Intracellular Rate-Limiting Steps in MHC
Class I Antigen Processing
María Montoya and Margarita Del Val
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The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
J Immunol 1999; 163:1914-1922; ;
http://www.jimmunol.org/content/163/4/1914
Intracellular Rate-Limiting Steps in MHC Class I Antigen
Processing1
Marı́a Montoya2 and Margarita Del Val3
Quantitative aspects of the endogenous pathway of Ag processing and presentation by MHC class I molecules to CD81 CTL were
analyzed over a wide range of Ag expression in recombinant vaccinia virus-infected cells expressing b-galactosidase as model Ag.
Only the amount of starting Ag was varied, leaving other factors unaltered. Below a certain level of Ag synthesis, increasing
protein amounts led to a sharp rise in recognition by CTL. Higher levels of Ag expression led to a saturation point, which
intracellularly limited the number of naturally processed peptides bound to MHC and thereby also CTL recognition. The ratelimiting step was located at the binding of the antigenic peptide to MHC inside the vaccinia virus-infected cell or before this
event. The Journal of Immunology, 1999, 163: 1914 –1922.
A
Centro Nacional de Biologı́a Fundamental, Instituto de Salud Carlos III, Madrid,
Spain
Received for publication December 7, 1998. Accepted for publication June 9, 1999.
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.
1
This work was supported by grants from the European Union, Dirección General de
Investigación Cientı́fica y Tecnológica, Comisión Interministerial de Ciencia y Tecnologı́a, and Comunidad de Madrid and by Ph.D. fellowships from Instituto de Salud
Carlos III and Comunidad de Madrid (to M.M.).
2
Current address: Edward Jenner Institute for Vaccine Research, Compton, Berkshire, U.K.
3
Address correspondence and reprint requests to Dr. Margarita Del Val, Centro Nacional de Biologı́a Fundamental, Instituto de Salud Carlos III, Ctra. Pozuelo, km 2,
E-28220 Majadahonda (Madrid), Spain. E-mail address: [email protected]
Abbreviations used in this paper: b2m, b2-microglobulin; b-gal, Escherichia coli
b-galactosidase; 9b-gal, 876 – 884 epitope (TPHPARIGL) from b-gal; ER, endoplasmic reticulum; rVV, recombinant vaccinia virus.
4
Copyright © 1999 by The American Association of Immunologists
the first studies with murine CMV showed an increase in mice
protection when the yield of antigenic peptides from vaccine constructs also increased (8). This and another report with three strains
of Listeria (10) suggest that the more Ag is expressed, the better
the recognition by CTL will be. On the other hand, other reports
indirectly suggest that other factors may limit the cell capacity to
present epitopes. Further studies with Listeria suggest that increased Ag processing does not result in better CTL induction in
vivo (11). Also, reports comparing the efficiency of peptide generation from either cytosolic or ER-targeted minigene products
with that from longer polypeptides show a remarkably higher efficiency of peptide production with the former constructs (12, 13),
suggesting a rate-limiting step either in the processing or in the
transport of antigenic peptides. Other studies report that substrate
ubiquitination or the proteasome activity are limiting for Ag presentation or MHC class I assembly, respectively, but only after
IFN-g stimulation (14, 15), a treatment that alters the basal balance
of the cellular components of the pathway by up-regulating several
of them (16, 17). Results with murine CMV show that IFN-g governs the yield of antigenic peptides also in vivo, demonstrating that
factors other than Ag synthesis limit the efficacy of Ag processing
(18). The last six reports (11–15, 18) thus suggest that the endogenous pathway of processing and presentation has a rate-limiting
step, in contrast with the first two reports mentioned above.
Our report contributes to the present knowledge by quantitatively studying the endogenous pathway of processing and presentation over a wide range of different amounts of starting Ag expressed in infected cells, thus aiming to reach saturation.
Recombinant vaccinia viruses (rVV) with point mutations in the
promoter were used to intracellularly express increasing amounts
of the model Ag b-galactosidase (b-gal) (19) that eventually can
enter the endogenous processing and presentation pathway. Because b-gal had the same sequence in all constructs tested (20), the
possible effect of flanking regions on the efficiency of processing
of the antigenic epitope (8, 9) was avoided. b-gal is proteolytically
cleaved by proteasomes, its TPHPARIGL (9b-gal) epitope binds
to the Ld allele (14, 21), and it is able to induce specific CTL when
BALB/c mice are immunized with an appropriate vector (22). The
MHC class I molecule Ld is characterized by its weak association
with b2m, its slow intracellular transport, and its low cell surface
expression (23). b2m plays a significant role in control of Ld expression (24, 25), but it is the processed peptide that induces the
proper folding and increases dramatically Ld surface expression
0022-1767/99/$02.00
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lthough CD81 CTL do not recognize intact proteins,
they interact with Ags on the APC surface as protein
fragments bound to MHC class I molecules. The antigenic peptides originate from proteins degraded mainly by the
multicatalytic complex proteasome in the cytosol (1) but also by
other proteases (2, 3). These peptides are transported by the MHCencoded transporter TAP to the endoplasmic reticulum (ER),4
where they may undergo proteolytic processing. Newly synthesized heavy chain/b2-microglobulin (b2m)/calreticulin complexes
associate with TAP and tapasin before peptide loading. Conformational changes in class I molecules associated with peptide
binding are postulated to result in their release from TAP (4).
Finally, the stable complex of heavy chain/b2m/peptide is transported through the Golgi apparatus to reach the cell surface where
it can interact with the TCR of the CTL (5, 6).
The capacity of MHC class I molecules to present epitopes is
linked, among other factors (7), to the efficient generation and
function of CTL. Little is known about what determines the quantity of pathogen-derived peptides presented by MHC class I molecules and the possible existence of any rate-limiting step in this
endogenous pathway of processing and presentation. Theoretically, there is a number of factors that may quantitatively affect the
outcome of this pathway. Some reports have indirectly addressed
this question, but their conclusions are only partial either because
few experimental points were tested, or because the possible effect
of regions flanking the epitope (8, 9) was not considered. One of
The Journal of Immunology
Materials and Methods
Cells, viruses, and infection conditions
P815 cells (H-2d) and their derivative P13.1, which are transfected with the
lacZ gene encoding b-gal (29), as well as the T2/Ld cells, which are transfected with the Ld gene (30), were maintained in Iscove’s modified Dulbecco’s medium with 10% FCS.
rVV from the vMJ series were provided by Dr. B. Moss (National
Institutes of Allergy and Infectious Diseases, Bethesda, MD). They express
varying amounts of b-gal due to the different point mutations introduced in
the early region of the VV early-late promoter 7.5 kDa (20). vSC8 is a rVV
with the b-gal gene under the control of the late promoter P11 (31), which
was used as a positive control in some experiments. As a negative control,
wild-type WR virus was used. rVV stocks were grown in CV1 cells in
DMEM supplemented with 10% FCS and consisted of clarified sonicated
extracts of pellets of infected cells. When used, purification involved centrifugation through a 36% sucrose cushion.
In all experiments described below, infection of P815 cells was performed as described (9), using a multiplicity of infection of 5–10 PFU/cell.
