MHC class I antigen processing distinguishes
endogenous antigens based on their translation
from cellular vs. viral mRNA
Brian P. Dolan, Aditi A. Sharma, James S. Gibbs, Tshaka J. Cunningham, Jack R. Bennink, and Jonathan W. Yewdell1
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Edited by Thierry Boon, Ludwig Institute for Cancer Research, Brussels, Belgium, and approved March 23, 2012 (received for review July 29, 2011)
To better understand the generation of MHC class I-associated
peptides, we used a model antigenic protein whose proteasome-
mediated degradation is rapidly and reversibly controlled by Shield-
1, a cell-permeant drug. When expressed from a stably transfected
gene, the efficiency of antigen presentation is ∼2%, that is, one cell-
source proteins degraded upon Shield-1 withdrawal. By contrast,
when the same protein is expressed by vaccinia virus, its antigen
presentation efficiency is reduced ∼10-fold to values similar to those
reported for other vaccinia virus-encoded model antigens. Virus in-
fection per se does not modify the efficiency of antigen processing.
Rather, the efficiency difference between cellular and virus-encoded
antigens is based on whether the antigen is synthesized from trans-
gene- vs. virus-encoded mRNA. Thus, class I antigen-processing ma-
their synthesis to modulate antigen presentation efficiency.
intracellular pathogens. Recognition is based on the interaction
of the clonally restricted T-cell receptor with MHC class I mol-
ecules bearing oligopeptides. Peptides are predominantly gener-
ated from defective ribosomal products (DRiPs), nascent
translation products that are degraded rapidly either by design
(i.e., dedicated for antigen presentation) or necessity (defective
forms of proteins that can interfere with cell function) (1). The
use of DRiPs greatly speeds recognition of virus-infected cells,
because many peptides derive from otherwise highly stable pro-
teins. For viral infections, the efficiency of recognition afforded
by DRiPs is of the essence, because viruses can replicate within
hours, and killing must be fast to be effective. Time is not limiting
for tumor cells but efficiency is critical, because T cells often
recognize antigens derived from gene products expressed at ex-
tremely low levels. Indeed, T cells may be the only means of
detecting expression of the source antigen (2).
Only a handful of studies have addressed the efficiency of pep-
tide generation, a critical issue with broad implications for the ef-
ficiency of protein synthesis and cytosolic compartmentalization of
antigen processing (3). Pamer and colleagues seminally reported
that class I–peptide complexes are generated with an efficiency of
3–25% (i.e., 3–25 complexes generated per 100 proteins degraded)
from Listeria monocytogenes proteins secreted into the cytosol of
mouse macrophage-like cells (4, 5). Using recombinant vaccinia
viruses (rVVs) to express a rapidly degraded full-length chimeric
protein, we reported that the efficiency of complex (Kb–SIIN-
FEKL) generation was much lower in a variety of mouse fibroblast
and macrophage/dendritic cell (DC)-like cell lines, 0.25%–0.05%
(1/400–1/2,000), with an efficiency of ∼2% for SIINFEKL synthe-
cytosol based on liberation from chimeric GFP-ubiquitin fusion
proteinsbyubiquitinhydrolases,reporting anefficiency of0.2%(1/
500) for EBV-transformed human B cells expressing permanently
demonstrated that ubiquitin hydrolase-liberated SIINFEKL is
D8+T lymphocytes play a key role in immunosurveillance by
eradicating tumor cells and cells harboring viruses and other
presented at high (15–20%) efficiency in a macrophage-like cell
that two very different systems of antigen introduction into the
cytosol need not differ widely in processing efficiency.
Taken together, these findings demonstrate system-dependent-
wide variation in the efficiencies of generating complexes from
full-length proteins and oligopeptides. To better understand these
differences, here we measure the context dependence of antigen-
processing efficiency using a number of antigens, cells, and ex-
Kb–SIINFEKL Is Generated from SCRAP at Remarkably High Efficiency
in EL4 Cells. Building on the work of the Wandless laboratory (9),
we recently described SCRAP, a chimeric antigen whose stability
is precisely controlled by the cell-permeant drug Shield-1 (10).
SCRAP consists of a Shield-1 interaction domain NH2-terminally
fused to SIINFEKL and GFP (10) (Fig. 1A). In the absence of
Shield-1, nascent SCRAP is degraded with a t1/2of 16 min. In the
presence of Shield-1, a stable pool of SCRAP accumulates that is
degraded with a t1/2of 30 min upon removal of Shield-1 (10).
