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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 952-962
Shaping the Repertoire of Cytotoxic T-Lymphocyte Responses:
Explanation for the Immunodominance Effect Whereby Cytotoxic T
Lymphocytes Specific for Immunodominant Antigens Prevent
Recognition of Nondominant Antigens
By
Stéphane Pion,
Gregory J. Christianson,
Pierre Fontaine,
Derry C. Roopenian, and
Claude Perreault
From the Research Center, Maisonneuve-Rosemont Hospital, Montreal,
Quebec, Canada; and The Jackson Laboratory, Bar Harbor, ME.
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ABSTRACT |
The immunodominance effect, whereby the presence of immunodominant
epitopes prevents recognition of nondominant determinants presented on
the same antigen-presenting cell (APC) considerably restricts the
repertoire of cytotoxic T lymphocyte (CTL) responses. To elucidate the
molecular basis of the immunodominance effect, we compared the
interactions of a dominant (B6dom1) and a nondominant
epitope (H-Y) with their restricting class I molecule
(H2-Db), and their ability to trigger cognate CTLs. We
found that B6dom1/Db complexes behaved as
optimal T-cell receptor (TCR) ligands and triggered a more rapid in
vivo expansion of cognate CTLs than H-Y/Db complexes. The
superiority of the dominant epitope was explained by its high cell
surface density (1,012 copies/cell for B6dom1 v 10 copies/cell for H-Y) and its optimal affinity for cognate TCRs. Based
on these results, we conclude that dominant class I-associated
epitopes are those that have optimal ability to trigger TCR signals in
CTLs. We propose that the rapid expansion of CTLs specific for dominant
antigens should enable them to compete more successfully than other
CTLs for occupancy of the APC surface.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
WHEN CONFRONTED with numerous peptides
presented in the context of self-major histocompatibility complex
(MHC), T cells usually respond only to a limited number of peptide
epitopes.1,2 The immunogenicity of such peptides is
determined by four parameters: (1) the rate of processing (proteolysis
and transport) of peptide epitopes, (2) their MHC binding affinity, (3)
the presence of specific T-cell precursors, and (4) the suppression of
T-cell responses to nondominant peptides by dominant epitopes
(immunodominance effect).3-15 The immunodominance effect
considerably restricts the repertoire of T-cell effectors. Indeed,
several peptides that are adequately presented on antigen-presenting
cells (APCs) are said to be nondominant because they elicit T-cell
responses when they are presented alone, but are neglected when they
are presented in conjunction with other peptides. Immunodominance has
been found to regulate cytotoxic T lymphocyte (CTL) responses to
viruses,16,17 bacteria,18 tumor
antigens,19-21 and minor histocompatibility antigens
(MiHAs).22-30 Based on detailed studies of in
vivo and in vitro CTL responses toward MiHAs, it appears that only a
very small proportion of epitopes, probably less than 10%, are
dominant.22-28 A priori, immunodominance may not appear
advantageous, as it signifies that by focusing exclusively on one or a
few epitopes, the immune system places all its eggs in the same (or a
few) basket(s). Theoretically, this should increase the risk that
pathogens or tumor cells can escape from
immunosurveillance.31 Therefore, it is of great importance
to decipher the rules that govern immunodominance to understand why the
repertoire of T-cell responses is restricted to only a few determinants
when confronted with numerous immunogenic epitopes and what are the
implications of this restriction.
When immunized against C57BL/6 cells, C3H.SW mice recognize a small set
of class I-associated dominant MiHAs collectively called
B6dom.32,33 To investigate the mechanisms of
dominance, we compared immune responses towards B6dom and
H-Y antigens because, in H2b mice, these MiHAs lie at
opposite ends on the dominance scale. Indeed, H-Y is always nondominant
when presented with one or numerous autosomal MiHAs, whereas
B6dom are dominant antigens when presented with other
MiHAs.32,34 Thus, when C3H.SW female mice were primed with
C57BL/6 male cells, they generated anti-B6dom CTLs, but
failed to respond to H-Y even though (1) C3H.SW female mice could
respond to H-Y when it was presented alone (on C3H.SW male cells), and
(2) C57BL/6 male cells express H-Y MiHA.32 B6dom did not suppress anti-H-Y responses by acting as a
T-cell receptor (TCR) antagonist for anti-H-Y CTLs because
B6dom and H-Y are not cross-reactive at the TCR level.