Infection of T2/Ld required a multiplicity of 20, as described (3, 32). After
1 h of adsorption, the virus inoculum was thoroughly washed; this was
taken as time 0 h postinfection. In all types of experiments, infection was
allowed to proceed for 7 h.
Protein analysis
For metabolic labeling, infected P815 cells were starved of methionine and
cysteine during the viral adsorption period and then labeled with 100
mCi/ml [35S]methionine 1 cysteine (Amersham, Arlington Heights, IL) in
medium free of both amino acids for the 7-h infection period. Proteins were
then resolved by 7.5% SDS-PAGE. Similar gels with unlabeled samples
were blotted to Immobilon (Millipore, Bedford, MA) and incubated with
rabbit Ab anti-b-gal (Cappel, Westchester, PA) at a 1/5,000 dilution and
goat anti-rabbit Ab labeled with horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AL), at 1/25,000. Bands were detected
with ECL (Amersham).
Quantitation of b-gal from rVV-infected P815 cells
For each rVV, lysates of pooled infected P815 cells and culture medium
were prepared. Quantitation of b-gal in serial dilutions of these lysates was
done by luminometry using the Luminescent b-Galactosidase Genetic Reporter System II (Clontech Laboratories, Palo Alto, CA) and measuring for
15 s in an Optocomp I luminometer (GEM Biomedical). Only values in
linear regions of the dose-response curve were taken for extrapolation on
the titration curve, which was prepared with serial dilutions of b-gal (Sigma, St. Louis, MO). Mean values and SEs of b-gal expressed in units were
transformed to molecules/cell by considering the m.w. and specific activity
of b-gal. Background resulting from enzyme present in the input virus was
measured on samples harvested immediately after the 1-h adsorption period. It had an average value of 14% of actual expression by each individual rVV after 7 h of infection. In sharp contrast, in some viruses (see
Results) this background was ;65%, so that we could not conclude that
there was significant evidence of expression by these rVV. This background, as well as the background detected in WR-infected cells (see legends to figures), were subtracted from each sample.
CTL cultures and cytotoxicity assays
Female 7-wk-old BALB/c (H-2d) mice were bred in our animal care facility
and were immunized by i.v. injection with 5 3 107 PFU of vMJ360 in
0.1– 0.2 ml PBS. Polyclonal b-gal-specific CTL lines were generated as
follows. At least 3 wk after immunization, 107 splenocytes/ml from immunized mice were restimulated in vitro with 105/ml mitomycin C-treated
P13.1 cells or with 1028 M 9b-gal peptide and cultured in a-MEM supplemented with 1% 2-ME and 10% FCS. IL-2, generously provided by
Hoffmann-LaRoche, was added after 5 days at a final concentration of 25
U/ml. Specificity of CTL cultures was tested starting 2 days later. Long
term CTL cultures were maintained in the presence of 100 U/ml IL-2 by
weekly restimulation with mitomycin C-treated P13.1 or with 1028 M 9bgal peptide and mitomycin C-treated splenocytes prepulsed for 20 min with
1025 M 9b-gal peptide. Following this protocol, no vaccinia-specific CTL
were ever selected. Occasionally, the b-gal-specific CTL line 0805B (22)
was used.
For cytolytic assays, after 4 h of infection, cells were pulsed with
Na51CrO4 for 90 min at 37°C in the presence or in the absence of exogenous peptide, washed, and combined with CTL. Total infection time until
the addition of CTL was 7 h. 51Cr release cytolytic assays were performed
for 3– 4 h as described (2, 33). When used, cycloheximide (Sigma) at 50
mg/ml, brefeldin A (Sigma) at 1 mg/ml, or lactacystin (E. J. Corey, Harvard
University, Cambridge, MA) at 200 mM was added after the adsorption
period, kept until targets were combined with CTL, and replaced by brefeldin A 1 mg/ml during the CTL assay itself.
Quantitation of naturally processed antigenic peptides from
rVV-infected P815 cells
P815 cells (5 3 108) were infected in parallel with different rVV, and 7 h
later cell extracts were prepared in parallel as described (3, 8). Reversed
phase HPLC was performed using a Smart equipment (Pharmacia, Piscataway, NJ) and a Sephasil C18-5 mm SC2.1/10 column and eluting with a
rather flat gradient of 12.95 to 28.7% CH3CN in 0.1% trifluoroacetic acid.
As internal standard in all HPLC runs, a b-gal-unrelated peptide was included. Fractions of 30 ml were collected and fully used to prepare triplicate serial dilutions that were added to 96-well plates at 4°C in medium
without FCS. Uninfected P815 cells that had been incubated overnight at
26°C to maximize expression of peptide-receptive Ld molecules (34) were
labeled with 51Cr at 26°C, washed, added to the fraction-containing wells,
and incubated for 15 min at room temperature in the presence of 2.5% FCS
before adding effector CTL. At this point, the resulting final dilution of the
fractions was at least 20-fold to avoid interference of solvents with the CTL
assay. The concentration of antigenic peptide recovered in the titrated
HPLC fractions was calculated by extrapolation from parallel titration
curves of purified synthetic peptide 9b-gal, synthesized, and sequenced in
Applied Biosystems (Foster City, CA) equipment.
Results
De novo synthesis of b-gal in rVV-infected P815 cells
rVV have been extensively used for both target formation and
induction of CTL and have been shown to present a large variety
of Ags both in vitro and in vivo (19). The vMJ series of rVV
expresses b-gal under the control of 7.5K-related promoters of
different strength, all of which drive the expression of the same
native b-gal gene (20). A set of 13 of these viruses that differ only
in point mutations in the early phase region of this promoter was
used in our study. Kinetics of expression from these promoters is
the same, and they only differ in the efficiency of transcription,
which results in varying amounts of expressed b-gal. Other viruses
that differ in the late phase part of this promoter were not used
because of possible interference by vaccinia late phase gene products with Ag presentation (35, 36). The widely used P815 cells
were chosen to express our model Ag b-gal, frequently used in
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(26). A weak interaction with peptides has been suggested from the
crystal structure of Ld (27). Indeed, the paucity of antigenic peptides able to bind to Ld is suggested as the main reason for retention of Ld molecules in the ER (26), in that there is a reservoir of
non-peptide-bound Ld molecules that can be specifically detected by
mAbs and that are retained in substantial amounts in the ER (28).
In this report, we have quantitatively compared the number of
molecules of synthesized protein expressed by a series of 13 rVV
with promoters of different strengths, with the number of molecules of processed antigenic peptide bound to MHC class I molecules at any place in the cell, and with surface recognition by CTL
of cells infected with each rVV. Our results show that with increasing amounts of starting protein, there was a rise in presentation to CTL, reaching a point above which saturation was revealed.
The same saturation point was found in the number of fully processed antigenic peptides bound to MHC class I molecules. The
limiting step was located in the binding of the antigenic peptide to
MHC or before this event, either in the processing or transport of
peptides or in the availability or avidity of MHC class I molecules.
The estimated maximum efficiency of the endogenous processing
and presentation pathway was 1/3900; i.e., one antigenic peptide
was produced of 3900 protein molecules, giving rise to a maximum of some 40 processed peptide molecules per infected cell.