Fig. 1 B and C illustrate the behavior of SCRAP when expressed
as a permanently transfected gene in EL4 cells, an H-2bT-cell
lymphoma. Via flow cytometry, properly folded fluorescent
SCRAP and Kb–SIINFEKL are measured simultaneously in live
cells based on, respectively, GFP and directly conjugated 25-D1.16
mAb (11). Because GFP fluorescence requires proper folding and
not all SCRAP is properly folded, only measuring fluorescent
SCRAP will underestimate the amount of SCRAP present in cells.
total cell extracts that interact with a mAb specific for GFP (Fig.
1D). Using purified GFP of known concentration to generate
a standard curve, we could calculate the average number of im-
munoreactive SCRAP molecules expressed per cell (Fig. 1D). We
simultaneously determined absolute numbers of directly conju-
beads as described in Materials and Methods. At the start of the
efficiency measurementperiod, we treated cells with mild acid (pH
3) to destroy preexisting Kb–SIINFEKL complexes, thereby im-
proving the signal-to-noise ratio.
The SCRAP system enabled us to determine the efficiency of
peptide generation from nascent proteins vs. retirees (12).
Retirees are folded, functional proteins that are selected for
degradation either stochastically according to the classical view
(13) or, in this case, due to unfolding based on Shield-1 with-
drawal. For nascent proteins, we determined the amount of
Author contributions: B.P.D., J.R.B., and J.W.Y. designed research; B.P.D. and A.A.S. per-
formed research; J.S.G. and T.J.C. contributed new reagents/analytic tools; B.P.D., A.A.S.,
J.R.B., and J.W.Y. analyzed data; and B.P.D., A.A.S., J.R.B., and J.W.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
| May 1, 2012
| vol. 109
| no. 18
SCRAP rescued over a 4-h period by Shield-1 and the concomi-
tant decrease in Kb–SIINFEKL expression (Fig. 1B). For retirees,
we accumulated a pool of SCRAP by treating for 4 h with Shield-
1, and then induced degradation by removing Shield-1 while
measuring the increase in Kb–SIINFEKL expression concomitant
with SCRAP degradation (Fig. 1C) in the presence of cyclohexi-
mide to prevent newly synthesized and rapidly degraded SCRAP
from entering the antigen presentation pathway.
The efficiency of generating Kb–SIINFEKL complexes was
statistically indistinguishable between Shield-1–sensitive nascent
and retiree pools (Tables 1 and 2), and was calculated to be
∼2%. Remarkably, this is 40-fold more efficient than generation
of Kb–SIINFEKL complexes from VV-expressed rapidly de-
graded nucleoprotein (NP)- SIINFEKL (S)-GFP [ubiquitin
(Ub)-R-S-GFP in Fig. 1A], and 10-fold more efficient than
misfolded NP-S-GFP due to the insertion of a KEKE sequence
in the NP domain (6, 14).
Theselargedifferences could be related todifferences inthe cell
type studied (EL4- vs. L-Kb), antigen (SCRAP vs. NP-S-GFP), or
expression system (transfection vs. VV infection). To distinguish
between the types of antigens, we measured the efficiency of anti-
gen presentation from VVs expressing SCRAP vs. NP-S-GFP (Fig.
2). L-Kbcells infected with VV SCRAP in the presence of Shield-1
expressed nearly identical amounts of GFP as detected by flow
cytometry as cells infected with rVV expressing NP-S-GFP, and
generated essentially identical amounts of Kb–SIINFEKL. In the
absence of Shield-1, nearly identical amounts of fluorescent GFP
were detected compared with Ub-R-S-GFP (6), and peptides
were generated at half the rate from SCRAP vs. rapidly degraded
for the high efficiency of peptide generation from SCRAP in
denotes the SIINFEKL peptide, influenza A virus (IAV) nucleoprotein is in orange, and the green “Ub” box represents ubiquitin. (B) EL4/SCRAP cells were
washed in a mild citric acid buffer (pH 3.0) to remove existing Kb–SIINFEKL complexes and cultured in the presence or absence of 5 μM Shield-1. At the
indicated times, Kb–SIINFEKL complexes and GFP levels were determined by flow cytometry. (C) Same as in A, except Shield-1 was removed following an initial
3.5-h incubation and, following a second acid wash, cells were cultured in the presence of cycloheximide (CHX). (D) An example of a Western blot for GFP
using recombinant GFP standards and lysates from EL4/SCRAP cells treated with or without Shield-1 for 3 h.