Importantly, dominance was not seen when mice (C3H.SW female) were
primed with the dominant and nondominant antigens on separate APCs (ie,
a mixture of C3H.SW male cells + C57BL/6 female
cells).32 These results indicated that dominance results
from competition for the APC surface between anti-B6dom and
anti-H-Y CTLs.2
Taking advantage of the recent elucidation of the sequence of the H-Y
and B6dom1 epitopes,33,35 both presented by
H2-Db, we evaluated the molecular basis of the
immunodominance effect. The results presented herein show that compared
with H2-Db/H-Y complexes,
H2-Db/B6dom1 complexes are more abundant at the
surface of APCs, likely interact with optimal affinity with their
specific TCR, and trigger a more rapid expansion of cognate CTLs.
Coupled with previous work,32 these findings provide strong
evidence that because of their rapid expansion, CTLs directed towards
immunodominant antigens occupy the surface of APCs and thereby prevent
the interaction of antinondominant CTLs with these same APCs. In this
way, CTL responses are focused on those antigens which are the most
effective at triggering T-cell activation.
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MATERIALS AND METHODS |
Mice.
Mice from the Jackson Laboratory (Bar Harbor, ME) were used between 6 and 16 weeks of age. They were maintained on acidified drinking water
and under normal housing conditions according to the standards of the
Canadian Committee for Animal Protection.
Cell lines.
The T2Db cell line (kindly provided by Dr P. Cresswell,
Yale University, New Haven, CT) was created by transfection of T2 cells with the H2-Db gene.36 T2 is a human hybrid
lymphoblastoid cell line with a large deletion in the MHC class II
region, including genes for the TAP1/2 and LMP2/7 products. RMA-S
(H2b) is a TAP2 mutant cell line expressing very low
surface amounts of MHC class I molecules. SW10/B, an established
AAPDNRETF-specific CD8+ cell line was generated by priming
intraperitoneally (IP) C3H.SW mice with C57BL/6 spleen cells followed
by four cycles of in vitro restimulation of effector cells with C3H.SW
cells coated with synthetic AAPDNRETF peptide (Christianson GJ, Pion S,
Roopenian DC, Perreault C, unpublished observation, October 1997).
Synthetic peptides.
Peptides were synthesized by Chiron Technologies (Victoria, Australia).
AAPDNRETF (B6dom1) is an H2-Db-restricted
dominant MiHA present on the surface of C57BL/6 spleen cells.33 WMHHNMDLI (H-Y) is a male specific
H2-Db-restricted MiHA derived from the UTY
protein.35 Purity, as determined by reversed
phase-high-performance liquid chromatography (HPLC), was above 97%
for both synthetic peptides.
Extraction and HPLC fractionation of natural MiHA peptides.
Natural MiHA peptides were eluted from C57BL/6 male cells by acid
extraction in the presence of protease inhibitors (25 mmol/L iodoacetamide, 1 mmol/L aprotinine, 1 mmol/L phenylmethyl sulfonyl fluoride [PMSF]).32,37 Thus, 2 × 108
cells were suspended in 5 mL of citrate-phosphate buffer (0.131 mol/L
citric acid/0.066 mol/L Na2HPO4, pH 3.3) for 1 minute at room temperature and centrifuged. Under these experimental
conditions (pH < 3.4), the efficiency of peptide extraction is
 95%.37 After a prepurification on a C18 Sep-Pak column
(Waters, Milford, MA), eluates were fractionationated on an HPLC system
using a Superpac Pep-S C18 column (5 µm, 4 × 250 mm, Pharmacia,
Uppsala, Sweden).32 Solvents used were 99.9% water/0.1%
trifluoro-acetic acid (solvent A) and 99.9% acetonitrile/0.1%
trifluoro-acetic acid (solvent B). The gradient consisted of the
following linear step intervals: 0% B (0 to 5 minutes), up to 20% B
at 10 minutes, up to 55% B at 55 minutes, plateau at 55% B from 55 to
60 minutes, and up to 100% B at 70 minutes. Flow rate was 1 mL/min,
and 1-mL fractions were collected and lyophilized.
CTL assays.