1915
1916
SATURATION IN THE MHC CLASS I Ag-PROCESSING PATHWAY
FIGURE 1. De novo synthesis of b-gal in rVV-infected P815 cells, as
assessed by metabolic labeling with [35S]methionine and cysteine (A) or
Western blot with b-gal-specific Ab (B) of cells infected with the indicated
rVV. Per Western blot lane, 106 cells were loaded. The lane labeled 0 h pi
(0 h postinfection) indicates that the cells were harvested right after the 1-h
virus adsorption period. The m.w. markers (MWM) included b-gal.
Cells infected with the vMJ series express a wide range of b-gal
Quantitation of the antigenic protein b-gal in infected cells was
performed by enzymatic luminometry. Enzymatic colorimetry,
previously used to detect expression in infected CV1 cells (20),
was not sensitive enough for P815. The results are shown in Fig.
2. Evidence of significant expression above virus input background
FIGURE 3. Endogenous processing and presentation of newly synthesized b-gal to CTL. P815 cells were infected with vMJ360 (squares) or
control WR (triangles) in the presence (open symbols) or in the absence
(filled symbols) of cycloheximide (CH) (A), brefeldin A (BFA) (B), or
lactacystin (LC) (C) and tested in a cytotoxicity assay with b-gal-specific
CTL. In all cases, recognition by CTL of peptide-loaded target cells (circles) was unaffected by the inhibitor treatment. In D, untreated TAP2
T2/Ld cells were used instead.
was detected for P815 infected with vMJ337, with the value of
93,000 6 19,000 b-gal molecules/cell, and for all viruses expressing higher amounts. Four other viruses, including vMJ102 shown
in Fig. 1A, are not included in Fig. 2 for clarity, because they
expressed no detectable b-gal above the background defined by
WR. The level of b-gal expression ranged between the value of
vMJ337 and the highest in the vMJ series, vMJ356, with 1,400,000
molecules/cell; i.e., a spectrum encompassing at least a 15-fold
range of Ag expression. This wide and continuous range of expression of the starting protein allowed us later to determine the
quantitative influence of the Ag synthesized in the infected cell on
the outcome of the processing and presentation pathway.
Endogenous processing and presentation of newly synthesized
b-gal to CTL
FIGURE 2. Cells infected with the vMJ series express a wide range of
b-gal. P815 cells were infected with the indicated rVV, and the levels of
b-gal protein expression detected by enzymatic luminometry. Background
present in the input virus, as well as background expression by WR, which
was 96 6 24 molecules/cell, were subtracted from each individual sample.
The mean values and SEs are shown.
The endogenous nature of the b-gal processing and presentation
pathway to CTL was next characterized. Although little evidence
of protein expression or enzymatic activity was detected in cells in
which viral replication was not allowed to proceed (see above),
this point was further checked at the level of target cell formation.
Indeed, the former assays would not have detected minor amounts
in the input virus inoculum of already processed b-gal or of b-gal
that could be processed exogenously. These minor amounts might
nevertheless result in exogenous loading of surface MHC class I
molecules. To this end, CTL recognition of cells harvested immediately after the 1-h virus adsorption period was checked and
found to be negative (data not shown). Also, no differences were
found when purified virus inoculum was used, when the virus inoculum was extensively washed after adsorption, or when this procedure was performed at 4°C to prevent virus internalization (data
not shown). Altogether, these data indicated that the virus inoculum was not a source of peptide recognized by CTL. In addition,
treatment with the protein synthesis inhibitor cycloheximide after
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infectious and tumor systems (36), and to quantitatively study the
endogenous Ag processing and presentation pathway.
Vaccinia virus replication in P815 cells proceeded at a low level,
because expression of neither b-gal nor vaccinia virus proteins was
detected after metabolic labeling of infected P815, which essentially synthesized the same proteins as uninfected cells (Fig. 1A).
The more sensitive Western blot analysis allowed detection of
b-gal expression (Fig. 1B). This was all the result of new synthesis,
because no b-gal was detected in cells harvested immediately after
the 1-h virus adsorption period (lane 0 h pi). Thus, rVV were able
to direct the synthesis of new b-gal molecules in infected P815
cells, without having a measurable effect on host macromolecular
synthesis and without representing a massive load to the cell-biosynthetic machinery.
The Journal of Immunology
1917
ecule Ld (Fig. 3D), as compared with infected P815 cells. As a
positive control, these TAP2 T2/Ld cells were able to present to
specific MHC class I-restricted CTL the rVV-expressed CMVderived chimeric protein sC-A9A, which is endogenously synthesized but fully processed in the secretory pathway by an alternative
route (3) (data not shown). In conclusion, recognition of b-gal by
CTL in rVV-infected P815 cells required endogenous synthesis of
the Ag and traffic through the Golgi apparatus to the plasma membrane of the presenting complex and involved the two main components of the endogenous pathway, proteasomes and TAP
transporters.
CTL specifically recognize and discriminate between different
Ag quantities
adsorption significantly decreased target formation (Fig. 3A). Residual recognition might be caused by synthesis initiated from the
first virus particles entering the cell during the 1-h adsorption period. Collectively, these results indicated that de novo-synthesized
b-gal was the source for processing and presentation to CTL.
Next, infected cells were treated with brefeldin A, which blocks
traffic of newly synthesized MHC class I molecules from the ER to
the cell surface (37). Because this prevented CTL recognition (Fig.
3B), it was concluded that presentation of b-gal followed the endogenous route. Further, the involvement of two components of
the classical endogenous processing pathway was confirmed. First,
proteasomes were involved in b-gal proteolytic cleavage, because
treatment of infected cells with the proteasome-specific inhibitor
lactacystin (38) completely blocked recognition by CTL (Fig. 3C).
Second, no presentation was detected in T2/Ld mutant cells lacking
TAP but transfected with the gene expressing the presenting mol-
Recognition by CTL of P815 target cells infected with each of the
13 rVV from the vMJ series was assayed next. Average results
from three experiments are shown in Fig. 4. The additional four
rVV that gave b-gal expression levels indistinguishable from that
of WR were also negative in the CTL assay and are not included
in the figure for clarity. The 100% value corresponded to lysis of
wild-type-infected target cells incubated with excess exogenous
peptide at the end of the infection time, just before combination
with CTL. The fact that no vMJ-infected sample reached a normalized value of 100% demonstrated that the capacity of CTL to
recognize antigenic peptide at the infected cell surface was not
saturated, because they were able to recognize higher quantities of
antigenic peptide when it was added in excess. The first samples
that differed statistically from background were P815 infected with
vMJ195, vMJ179, or vMJ337, with values of ;7% normalized
lysis. In the t test, p was ,0.05 in each of the three separate
experiments, when compared with wild-type WR. CTL recognition was thus significantly more sensitive than enzymatic detection
of the protein. Half-maximal lysis among virus-infected cells was
marked by the vMJ172 and vMJ177 viruses. Fig. 4 also shows that
the CTL exhibited different degrees of recognition of the cells
infected with the different rVV, which implies that different quantities of antigenic peptides were exposed to CTL recognition and
that the CTL were able to discriminate between them within a
certain range.