Model antigens and a schematic of SCRAP presentation and quantification. (A) The various constructs used in this study are depicted. The red “S” box
from rapidly degraded self-proteins
Efficient antigen presentation of peptides derived
2.54 × 105
5.62 × 105
6.83 × 104
EL4/SCRAP cells were treated as in Fig. 1. Peptide–MHC complexes were
determined by quantitative flow cytometry. Shield-1–sensitive nascent
SCRAP molecules were quantitated via immunoblotting. Efficiencies were
calculated at 4 h after addition of Shield-1.
| www.pnas.org/cgi/doi/10.1073/pnas.1112387109 Dolan et al.
Are there cell type-related differences in antigen-processing ef-
ficiency? Because EL4 cellsare highlyresistant to VVinfection,we
compared the antigen-processing efficiencies of EL4 vs. L-Kbcells
using recombinant vesicular stomatitis viruses (rVSVs) expressing
SIINFEKL peptide (VFP-Ub-S). Following infection with VSV-
VFP-Ub-S, thecelllinesexpressed nearlyidentical amounts ofKb–
SIINFEKL (Fig. 3A, red trace), although EL4 cells expressed al-
peptidesare inexcessunder theseconditions(3),thisdemonstrates
that EL4 and L-Kbcells have a similar overall capacity to generate
surface Kb–SIINFEKL complexes from a cytosolic SIINFEKL
pool. This implies similar functional levels of the transporter
associated with antigen processing (TAP), Kb, and intracellular
trafficking machinery in EL4 and L-Kbcells.
We next compared the abilities of EL4 and L-Kbcells to
generate Kb–SIINFEKL complexes from a full-length stable
protein expressed by VSV. Following infection with rVSV-NP-S-
GFP, EL4 and L-Kbcells expressed nearly identical levels of
fluorescent NP-S-GFP between 1 and 3 h postinfection (hpi) (Fig.
3A). Levels of Kb–SIINFEKL were similar, although L-Kbcells
demonstrated a more complicated pattern, with increased im-
mediate presentation and a 50% decrease in complex generation
between 2 and 3 hpi Overall, however, the efficiency of gener-
ating Kb–SIINFEKL complexes from DRiPs derived from this
full-length stable protein was similar between the two cell types.
We also examined the efficiency of Kb–SIINFEKL generation
in L-Kbcells infected with rVV-NP-S-GFP or rVSV-NP-S-GFP
with virus dose adjusted to yield equivalent levels of NP-S-GFP
fluorescence. Generation of Kb–SIINFEKL complexes was su-
perimposable (Fig. 3B), demonstrating that antigen presentation
canoccurat similarefficiency fromtwovery differentviralvectors.
Antigen Processing Efficiency is Dependent on the mRNA Source of
the Antigen. The findings demonstrate that the efficiency of gen-
erating Kb–SIINFEKL complexes is similar between EL4 and L-
Kbcells and between rVV and rVSV expressing NP-S-GFP.
Could the specifics of gene expression related to host- vs. virus-
To explore this possibility, we transiently transfected SCRAP in
HeLa Kbcells and compared their antigen-processing efficiency to
HeLa Kbcells infected with rVV SCRAP at a dose titrated to
and B). Although the cells were synthesizing near-identical
amounts of SCRAP from host vs. VV mRNA, transfectants gen-
erated Kb–SIINFEKL complexes at a much higher rate from the
Shield-1–sensitive pool (Fig. 4 A and B). In five separate experi-
ments (Table 3), we determined that the average fold increase in
antigen presentation efficiency of transfect-encoded protein com-
pared with VV-encoded protein was 8.3 (±1.6 SD units, P < 0.05).
Notably, the efficiency of Kb–SIINFEKL generation from
transfected SCRAP in HeLa vs. EL4 cells was similar (0.6% vs.
2.4%), particularly when considering that a mismatch between the
mouse class I molecule (Kb) and human antigen-processing ma-
chinery (TAP, tapasin, other chaperones, and endoplasmic re-
ticulum aminopeptidase) likely lowers peptide loading efficiency.