Standard 51Cr-release assays were performed as previously
described.32,38,39 Analysis of the expression of
B6dom1 by various strains of H2b mice and
quantification of natural AAPDNRETF epitopes expressed by C57BL/6 and
129 cells were performed with SW10/B effectors (see Fig 3). In all
other experiments, effectors were polyclonal CTLs obtained after a
single in vivo priming followed by a single in vitro mixed leukocyte
culture (MLC).32 Target cells were either concanavalin A
(Con A) blasts or T2Db cells that were either
untreated or preincubated with an exogenous source of peptides in the
form of HPLC fractions or synthetic peptides. In peptide sensitization
assays, target cells were incubated at 26°C overnight, then
sensitized with synthetic peptides or HPLC fractions for 90 minutes
before being used in CTL assays. Effectors were incubated with targets
for 3 hours in standard CTL assays and for 4 hours in peptide
sensitization assays. All tests were performed in triplicate.
Spontaneous release values varied from 5% to 20% of total release.
Interactions between H2-Db and MiHA peptides.
The binding affinity of WMHHNMDLI and AAPDNRETF for the Db
molecule was evaluated by fluorescence-activated cell sorting (FACS) analysis with anti-H2-Db antibody (Ab) (cat
no. 06114D; Cedarlane, Hornby, Canada) using T2Db and RMA-S cells, as previously
described.32 The anti-H2-Db Ab used recognizes
an epitope whose expression is not modified by the nature of the
specific peptide bound to Db molecules.40
Briefly, T2Db and RMA-S cells were sensitized with
different concentrations of synthetic peptides to stabilize surface
H2-Db expression. Afterwards, cells were stained with
anti-H2-Db Ab, washed, and analyzed by FACS. To evaluate
the half-life of H2-Db/peptide complexes, T2Db
cells were incubated for 12 hours in the presence of B6dom1
(10 7 mol/L) or H-Y (105 mol/L)
synthetic peptides at 26°C. Brefeldin A (10 µg/mL) (Sigma, Oakville, Canada) was added to the culture for the last 2.5 hours to
prevent the appearance of newly synthesized class I molecules. Cells
were washed five times, resuspended in Brefeldin A supplemented medium,
and transferred to a 37°C incubator. Then, H2-Db
expression was evaluated by flow cytometry at different time points.
Mean fluorescence intensity (MFI) was evaluated with the MFI software
program (E. Martz, University of Massachusetts, Amherst). Corrected MFI = MFIexp  MFImin, where
MFIexp is the fluorescence intensity of cells incubated
with one of the synthetic peptides and MFImin is the
fluorescence intensity of cells that had been pulsed with medium alone
before labeling with the anti-H2-Db Ab.
Limiting dilution analysis (LDA) of CTL precursor frequencies.
Limiting numbers of responder spleen cells from C3H.SW female mice
primed either with C57BL/6 female (B6dom1+) or C3H.SW male
(H-Y+) splenocytes (20 × 106 cells
injected IP) were restimulated in vitro on days 5, 10, 15, or 20 postimmunization with 3 × 105 irradiated stimulator
cells in the presence of 2.5 or 20 U/mL of interleukin-2 (IL-2). After
9 days, cultures were evaluated in a standard 4-hour 51Cr
release assay. Wells were scored positive if 51Cr release
exceeded the spontaneous release by more than 3 SD. The frequency of
CTL precursors was determined by the  2 minimization procedure.
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RESULTS |
Relative affinities of AAPDNRETF (B6dom1) and
WMHHNMDLI (H-Y) for Db.
To evaluate the binding affinity for Db molecules, we
compared the ability of synthetic B6dom1 and H-Y peptides
to upregulate Db expression on T2Db and RMA-S
cells. As they are deficient in TAP1/2 and LMP2/7 proteins (T2Db) or TAP2 (RMA-S), these cells express only low levels
of unstable Db molecules on the cell surface.36
However, when exogenous Db-binding peptides are added, they
stabilize "empty" Db molecules, thereby causing an
increased cell surface expression of this class I molecule that can be
measured by flow cytometry. Studies involving a large variety of
peptides have demonstrated that MHC stabilization assays can be taken
as a measure of the relative peptide affinity.4,5,7,13
Thus, both types of cells were incubated with titrated amounts of H-Y
and B6dom1 peptides and stained with an anti-Db
Ab. In three experiments with T2Db cells, the concentration
of H-Y required for half-maximal Db upregulation was one to
two logs greater than that of B6dom1. In the representative
example depicted in Fig 1B, the peptide concentrations required for similar Db expression were 2 × 106 mol/L for B6dom1 and
104 mol/L for H-Y. Similar results were observed
using RMA-S cells (Fig 1D). In agreement with these findings,
B6dom1 was previously shown to have a very high binding
affinity for Db, even superior to that of the
immunodominant Db-associated epitope of influenza
nucleoprotein (ASNENMDAM).32,41,42
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| Fig 1.