Finally, when the number of synthesized b-gal molecules per
cell was compared with the specific recognition by CTL in each
infected sample (Table I), it was found that the increasing Ag
Table I. Summary of values determined for nine selected rVV of three different intermediate products or stages of the endogenous Ag-processing and
presentation pathway
Virus
b-gal Expression
(molecules/cell 3 1023)a
CTL Recognition
(% normalized lysis)b
Processed Peptide
(molecules/cell)c
WR
vMJ195
vMJ179
vMJ337
vMJ172
vMJ177
vMJ243
vMJ166
vMJ360
vMJ356
0 (18)e
0.5 6 0.10 (17)
0.75 6 0.16 (17)
93 6 19 (19)
0.14 6 0.04 (15)
7.8 6 1.9 (17)
140 6 20 (20)
490 6 48 (18)
660 6 120 (21)
1,400 6 130 (7)
0 6 1.3 (21)f
7 6 1.4 (21)
10 6 3.7 (21)
5 6 3 (21)
30 6 3.9 (21)
31 6 4.6 (21)
58 6 3.9 (21)
68 6 7.5 (21)
61 6 3.9 (21)
63 6 5.4 (21)
0 (4)e
Processing Efficiencyd
NDg
36 6 0.8 (2)
40 6 0 (2)
37 6 17 (5)
29 6 6 (5)
1/3,900
1/12,000
1/18,000
1/50,000
P815 cells were infected with the indicated rVV, and the levels of b-gal protein expression were detected by enzymatic luminometry. See legend to Fig. 2.
P815 cells were infected with the indicated rVV and tested in a standard CTL assay at different E:T ratios. See legend to Fig. 4.
P815 cells were infected with the indicated rVV, and whole cells were then subjected to acid extraction and HPLC separation. The number of 9b-gal antigenic peptide
molecules per cell was measured by titration with specific CTL, as described in Materials and Methods.
d
Ratio of processed peptide molecules to starting b-gal molecules.
e
Mean 6 SE (number of experimental data).
f
Mean 6 SEM (number of experimental data).
g
ND, not detected.
a
b
c
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FIGURE 4. CTL specifically recognize and discriminate between different Ag amounts. P815 cells were infected with the indicated rVV and
tested in a standard CTL assay at different E:T ratios. To compare the
results from three separate experiments, data were normalized as follows.
Only data from E:T ratios giving plateau specific lysis were taken for
further calculations. Specific lysis of WR-infected P815 cells to which an
excess of 1027 M synthetic 9b-gal was exogenously added, which was
85 6 4% lysis in average, was taken in each experiment as the 100%
normalized value. Specific lysis obtained on WR-infected P815, which was
4 6 1% lysis, was taken as the normalized 0% value. The mean values and
SEs of the mean are shown.
1918
SATURATION IN THE MHC CLASS I Ag-PROCESSING PATHWAY
recognition obtained with growing b-gal protein expression
reached a point where saturation in recognition by CTL occurred
(see also Fig. 6). In other words, within a range of differences in
Ag expression, up to vMJ243, CTL were able to respond with
increasing lysis. From here on, as few as 140,000 molecules of
synthesized protein in vMJ243-infected cells sufficed to give the
maximal CTL response, and up to 10-fold higher synthesis rates in
vMJ356 did not contribute to a better recognition of infected cells
at the cell surface. These data fitted very well with the standard
saturation curve (Fig. 6, curved line). Because the CTL were not
saturated, as discussed above, it was concluded that there was a
saturation point in the endogenous pathway of b-gal processing
and presentation and that this was located in the infected cell.
Isolation of naturally processed b-gal peptides from whole cells
FIGURE 5. Naturally processed b-gal peptides from whole cells. Recognition by b-gal-specific CTL of uninfected P815 cells incubated with the
indicated fractions of a representative set of reversed phase HPLC runs of
naturally processed peptides that had been acid extracted from the following samples. In all cases except in A, whole cells were extracted. A, Synthetic 9b-gal peptide, that was not acid-extracted (109 initial peptide molecules was tested per assay well (Œ), as well as two 2-fold serial dilutions
thereof); B, uninfected P815 cells (1.4 3 107 initial cell equivalents per
assay well); C, b-gal-transfected P13.1 cells (1.2 3 107); D, WR-infected
P815 cells (4.2 3 107); E, WR-infected P815 cells that had received synthetic 9b-gal just before acid extraction (4.2 3 107 initial cell equivalents
and 5 3 109 initial peptide molecules per assay well, corresponding to a
ratio of 120 peptide molecules per cell (Œ) and two 3-fold dilutions thereof); F, vMJ166-infected P815 cells (4.2 3 107 initial cell equivalents per
assay well, and a 2-fold dilution thereof); G, vMJ360-infected P815 cells
(4.2 3 107, and two 2-fold dilutions thereof); H, vMJ356-infected P815
cells (4.2 3 107, and a 2-fold dilution thereof). One fraction corresponds to
9 s of the HPLC run and an increase of 0.25% acetonitrile in the flat
gradient used. Only the few relevant fractions are depicted.
pected to contain less total peptide than vMJ243, because if they
were to have the same amount, one would reasonably expect them
to be recognized by CTL to the same extent as those viruses in the
saturation region. Attempts were made to quantify peptides from
samples not located in the saturation region of CTL recognition,
but they were unsuccessful, in spite of using all the material from
the HPLC runs for the assay.
Finally, data on whole cell natural peptide quantitation are plotted vs the number of synthesized b-gal molecules in Fig. 6. These
results indicated that the saturation point found at the vMJ243
virus by probing the cell surface with CTL was also found at the
level of intracellularly processed peptides. Also, the results confirmed that saturation occurred intracellularly, because several viruses that expressed increasing amounts of protein nevertheless
produced the same amount of processed peptide. Further, these
data suggested that the rate-limiting step was located in the binding
of peptide to the available MHC class I molecules or before this
event, as will be discussed below.
Efficiency of endogenous Ag processing
Once the values of extracted antigenic peptides were obtained, the
rate of Ag processing was determined by comparing the value of
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A likely hypothesis is that the saturation detected occurred intracellularly. Besides testing it, we also wanted to study whether or
not the rate-determining step was placed before or after the binding
of peptide to MHC class I in the ER. Isolation of the previously
described (21) b-gal antigenic peptide 9b-gal from infected cells
could help solve these questions. Such isolation is dependent on
9b-gal binding to the MHC class I molecule Ld (39). Particularly,
we wanted to know whether the saturation point defined by
vMJ243 at the target cell surface also applied when the naturally
processed peptides were quantified or whether a virus with higher
expression would now mark the saturation point. In the latter case,
intracellular saturation would be located after complex formation
between MHC and the fully processed peptide. Therefore, the biochemical analysis of endogenous 9b-gal was performed on
vMJ243 and viruses of higher expression.