There was a reasonably good match between the efficiency of
generating Kb–SIINFEKL from the Shield-1–sensitive pool of
VV-encoded SCRAP (1/1,250) and our previous determination
of the efficiency of generating Kb–SIINFEKL from rapidly de-
graded NP in various mouse cell types (as high as 1/1,400 in DC
2.4 cells) or slowly degraded, misfolded NP (1/440) (6).
What is the contribution of virus-induced alterations in cell
function to modulating antigen presentation efficiency? We
infected EL4/SCRAP cells with VSV or HeLa Kb/transient
SCRAP cells with rVV, acid-stripped preexisting Kb–SIINFEKL
complexes, and measured the efficiency of Kb–SIINFEKL gener-
ation from nascent, Shield-1–sensitive SCRAP synthesized during
viral infection (Fig. 4C). Infection with VSV or VV resulted in
a partialdecreasein both Kb–SIINFEKL andGFP expression,but
had only a minor (less than 20%) inhibitory effect on the overall
efficiency of antigen presentation.
Taken with the data in Table 3, these findings point to the
remarkable conclusion that the antigen-processing machinery
has the capacity to distinguish folded proteins based on their
synthesis from cell- vs. virus-encoded mRNA. The same peptide
from the same folded protein pool in the same cell line is gen-
erated approximately eightfold more efficiently when synthesized
from cell- vs. VV-encoded RNA.
What about DRiP efficiency? The SCRAP system allows us to
separate the presentation of peptides from retired vs. DRiP sub-
strates, which are the only source of peptides when saturating
amounts of Shield-1 are added to SCRAP-expressing cells (10).
We compared the amount of GFP to Kb–SIINFEKL synthesized
in acid-stripped HeLa cells treated with Shield-1 and expressing
SCRAP from transfected vs. VV-expressed genes. This revealed
that VV infection of HeLa Kbcells per se has little effect on Kb–
SIINFEKL processing from DRiPs derived from cell-encoded
SCRAP (Fig. 4D, infected), whereas DRiPs derived from
nascent, rapidly degraded SCRAP
Retired SCRAP is presented with similar efficiency as
EL4/SCRAP cells were treated as in Fig. 1C. Peptide–MHC complexes were
determined by quantitative flow cytometry. Retired SCRAP molecules were
quantitated via immunoblotting. Efficiencies were calculated 2 h following
the removal of Shield-1.
cells. Cells were cultured with or without Shield-1 (for SCRAP infection) and analyzed at the indicated times for GFP (Left) or Kb–SIINFEKL (Right) by FACS
SCRAP protein expressed by rVV is similar to other model antigens. rVV expressing either SCRAP, NP-S-GFP, or Ub-R-NP-S-GFP was used to infect L-Kb
Dolan et al. PNAS
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VV-encoded SCRAP have approximately twofold reduced effi-
ciency. Thus, viral infection is either associated with fewer
SCRAP DRiPs synthesized or diminished efficiency of DRiP
conversion to Kb–SIINFEKL complexes.
in our understanding of the class I antigen-processing pathway.
First, we show that cellular gene products in the form of the
model antigen SCRAP can be processed from a folded state at
remarkably high efficiency, at 2% (1 Kb–SIINFEKL complex
generated per 50 precursors degraded). This value is ∼40 times
higher than our previous determination of Kb–SIINFEKL gen-
eration from a rapidly degraded VV-encoded protein (6). All
things being equal, a 2% antigen-processing efficiency is ex-
tremely difficult to square with cellular protein economy (6, 12,
15). Even in the absence of DRiPs and other rapidly degraded
polypeptides (16), normal cellular protein turnover amounts to
∼109d−1, or 7 × 105proteins min−1, potentially spawning
peptides that bind a given class I allomorph at 3.5 × 106min−1
Ub-SIINFEKL (Left) or NP-S-GFP proteins (Right), and antigen presentation as well as fluorescent protein expression were monitored by FACS. (B) L-Kbcells
were infected with rVV and rVSV viruses expressing NP-S-GFP and monitored as in A.