Relative affinities of B6dom1 (AAPDNRETF) and
H-Y (WMHHNMDLI) for Db. T2Db (A and B) and
RMA-S cells (C and D), precultured at 26°C, were incubated in
serum-free medium with various concentrations of synthetic
B6dom1 or H-Y peptide, stained for Db surface
expression, and analyzed by flow cytometry. (A) H2-Db
surface expression of T2Db cells incubated alone (dashed
line), with 104 mol/L H-Y (solid black line, grey
background), or with 104 mol/L B6dom1 (solid
black line). (B) Corrected MFI of T2Db cells stained with
anti-Db Ab after incubation with graded concentrations of
peptides. (C) H2-Db surface expression of RMA-S cells
incubated alone (dashed line), with 10-4 mol/L H-Y (solid
black line, grey background), or with 104 mol/L
B6dom1 (solid black line). (D) Corrected MFI of RMA-S cells
stained with anti-Db Ab after incubation with graded
concentrations of peptides. One representative experiment of two is
shown.
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Stability of Db/peptide complexes.
T2Db cells, precultured overnight at 26°C, were
incubated with B6dom1 or H-Y peptide. Peptides were used at
different molar concentrations selected to obtain an MFI of 15.
Under these conditions, the level of Db expression at time
0 was similar for both peptides. Then, T2Db cells were
washed five times, incubated at 37°C in serum-free medium
supplemented with Brefeldin A, and the density of residual Db molecules was monitored over a 12-hour period. The
half-life of B6dom1/Db and H-Y/Db complexes was
remarkably similar (8 hours, Fig 2C) and
longer than that of most viral peptides.32,41,42 As these
estimates of Db/peptide stability reflect relative
dissociation rates,5 we conclude that the greater affinity
of B6dom1 for Db (Fig 1) is due to a faster
association rate rather than to a slower dissociation rate. It has
previously been found that for a number of peptides, a close
correlation existed between the MHC-binding affinity and the
dissociation rate32,41. Therefore, it is
interesting to note that in the case of B6dom1 and H-Y, the
major difference in Db-binding affinity can be ascribed to
different association rates. However, this is not unprecedented, as
similar findings have been reported with Kb-binding
ovalbumin (OVA) peptides.5
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| Fig 2.
Stability of Db/peptide complexes.
T2Db cells, precultured at 26°C in the presence of
Brefeldin A, were incubated with peptides in complete medium. Peptides
were used at different molar concentrations selected to obtain a
similar MFI value. In these conditions, the level of H2-Db
expression at time 0 was equivalent for both B6dom1 and
H-Y. T2Db cells were then washed five times, incubated at
37°C in serum-free medium supplemented with Brefeldin A, and the
density of residual Db molecules was measured by staining
with anti-Db Ab at various time points. (A)
B6dom1/Db expression at 0 and 12 hours. (B)
H-Y/Db expression at 0 and 12 hours. (C) Downregulation of
peptide/Db complexes over a 12-hour time frame. One
representative experiment of two is shown.
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Cell surface density of naturally processed B6dom1 and
H-Y epitopes.
Peptides associated with cell surface MHC molecules were obtained by
acid elution from C57BL/6 male splenocytes, fractionated by HPLC, and
their biologic activity was compared with that of synthetic peptides
(AAPDNRETF and WMHHNMDLI) in CTL sensitization assays using
T2Db targets with anti-H-Y- and
anti-B6dom1-specific effectors.32,37
Polyclonal anti-H-Y CTLs, obtained after a single in vivo priming and
in vitro restimulation of C3H.SW female cells with C3H.SW male cells,
were used for quantification of natural H-Y epitopes because such
effectors recognize a single peptide-Db
complex.43 B6dom1 activity was detected with an
AAPDNRETF-specific C3H.SW-derived CTL line (SW10/B). The retention
times of the natural B6dom1 and H-Y peptides were identical
to those of the synthetic peptides: 18 minutes for AAPDNRETF and 27 minutes for WMHHNMDLI (data not shown). As a control, peptides
extracted from C3H.SW female cells did not sensitize target cells to
lysis by anti-B6dom1 or anti-H-Y effectors (data not shown).