First of all, specificity and yield of the peptide extraction and
HPLC procedures were tested. As shown in Fig. 5A, the synthetic
9b-gal peptide was resolved in our HPLC gradient system and
specifically recognized by CTL, whereas P815 cells did not generate any antigenic peptide in detectable amounts (Fig. 5B). In
contrast, an antigenic peak was detected from P13.1 cells, which
constitutively express, accumulate, and present b-gal. This peak
coeluted with the synthetic 9b-gal, suggesting that the endogenously generated peptide was indeed 9b-gal (Fig. 5C). A sample
of P815 infected with WR (Fig. 5D), to which a known amount of
synthetic peptide was added just before starting the biochemical
isolation of cellular peptides (Fig. 5E), was used to calculate the
efficiency of the experimental procedure. Recovery was 1.8% of
the starting antigenic activity. Care was taken to use an initial ratio
of synthetic peptide molecules to WR-infected cell of 120, close to
that actually found in vMJ-infected cells (see below), so as to
mimic closely the conditions met by the natural peptides. It was
thus also confirmed that the procedures did not alter the HPLC
behavior of 9b-gal.
Next, whole cell naturally processed b-gal peptides were isolated from vMJ-infected samples that were in the saturation region
of recognition by CTL. The results are shown in Fig. 5, F–H. The
antigenic peak from all infected samples coeluted with synthetic
9b-gal, within the range of experimental error. Pure synthetic peptides repeatedly injected in the HPLC gave a similar range of variation. Quantitation of 9b-gal in the samples was next performed by
serial dilution followed by extrapolation on a parallel titration
curve of synthetic peptide. Peptide recovery as measured earlier
was taken into account. Peptide yield was similar in all analyzed
samples with a maximum value of ;40 (average, 36 6 4) molecules of antigenic peptide per infected cell (Table I). The vMJ360
sample depicted in Fig. 5G showed the lowest repeatability. Cells
infected with viruses such as vMJ172 and vMJ177 would be ex-
The Journal of Immunology
steady state b-gal molecules with the number of antigenic peptides
obtained in each infected sample. Because peptide content from
viruses in the nonsaturated region of the curve was below our
detection limit, we could not accurately determine the maximum
efficiency of processing. Our closest estimation is to assume that
vMJ243 is just the turning point from the nonsaturated to the saturated segments of the curve. Based on this assumption, the total
efficiency of the endogenous processing pathway had an estimated
maximum value of 0.03% (Table I). In other words, the cell needed
3900 molecules of steady state starting Ag to generate 1 antigenic
peptide bound to MHC class I molecules. The efficiency in rVV
expressing more b-gal decreased accordingly, because the processing pathway was saturated.
In the nonsaturated segment of the saturation curve shown in
Fig. 6, one might expect a linear relationship between the amount
of 9b-gal peptide in infected cells, and the percentage specific lysis
by CTL of the same cells. Based on this assumption, the value of
recognition by CTL of P815 cells infected with vMJ172 or
vMJ177, which were the samples giving half-maximal lysis, was
extrapolated in the antigenic peptide scale. This extrapolation
showed that about 18 molecules of total antigenic peptide would
be needed for recognition by CTL of one-half of the cells. The
same approach, applied to vMJ195-, vMJ179-, or vMJ337-infected
P815, which were the first positive samples, suggested that ;4
molecules of total antigenic peptide in the infected cell would be
needed to trigger recognition by CTL. This is the first time that the
minimum quantity of total antigenic peptide in a VV-infected cell
needed to detect a CTL response is roughly estimated.
Discussion
We have studied quantitative aspects of the endogenous pathway
of Ag processing and presentation by MHC class I molecules to
CTL. By using rVV that expressed a wide range of amounts of the
model Ag b-gal in P815 cells, both the region of rising response
and the saturation of the pathway were covered. Our experimental
system allowed us to vary only the amount of starting Ag, leaving
other factors unaltered. Indeed, b-gal protein stability, intracellular
location, accessibility to the processing pathway, and the residues
flanking the 9b-gal epitope were all kept constant. Also, in contrast
to reports of other authors, no cytokines that elevate the normal
levels of some of the cellular components of the pathway were
used. We conclude that there is an intracellular rate-limiting step in
the endogenous processing pathway, located either at binding of
peptide to MHC or before this event, at the steps of processing or
transport to the ER. A maximum of ;40 processed 9b-gal peptide
molecules were bound to MHC per infected cell, giving an estimated maximal rate of processing of 1/3900 in this system.
Few studies have fully addressed quantitative aspects of the endogenous pathway of Ag presentation as a whole in living cells.
Although in an earlier study by us with two murine CMV antigenic
constructs (8) and in another report with three different expression
levels of a Listeria Ag (10), saturation of their presentation to CTL
was not reached, several other reports are compatible with a saturation of the pathway (11–15, 18). At present it is not clear
whether the different infectious agents used in all of these reports,
including ours, contribute themselves to potentiate or diminish the
saturation of the pathway. Saturation in our system was detected
by CTL at the infected cell surface and independently revealed to
occur intracellularly after biochemical purification of processed
peptides from whole infected cells. The fact that we now did find
saturation probably relates to the wider range of Ag expression
levels studied by us, but might also be explained by the different
systems used.
Location of the intracellular rate-limiting step
When varying amounts of a protein are synthesized in the cytosol,
a given fraction will be processed to yield peptides bound to MHC
class I molecules, following a dose-response curve that eventually
may reach saturation levels, as would any biological process. CTL
that recognize this processed peptide at the cell surface will follow
this response; i.e., the more peptide is processed, the more recognition and lysis will be detected. Theoretically, if transit of peptide/
MHC complexes from the ER to the plasma membrane were a
rate-limiting step, then CTL would reach their maximal recognition at protein expression levels lower than those that lead to saturation of the whole cell content of processed peptides, including
both intracellular and cell surface peptides. In other words, in this
scenario, even if more processed peptides were available intracellularly, they would not be presented to the CTL. On the contrary,
if traffic through the secretory pathway is not a rate-limiting step,
CTL recognition parallels the amount of processed peptide, and if
saturation is ever achieved, both reach saturation levels at the same
level of original antigenic protein, as we have found in our model.
Indeed, the levels of peptide molecules per infected cell for all
viruses in the CTL saturation area were the same. Therefore, we
conclude that the rate-limiting step is located either at binding of
MHC class I and peptide, or before this event, whereas complex
migration to the plasma membrane is not saturated.
Our data indicated that there was no limitation in peptide/MHC
class I complex traffic through the secretory pathway. A recent
report indicated that such a control step exists for Kb and Db molecules (40). It may well exist also for Ld as suggested from earlier
experiments (41) but, at least for b-gal in VV-infected cells, this
second control point would not be operative because saturation
occurs at a lower Ag level at an earlier step. Because the level of
processed peptides was saturated, the rate-limiting step was unequivocally located within the infected cell. Any cellular component involved in the pathway until or in the ER may contribute to
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
FIGURE 6. Intracellular saturation of the endogenous b-gal processing
pathway. The levels of normalized lysis by CTL of P815 cells infected with
the indicated rVV (Fig. 4 and Table I), on the left vertical axis (f), and the
number of whole cell naturally processed peptide molecules from each
rVV-infected sample (Table I), on the right ordinate (E), are plotted against
the measured number of b-gal molecules per infected cell (Fig. 2 and Table
I), on the abscissa. The solid horizontal line at 100% lysis represents the
normalized value of WR-infected P815 cells to which an excess of synthetic 9b-gal was exogenously added. Each symbol represents a given vMJ
virus-infected sample, as indicated. The curved line following the experimental data of the CTL recognition values (f]) represents the fit to a
rectangular hyperbole, the classical saturation curve.