Similar antigen presentation kinetics between different cell types and viruses. (A) L-Kband EL4 cells were infected with rVSV expressing either Venus-
with SCRAP DNA constructs, and antigen presentation as well as GFP expression were determined 4 hpi or post-acid wash. (B) Representative histograms after 4
h of Shield-1 treatment of SCRAP-expressing cells (blue trace) compared with non-SCRAP-expressing cells (red trace). Kb–SIINFEKL staining with 25-D1.16 mAb
(Right) is restricted to GFP-positive cells. (C) EL4/SCRAP or HeLa Kbcells transfected with SCRAP DNA were infected with VSV or rVV, respectively, and both Kb–
SIINFEKL and GFP levels were determined 4 hpi. The efficiency of presentation was determined by dividing the MFI of Kb–SIINFEKL staining by the MFI of GFP.
Values were compared with uninfected cells and are plotted as a percentage. (D) Levels of Kb–SIINFEKL derived from DRiP substrates (in the presence of Shield-
1) from SCRAP expressed as transfected DNA or by rVV in HeLa Kbcells were normalized to GFP expression. Cells (both transfected and nontransfected) were
briefly washed in mild acid and cultured for 2 h in complete media. Cells were then harvested and were either left uninfected or infected with control rVV at an
MOI of 10, followed by an additional 4 h in culture before FACS analysis. Background levels of Kb–SIINFEKL were determined immediately following rVV
infection. These data are representative of three independent experiments.
Virus-expressed antigens are presented at lower efficiencies than self-antigens. (A) HeLa Kbcells were either infected with rVV SCRAP or transfected
| www.pnas.org/cgi/doi/10.1073/pnas.1112387109Dolan et al.
[a 500-residue protein possesses 500 potential n-mer peptides, of
which 5 (1%) bind with immunogenic affinity (17)]. A 2% effi-
ciency would mean that greater than 7 × 104peptides are loaded
onto class I molecules min−1, which would competitively preclude
Kb–SIINFEKL generation, because there are only ∼102class I
molecules exported min−1per cell. One or more of these num-
bers, therefore, must either be in error or unrepresentative.
Two factors potentially contribute to the remarkably high ef-
ficiency of SCRAP retiree processing. First, SIINFEKL may be
a far above average peptide. Many other defined antigenic pep-
tides demonstrate a similar affinity for class I molecules and copy
number (18), but these may all be far above average (the Mas-
sachusetts Institute of Technology student-body effect). Second,
there may be something peculiar about SCRAP that enables it to
access the class I pathway at high efficiency. As a chimeric protein
derived from several sources originating in different organisms,
SCRAP did not experience the honing forces of evolution that
assure its integration into the cellular landscape. In some manner,
cells may be able to detect SCRAP as a foreign entity and shunt it
for high-efficiency antigen presentation. This may also contribute
to the similarly high efficiency of antigen processing from Listeria
proteins secreted into the cytosol reported in the seminal work of
Pamer and colleagues (4, 5), the high efficiency of SIINFEKL
presentation when liberated from Listeria- or VV-synthesized Ub
fusion proteins (8), and the remarkably high class I occupancy
exhibited by a some peptides (19–21).
Our second startling finding is the approximately eightfold
difference in efficiency of Kb–SIINFEKL generation from retired
SCRAP synthesized from cellular vs. VV mRNA. Ironically, from
the perspective of immunosurveillance, viral SCRAP is less effi-
ciently processed. As discussed above, however, this may in part
relate to the artificial nature of SCRAP, and not to an intrinsic
lower efficiency of viral antigen processing. Indeed, Reits et al.
(22) reported that influenza A virus infection rapidly increases the
overall supply of TAP-transported peptides, a critical finding for
immunosurveillance that still begs an explanation.
However artificial, it is intriguing and potentially important
that cells can distinguish cellular from viral SCRAP. We cannot
attribute this to VV-induced changes in cellular physiology, be-
cause VV infection does not greatly modulate the efficiency of
cellular Shield-1–sensitive SCRAP presentation. How can the
antigen-processing machinery distinguish between these osten-
sibly identical antigen sources?
VV SCRAP is likely synthesized in regions of the cytosol or-
ganized specifically for viral translation that serve as the precursors
for viral factories (23, 24), whereas cellular SCRAP is synthe-
sized on “normal” ribosomes. It is therefore possible that there
are subtle (or even not so subtle) differences between the two
in their folding/posttranslational modifications. Indeed, because
tRNA aminoacyl synthetases are recruited to locally translating
ribosomes (24), alterations in their specificity (25) could generate
SCRAP with altered primary sequences that enhance antigen
processing. Still, there is nothing known about antigen processing
to explain why one form of a folded gene product would be a
superior source of a given peptide, pointing to a significant lacuna
in our understanding.
