obtained in vivo. Finally, it is also possible that
the variability in responses between individual mice could obscure
the detection of small differences.
Taken together, we conclude that TPPII is not required for MHC
class I Ag presentation in vivo. This conclusion is consistent with
recent papers using cultured cells (23) or TPPII KO mice (24) and
adds to the weight of evidence against the model proposed by Reits
et al. (16). This could in principle be because TPPII is not required
for trimming precursors with long N-terminal extensions, and/or
because such precursors are not generated very often in cells.
A substantial amount of trimming of Ag precursor peptides oc-
curs in the cytosol (10). Although a number of cytosolic amino-
peptidase (LAP, BH, PSA, and TPPII) can trim such precursors in
cell extracts, their elimination from cells and in KO mice does not
inhibit peptide trimming in vivo (Refs.25, 46, 47 and this paper).
This raises the possibility that there may be other cytosolic amin-
opeptidases that are important to Ag presentation and remain to be
identified. Alternatively, since the known cytoplasmic aminopep-
tidases generally have broad substrate specificity, the enzymes’
function may be sufficiently redundant so that the lack of one pep-
tidase could be readily compensated for by other ones. Crossing of
several kinds of KO mouse strains will help to define the func-
tional redundancy or a unique role of aminopeptidases.
We thank Dr. Raymond Welsh for providing LCMV and Dr. Eicke Latz for
providing lentiviral vectors.
The authors have no financial conflicts of interest.
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6077 The Journal of Immunology