1919
1920
SATURATION IN THE MHC CLASS I Ag-PROCESSING PATHWAY
Ld molecules in the ER (28), and addition of antigenic peptide can
induce proper folding and increase Ld surface expression (26).
Also, a lack of limitation in TAP and MHC availability is suggested from the experiments where very high numbers of peptides
are presented from minigene products (12).
Finally, in vivo experiments with murine CMV show that IFN-g
governs the yield of CMV peptides (18), implying that IFN-ginducible cellular components are rate limiting in Ag processing.
These include the three that we have just discussed, namely some
proteasome subunits, TAP, and MHC class I molecules (16, 17).
Our results are consistent with this idea because the limiting step
was located at the binding of antigenic peptide to MHC class I or
before this point, either in the proteolytic cleavage, transport, or
availability or avidity of MHC class I.
Efficiency of the endogenous Ag processing and presentation
pathway
We estimated that the maximum efficiency of Ag processing in our
system was 1/3900, i.e., that one correctly processed peptide molecule was produced of 3900 steady state b-gal protein molecules.
This low number may represent a low energetic cost for the cell
and thus allow that not only nonproperly folded proteins, as has
been suggested (55), but also functional proteins enter the endogenous processing pathway. In the fully different Listeria system,
efficiency was defined as the number of correctly processed
epitopes produced from the subset of protein molecules that are
degraded, and expectedly found to be higher, of 1/35 (10). In our
system, with the widely used and potent P815 targets, it was surprising that as few as 40 processed 9b-gal peptides were produced.
This number of peptide molecules bound to MHC per infected cell
lies at the lower end but is within the range that has been found for
other Ags in other systems, i.e., between 10 and 85,000 molecules/
cell (10, 12, 13, 56)., and even with this limited Ag expression and
peptide production, it was remarkable that saturation was reached.
This low number found in cell culture allows nevertheless efficient
induction of CTL in vivo. In contrast, in the Listeria system, constructs that produce at least 1000 molecules in vitro are needed in
vivo (11). Finally, our results suggest that triggering of our CTL
may require at least ;4 antigenic peptides in the whole cell, both
inside and at the cell surface, a result in agreement with the recently estimated minimum triggering number of 1 antigenic molecule presented by Ld at the cell surface (57).
We think that for different combinations of presenting cells (58),
Ags, expression vectors, MHC class I alleles, etc., slightly different
rate-limiting or saturation conditions may apply. However, our results probably truly reflect the situation in vaccinia virus-infected
cells in vivo and may thus probably apply for vaccine development. Indeed, the results are in agreement with data obtained with
two of these recombinant viruses in vivo, because vMJ360, a virus
in the saturation region, was found to confer better protection than
vMJ177, a virus giving half-maximal detection by CTL, when assayed in a tumor system using b-gal as surrogate Ag (36).
Also, given the moderate to high sensitivity to Ag sequence
differences of many cellular components of the processing pathway, it is likely that functionally saturating conditions may occur
with certain frequency. Thus, one implication of this findings is
that competition between Ags.5 is expected whenever there is a
5
D. López, Y. Samino, U. H. Koszinowski, and M. Del Val. Submitted for publication.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
the limitation. These include ubiquitination (14), proteasomes (15)
or other enzymes involved in proteolytic cleavage (2, 3), the TAP
complex, including calnexin, calreticulin, and tapasin (4), chaperonins gp96 and protein disulfide isomerase (42), b2m, and MHC
class I molecules themselves. Little can be said about other processing enzymes and tapasin, because they have been described
recently and little is known about their availability in cells. Protein
disulfide isomerase, gp96, and calreticulin have been shown to
bind peptides, although none of them yet with high selectivity (42,
43). On the other hand, gp96 and calnexin have been already
shown not to determine the rate of Ag presentation (44, 45), so that
the implication of all these molecules in limiting Ag presentation
can be taken as unlikely. On the contrary, it cannot be excluded
that b2m may be the limiting factor (46).
Proteasomes and TAP have been reported as being sequence
selective in that certain sequences in certain contexts make processing by proteasomes in vitro and in vivo (47, 48) and transport
by TAP (49) less efficient. That makes them good candidates as
cellular components that may prevent unlimited presentation of the
b-gal epitope. Because proteasome activity is very tightly regulated (1), it is not difficult to believe that it might represent a
bottleneck in the pathway, particularly when its primary function
is probably not Ag processing. Some articles support this idea, as
they show increased yields of MHC-bound peptides when proteolytic cleavage is by-passed with minigene constructs in rVV-infected cells (12, 13) or that ubiquitination and the proteasome activity
limit presentation (50) and the assembly of MHC class I molecules,
respectively, albeit only under IFN-g stimulation (14, 15).
On the other hand, TAP appears at first sight not to be quantitatively limiting. Thus, the amount of peptides that TAP can transport to the ER is well above the numbers that can be bound by
MHC class I molecules (51), and the recovery of peptides bound
to MHC class I from constructs with and without signal sequence
was the same (12). However, TAP shows a particular difficulty in
transporting peptides with proline in the second position (49). Such
peptides, which include the 9b-gal epitope, TPHPARIGL, happen
to be those with highest affinity for Ld, the anchor residues of
which are proline at position 2 and a hydrophobic amino acid at
position 9 (52). Thus, TAP might indeed be functionally rate limiting for transporting peptides for Ld. This is in line with speculations as to why unfolded Ld molecules accumulate in the ER (28),
an accumulation that can be overcome with an adequate excess supply
of peptides (26).
Whether MHC class I molecules can represent a rate-limiting
step in Ag processing and presentation is under some controversy.
Several findings favor this possibility. In some tumor systems, increased elimination in vivo is correlated with increased MHC class
I levels (53). Also, TAP can transport to the ER more peptides than
MHC can bind (51). Additionally, MHC class I displays a broad
difference in affinities to peptides (54). Finally, the Ld molecule
exhibits some peculiarities. Its recently published crystallographic
structure reveals an unusually weak interaction with b2m and peptide (27), which suggests that its affinity to peptides could be relatively low, and even in a situation of high antigenic peptide concentration in the ER, the interaction would be poorly efficient, and
therefore Ld could be functionally rate limiting. Because this is the
first quantitative report on endogenous levels of an Ld-presented
peptide, further studies with other Ld-restricted epitopes are
needed to shed light on the issue of whether the limiting step and
low endogenous numbers found for the b-gal epitope are a property of Ld or of the 9-bgal/Ld complex, such as a putative low
binding affinity.