One explanation is that SCRAP synthesized from cellular
mRNA is better-partitioned into a local antigen-processing com-
partment(s) defined by lack of competition (26) or localized pre-
sentation (27). Whereas it is hard to imagine compartmentalizing
folded proteins in a manner kinetically dissociated from their
synthesis by hours, it is possible that nascent proteins are imme-
diately sequestered in small amounts in regions that favor antigen
processing when the protein is eventually degraded by protea-
we still have a lot to learn about MHC class I antigen processing.
Regardless of the mechanism(s), the present findings point to
features of antigen processing that could be handy in discrimi-
nating peptides encoded by innocuous vs. danger-associated
mRNAs. This would be of obvious utility in immunosurveillance
of tumors. Here the immune system often succeeds in finding the
needle in the haystack, by recognizing peptides generated at high
efficiency from genes whose translation is minimal (2, 28).
Materials and Methods
Cells, Antibodies, and Viruses. L-Kb, EL4, EL4/SCRAP, and HeLa Kbcell culture
has been previously described (10, 29). Recombinant vaccinia virus expressing
influenza nucleoprotein fused to the SIINFEKL peptide and GFP (NP-S-GFP) or
its rapidly degraded counterpart (Ub-R-NP-S-GFP), SCRAP, or recombinant vi-
rus expressing no protein were previously described (10, 3). Recombinant ve-
sicular stomatitis virus expressing NP-S-GFP hasbeen previously described (30).
Generation of rVSV expressing Venus-Ub-SIINFEKL (VFP-Ub-S) was as follows.
CT). Venus-Ub-SIINFEKL was polymerase chain-amplified from recombinant
vaccinia virus (3) with the primers Venus 5′ XhoIXmaIMluI (5′-CTCGAGCCC-
GGGACGCGTCCATGGTGAGCAAGGGCGAGGAGC-3′) and Ub-SIINFEKL 3′ NheI
AAG-3′)usingPlatinumTaqHigh Fidelity DNApolymerase(LifeTechnologies).
PCR product was then cloned into pCR4-topo (Life Technologies) according to
the manufacturer’s instructions. Insert DNA containing Venus-Ub-SIINFEKL
was excised with XhoI and NheI restriction enzymes (Roche) and ligated with
similarly digested pVSV-XN2. Final plasmid pVSV-Venus-Ub-SIINFEKL was
verified by restriction digestion patterns and DNA sequencing of the inserted
gene. Baby hamster kidney cells (BHK-21; American Type Culture Collection)
were maintained in DMEM supplemented with 10% (vol/vol) FBS. Cells were
infection (MOI) of 10 for 1 h, followed by transfection with plasmids pBS-N,
pBS-P, pBS-L (31, 32), and pVSV-VFP-Ub-S using Lipofectamine 2000 (Life
Technologies) according to the manufacturer’s instructions. Supernatant me-
dia containing recombinant virus were recovered at 48 hpi and used to make
viral stocks. Viral titers were determined by plaque assays on BHK cells. The
mAb 25D-1.16 was previously described (11). Anti-GFP antibodies were from
Roche. Infrared secondary antibodies were from LI-COR.
Table 3. Transfected SCRAP is presented more efficiently than SCRAP expressed by rVV infection
Transfected SCRAP rVV-infected SCRAP
ExperimentKb–SIINFEKL GFP-degradedEfficiency (%)Kb–SIINFEKL GFP-degraded Efficiency (%)
2.9 × 105
1.1 × 105
3.5 × 105
2.0 × 105
2.5 × 105
8.9 × 105
7.1 × 105
9.4 × 105
5.9 × 105
6.0 × 105
Average8.3 ± 1.6
HeLa Kbcells were transfected with DNA constructs encoding SCRAP or infected with rVV expressing SCRAP and treated with or without Shield-1 for 4 h.
The number of Kb–SIINFEKL complexes derived from a defined number of SCRAP molecules, determined by quantitative immunoblotting for GFP, is shown.
The difference in efficiencies between transfected and infected cells is statistically significant (Student’s t test, P < 0.05).