On the contrary, some pieces of evidence discard the MHC as
the limiting factor. First, there is a reservoir of non-peptide-bound
The Journal of Immunology
shortage of any given component of the pathway. Another important point is that our studies can propose ways to overcome this
deficit in presentation. It is very possible that codelivering or coexpressing cytokines such as IFN-g with different types of vaccines may improve their efficacy, because in vivo IFN-g can enhance both presentation by infected cells and the function of
professional APC (18) by up-regulating the steps that we have
identified to be rate limiting. Our results also open the way to
characterize in more detail the precise step that is rate determining
for the whole pathway, and thus to the development of more specifically targeted strategies for vaccine improvement.
Acknowledgments
We thank Dr. M. J. Bevan for the P13.1 cells, Dr. H.-G. Rammensee for the
0805B CTL cell line, Dr. B. Moss for rVV, Dr. P. Cresswell for the T2/Ld
cells, A. R. Castaño and D. López for critically reading the manuscript, and
B. Gómez and F. Vélez for technical assistance. We thank Hoffmann-La
Roche for the generous gift of IL-2.
1. Groettrup, M., A. Soza, U. Kuckelkorn, and P. M. Kloetzel. 1996. Peptide antigen
production by the proteasome: complexity provides efficiency. Immunol. Today
17:429.
2. Lopez, D., and M. Del Val. 1997. Selective involvement of proteasomes and
cysteine proteases in MHC class I antigen presentation. J. Immunol. 159:5769.
3. Gil-Torregrosa, B., A. R. Castano, and M. Del Val. 1998. MHC class I viral
antigen processing in the secretory pathway defined by the trans-Golgi network
protease furin. J. Exp. Med. 188:1.
4. Elliott, T. 1997. How does TAP associate with MHC class I molecules? Immunol.
Today 18:375.
5. York, I. A., and K. L. Rock. 1996. Antigen processing and presentation by the
class I major histocompatibility complex. Annu. Rev. Immunol. 14:369.
6. Koopmann, J. O., G. J. Hammerling, and F. Momburg. 1997. Generation, intracellular transport and loading of peptides associated with MHC class I molecules.
Curr. Opin. Immunol. 9:80.
7. Iezzi, G., K. Karjalainen, and A. Lanzavecchia. 1998. The duration of antigenic
stimulation determines the fate of naive and effector T cells. Immunity 8:89.
8. Del Val, M., H. J. Schlicht, T. Ruppert, M. J. Reddehase, and U. H. Koszinowski.
1991. Efficient processing of an antigenic sequence for presentation by MHC
class I molecules depends on its neighboring residues in the protein. Cell 66:
1145.
9. Eisenlohr, L. C., J. W. Yewdell, and J. R. Bennink. 1992. Flanking sequences
influence the presentation of an endogenously synthesized peptide to cytotoxic T
lymphocytes. J. Exp. Med 175:481.
10. Villanueva, M. S., P. Fischer, K. Feen, and E. G. Pamer. 1994. Efficiency of MHC
class I antigen processing: a quantitative analysis. Immunity 1:479.
11. Vijh, S., I. M. Pilip, and E. G. Pamer. 1998. Effect of antigen-processing efficiency on in vivo T cell response magnitudes. J. Immunol. 160:3971.
12. Anton, L. C., J. W. Yewdell, and J. R. Bennink. 1997. MHC class I-associated
peptides produced from endogenous gene products with vastly different efficiencies. J. Immunol. 158:2535.
13. Porgador, A., J. W. Yewdell, Y. P. Deng, J. R. Bennink, and R. N. Germain.
1997. Localization, quantitation, and in situ detection of specific peptide MHC
class I complexes using a monoclonal antibody. Immunity 6:715.
14. Grant, E. P., M. T. Michalek, A. L. Goldberg, and K. L. Rock. 1995. Rate of
antigen degradation by the ubiquitin-proteasome pathway influences MHC class
I presentation. J. Immunol. 155:3750.
15. Benham, A. M., and J. J. Neefjes. 1997. Proteasome activity limits the assembly
of MHC class I molecules after IFN-g stimulation. J. Immunol. 159:5896.
16. White, L. C., K. L. Wright, N. J. Felix, H. Ruffner, L. F. L. Reis, R. Pine, Ting,
and JPY. 1996. Regulation of LMP2 and TAP1 genes by IRF-1 explains the
paucity of CD8(1) T cells in IRF-1(2/ 2) mice. Immunity 5:365.
17. David-Watine, B., A. Israel, and P. Kourilsky. 1990. The regulation and expression of MHC class I genes. Immunol. Today 11:286.
18. Geginat, G., T. Ruppert, H. Hengel, R. Holtappels, and U. H. Koszinowski. 1997.
IFN-g is a prerequisite for optimal antigen processing of viral peptides in vivo.
J. Immunol. 158:3303.
19. Moss, B. 1996. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc. Natl. Acad. Sci. USA 93:11341.
20. Davison, A. J., and B. Moss. 1989. Structure of vaccinia virus early promoters.
J. Mol. Biol. 210:749.
21. Dick, L. R., C. Aldrich, S. C. Jameson, C. R. Moomaw, B. C. Pramanik,
C. K. Doyle, G. N. DeMartino, M. J. Bevan, J. M. Forman, and C. A. Slaughter.
1994. Proteolytic processing of ovalbumin and b-galactosidase by the proteasome to yield antigenic peptides. J. Immunol. 152:3884.
22. Rammensee, H. G., H. Schild, and U. Theopold. 1989. Protein-specific cytotoxic
T lymphocytes. Recognition of transfectants expressing intracellular, membraneassociated or secreted forms of b-galactosidase. Immunogenetics 30:296.
23. Beck, J. C., T. H. Hansen, S. E. Cullen, and D. R. Lee. 1986. Slower processing,
weaker b2-M association, and lower surface expression of H-2Ld are influenced
by its amino terminus. J. Immunol. 137:916.
24. Smith, J. D., N. B. Myers, J. Gorka, and T. H. Hansen. 1993. Model for the in
vivo assembly of nascent Ld class-I molecules and for the expression of unfolded
Ld molecules at the cell surface. J Exp. Med 178:2035.
25. Cook, J. R., J. C. Solheim, J. M. Connolly, and T. H. Hansen. 1995. Induction of
peptide-specific CD81 CTL clones in b2-microglobulin-deficient mice. J. Immunol. 154:47.
26. Lie, W. R., N. B. Myers, J. Gorka, R. J. Rubocki, J. M. Connolly, and
T. H. Hansen. 1990. Peptide ligand-induced conformation and surface expression
of the Ld class I MHC molecule. Nature 344:439.
27. Balendiran, G. K., J. C. Solheim, A. C. Young, T. H. Hansen, S. G. Nathenson,
and J. C. Sacchettini. 1997. The three-dimensional structure of an H-2Ld-peptide
complex explains the unique interaction of Ld with b2 microglobulin and peptide.
Proc. Natl. Acad. Sci. USA 94:6880.
28. Lie, W. R., N. B. Myers, J. M. Connolly, J. Gorka, D. R. Lee, and T. H. Hansen.
1991. The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure. J. Exp. Med. 173:
449.
29. Carbone, F. R., and M. J. Bevan. 1990. Class I-restricted processing and presentation of exogenous cell-associated antigen in vivo. J Exp. Med. 171:377.