Dolan et al. PNAS
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Cloning and Transfections. To enhance SCRAP expression in transient trans- Download full-text
fectants, a SCRAP construct was generated by PCR amplifying the original
SCRAP cassette with the primers upstream SacI 5′-TCTAGAGAGCTCCCACC-
ATGGGAGTGCAGGTGGAAACCA-3′ and downstream XhoI 5′-AGATCTCTC-
GAGTTACTTGTACAGCTCGTCCATGCCCAG-3′ to introduce an upstream SacI
and downstream XhoI restriction site. The PCR product was cut with both
enzymes and ligated with similarly digested pCAGGS (a gift from Ronald
Harty, University of Pennsylvania, Philadelphia, PA). Plasmid DNA was puri-
fied (HiSpeed Midi Kit; Qiagen) and used for transfections. HeLa Kbcells (4 ×
105) were resuspended in 20 μL of Amaxa Solution SF (Lonza) and mixed
with 200 ng DNA. Cells were transfected using the Amaxa 96-well Shuttle
System using the program CN-114. Following transfection, cells were cul-
tured overnight and used the following day.
Antigen Presentation Assays and in Vitro Viral Infections. MHC class I antigen
presentation in EL4/SCRAP cells in the presence, absence, or following re-
moval of Shield-1 was examined as previously reported (10). All flow
cytometry experiments were conducted using a BD LSR II flow cytomter. To
quantify the number of peptide–MHC complexes on the cell surface, acid-
washed EL4/SCRAP cells were stained with FITC-coupled 25D-1.16 at differ-
ent times postwash, and the mean fluorescence intensity (MFI) of the FITC
signal was converted to molecular equivalents using FITC-coupled beads
(Spherotech) as previously described (6). Cells were also stained in parallel
with Alexa 647-coupled 25D-1.16, and the number of peptide–MHC com-
plexes calculated from FITC-labeled cells was used to generate a standard
curve for Alexa 647-labeled cells for comparison of Shield-1–treated and –
untreated cells. For all other antigen presentation experiments, cells were
stained with Alexa 647-coupled 25D-1.16 monoclonal antibody and analyzed
by flow cytometry. For rVV and rVSV infections, cells were resuspended at
a concentration of 2 × 106in the appropriate solution (saline solution with
0.1% BSA for rVV, serum-free MEM for rVSV) and virus was added at an MOI
of 10. Cells were incubated at 37 °C for 30 min with occasional agitation,
washed, and cultured at 106cells/mL in complete media. When transiently
transfected HeLa Kbcells were to be infected with rVV, cells were first acid-
washed as described above and recultured for 2 h before infection. In this
scenario, the background levels of Kb–SIINFEKL were determined immedi-
ately following rVV infection. In transiently transfected cells, Kb–SIINFEKL
levels were determined on GFP+cells rather than on the entire population.
Quantitative Western Blot and Antigen Presentation Efficiency. Quantitative
Western blots were performed based on previous experiments (6). Briefly,
total cell lysates were prepared from cells at various times after the indicated
treatments by boiling cells in SDS sample buffer (Quality Biological) at 107
cells/mL for 20 min, and then an equal volume of 0.05 M DTT (in water) was
added to the lysates and boiled for an additional 10 min. Recombinant GFP
(Clontech) was added to similarly prepared EL4 cell lysates (ranging in con-
centration from 1 to 10 nM), and both experimental samples as well as
recombinant GFP were resolved by SDS/PAGE, blotted onto nitrocellulose,
and analyzed by Western blot using the Odyssey Imaging System (LI-COR).
Odyssey software was used to quantitate GFP signal in samples and stand-
ards. A standard curve was generated to determine the concentration of
GFP in each experimental sample. This concentration was then divided by
the number of cell equivalents in the sample (generally 100,000 cells) and
multiplied by Avogadro’s number to determine the average number of GFP
molecules present in each cell. The difference between Shield-1–treated and
–untreated samples is reported. For transiently transfected cells, the cell
equivalent was adjusted based on the transfection efficiency, the number of
GFP+cells as determined by flow cytometry. The efficiency of antigen pre-
sentation was determined by dividing the number of peptide–MHC com-
plexes calculated above by the number of GFP molecules per cell. A two-
tailed Student’s t test was used to statistically analyze the datasets using
GraphPad Prism software.
ACKNOWLEDGMENTS. Glennys Reynoso provided outstanding technical
support. This work was generously supported by the Division of Intramural
Research, National Institute of Allergy and Infectious Diseases.
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