30. Crumpacker, D. B., J. Alexander, P. Cresswell, and V. H. Engelhard. 1992. Role
of endogenous peptides in murine allogenic cytotoxic T cell responses assessed
using transfectants of the antigen-processing mutant 174xCEM. T2. J. Immunol.
148:3004.
31. Chakrabarti, S., K. Brechling, and B. Moss. 1985. Vaccinia virus expression
vector: coexpression of b-galactosidase provides visual screening of recombinant
virus plaques. Mol. Cell. Biol. 5:3403.
32. Eisenlohr, L. C., I. Bacik, J. R. Bennink, K. Bernstein, and J. W. Yewdell. 1992.
Expression of a membrane protease enhances presentation of endogenous antigens to MHC class I-restricted T lymphocytes. Cell 71:963.
33. Del Val, M., H. Volkmer, J. B. Rothbard, S. Jonjic, M. Messerle, J. Schickedanz,
M. J. Reddehase, and U. H. Koszinowski. 1988. Molecular basis of cytolytic
T-lymphocyte recognition of the murine cytomegalovirus immediate-early protein pp89. J. Virol. 62:3965.
34. Ljunggren, H. G., N. J. Stam, C. Ohlen, J. J. Neefjes, P. Hoglund, M. T. Heemels,
J. Bastin, T. N. Schumacher, A. Townsend, K. Karre, et al. 1990. Empty MHC
class I molecules come out in the cold. Nature 346:476.
35. Townsend, A., J. Bastin, K. Gould, G. Brownlee, M. Andrew, B. Coupar,
D. Boyle, S. Chan, and G. Smith. 1988. Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen. J. Exp. Med. 168:1211.
36. Bronte, V., M. W. Carroll, T. J. Goletz, M. Wang, W. W. Overwijk, F. Marincola,
S. A. Rosenberg, B. Moss, and N. P. Restifo. 1997. Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant
poxvirus tumor vaccine. Proc. Natl. Acad. Sci. USA 94:3183.
37. Yewdell, J. W., and J. R. Bennink. 1989. Brefeldin A specifically inhibits presentation of protein antigens to cytotoxic T lymphocytes. Science 244:1072.
38. Fenteany, G., R. F. Standaert, W. S. Lane, S. Choi, E. J. Corey, and
S. L. Schreiber. 1995. Inhibition of proteasome activities and subunit-specific
amino-terminal threonine modification by lactacystin. Science 268:726.
39. Rotzschke, O., K. Falk, H. J. Wallny, S. Faath, and H.-G. Rammensee. 1990.
Characterization of naturally occurring minor histocompatibility peptides including H-4 and H-Y. Science 249:283.
40. Joyce, S. 1997. Traffic control of completely assembled MHC class I molecules
beyond the endoplasmic reticulum. J. Mol. Biol. 266:993.
41. Weis, J. H., and J. G. Seidman. 1985. The expression of major histocompatibility
antigens under metallothionein gene promoter control. J. Immunol. 134:1999.
42. Schild, H., D. Arnold-Schild, E. Lammert, and H.-G. Rammensee 1999. Stress
proteins and immunity mediated by cytotoxic T lymphocytes. Curr. Op in Immunol. 11-109.
43. Arnold, D., C. Wahl, S. Faath, H. G. Rammensee, and H. Schild. 1997. Influences
of transporter associated with antigen processing (TAP) on the repertoire of peptides associated with the endoplasmic reticulum- resident stress protein gp96.
J. Exp. Med. 186:461.
44. Lammert, E., D. Arnold, H. G. Rammensee, and H. Schild. 1996. Expression
levels of stress protein gp96 are not limiting for major histocompatibility complex
class I-restricted antigen presentation. Eur. J. Immunol. 26:875.
45. Prasad, S. A., J. W. Yewdell, A. Porgador, B. Sadasivan, P. Cresswell and J. R.
Bennink, 1998. Calnexin expression does not enhance the generation of MHC
class I-peptide complexes. Eur. J. Immunol. 28:907.
46. Myers, N. B., E. M. Wormstall, and T. H. Hansen. 1996. Differences among
various class I molecules in competition for b2m in vivo. Immunogenetics 43:
384.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
References
1921
1922
SATURATION IN THE MHC CLASS I Ag-PROCESSING PATHWAY
47. Niedermann, G., S. Butz, H. G. Ihlenfeldt, R. Grimm, M. Lucchiari,
H. Hoschutzky, G. Jung, B. Maier, and K. Eichmann. 1995. Contribution of
proteasome-mediated proteolysis to the hierarchy of epitopes presented by major
histocompatibility complex class 1 molecules. Immunity 2:289.
48. Eggers, M., B. Boesfabian, T. Ruppert, P. M. Kloetzel, and U. H. Koszinowski.
1995. The cleavage preference of the proteasome governs the yield of antigenic
peptides. J Exp. Med. 182:1865.
49. Neefjes, J., E. Gottfried, J. Roelse, M. Gromme, R. Obst, G. J. Hammerling, and
F. Momburg. 1995. Analysis of the fine specificity of rat, mouse and human TAP
peptide transporters. Eur. J. Immunol. 25:1133.
50. Wu, Y., and T. J. Kipps. 1997. Deoxyribonucleic acid vaccines encoding antigens
with rapid proteasome-dependent degradation are highly efficient inducers of cytolytic T lymphocytes. J. Immunol. 159:6037.
51. Momburg, F., J. J. Neefjes, and G. J. Hammerling. 1994. Peptide selection by
MHC-encoded TAP transporters. Curr. Opin. Immunol. 6:32.
52. Corr, M., L. F. Boyd, S. R. Frankel, S. Kozlowski, E. A. Padlan, and
D. H. Margulies. 1992. Endogenous peptides of a soluble major histocompatibility complex class I molecule, H-2Lds: sequence motif, quantitative binding,
and molecular modeling of the complex. J. Exp. Med. 176:1681.
53. Rivoltini, L., K. C. Barracchini, V. Viggiano, Y. Kawakami, A. Smith, A. Mixon,
N. P. Restifo, S. L. Topalian, T. B. Simonis, S. A. Rosenberg, and
F. M. Marincola. 1995. Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes. Cancer Res. 55:3149.
54. Luescher, I. F., P. Romero, J. C. Cerottini, and J. L. Maryanski. 1991. Specific
binding of antigenic peptides to cell-associated MHC class I molecules. Nature
351:72.
55. Yewdell, J. W., L. C. Anton, and J. R. Bennink. 1996. Defective ribosomal
products (DRiPs): a major source of antigenic peptides for MHC class I molecules? J. Immunol. 157:1823.
56. Malarkannan, S., F. Gonzalez, V. Nguyen, G. Adair, and N. Shastri. 1996. Alloreactive CD8(1) T cells can recognize unusual, rare, and unique processed
peptide/MHC complexes. J. Immunol. 157:4464.
57. Sykulev, Y., M. Joo, I. Vturina, T. J. Tsomides, and H. N. Eisen. 1996. Evidence
that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell
response. Immunity 4:565.
58. Butz, E. A., and M. J. Bevan. 1998. Differential presentation of the same MHC
class I epitopes by fibroblasts and dendritic cells. J. Immunol. 160:2139.
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