Theoretically, an assay based on the comparison of the CTL-sensitizing
activity of a synthetic peptide with the activity in cell extracts is
prone to underestimate copy number because other peptides present in
the extract can act as competitive inhibitors of MHC
binding.44 Therefore, we modified the assay to eliminate this bias which, in principle, could be more important for H-Y than for
B6dom1 because the former peptide has a lower MHC binding
affinity (Fig 1). Thus, we compared the activity of peptide extracts
from a given number (N) antigenpos cells with the activity
of various concentrations of synthetic peptide mixed with peptide
extracts from N antigenneg cells. Cell extracts and
synthetic peptides were subjected to the same HPLC fractionation
procedure and the fraction of interest (no. 18 for B6dom1
and no. 27 for H-Y) was used in CTL assays. For H-Y, the
CTL-sensitizing activity of natural peptides extracted from 1.6 × 107 C57BL/6 male cells was compared with a dose response
curve of graded concentrations of synthetic H-Y peptide mixed with a
fixed amount of nonantigenic peptide extract from 1.6 × 107 C57BL/6 female cells. In the representative experiment
shown in Fig 3A, the activity of fraction
27 extracted from 1.6 × 107 C57BL/6 male cells was
equivalent to that of 2 × 1012 mol/L of
synthetic H-Y peptide. By multiplying the molar amount of synthetic
peptide in a given well by Avogadro's number and dividing by the
number of H-Ypos cells required to have the same percent
killing, we calculated that the number of H-Y epitopes per cell ranged
from 3 to 12 in 12 experiments. The same strategy was used for
B6dom1. The activity of natural peptides extracted from 4 × 106 C57BL/6 male cells was compared with the
titration curve of synthetic B6dom1 peptide mixed with a
nonantigenic peptide extract from 4 × 106 C3H.SW male
cells (Fig 3B). Here we found that the number of B6dom1
epitopes/C57BL/6 cell was 824 to 1,200 (12 experiments). Interestingly, the epitope density of B6dom1 was decreased by 50% on
(C57BL/6 × C3H/HeJ)F1 hybrids relative to C57BL/6
cells (Fig 3C). Epitope density of B6dom1 as expressed on
C57BL/6 cells place this peptide among the most abundant natural class
I-restricted epitopes44-48 and show that it is much more
abundant than H-Y. The low H-Y epitope density is similar to that of
the H13 MiHA whose structure has recently been
discovered.45
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| Fig 3.
Abundance of naturally expressed B6dom1 and
H-Y epitopes. Synthetic B6dom1 and H-Y peptides, as well as
cell extracts, were fractionated by RP-HPLC. Both synthetic and natural
B6dom1 peptides were recovered in HPLC fraction no. 18, while synthetic and natural H-Y peptides were recovered in fraction no.
27. (A) The activity of natural H-Y peptide extracted from 1.6 × 107 C57BL/6 male cells is plotted on a dose response curve
showing the activity of graded concentrations of synthetic H-Y peptide
mixed with competitor peptides extracted from 1.6 × 107
C57BL/6 female cells. Also evaluated were the activity of 4 × 106, 8 × 106, and 1.2 × 107
cell extracts from C57BL/6 male cells giving 3, 7, and 12 copies per
cell, respectively (data not shown). One representative experiment of
three is shown. (B) The activity of natural B6dom1 peptide
extracted from 4 × 106 C57BL/6 male cells or 1.6 × 107 129 male cells is plotted on dose response curves
depicting the activity of graded concentrations of synthetic
B6dom1 peptide mixed with competitor peptides extracted
from 4 × 106 ( ) or 1.6 × 107 ( )
C3H.SW male cells, respectively. Also evaluated were the activities of
extracts from 8 × 106, 1.2 × 107, or 1.6 × 107 C57BL/6 male cells and 4 × 106, 8 × 106, or 1.2 × 107 129 male cells. The number
of epitope copies per cell was established at 824, 903, and 1,200, respectively for C57BL/6, and at 112, 126, and 107 copies for 129 cells
(data not shown). One representative experiment of three is shown. (C)
The activity of natural B6dom1 peptide extracted from
8 × 106 (C57BL/6J × C3H.HeJ)F1
cells is plotted on a dose response curve depicting the activity of
graded concentrations of synthetic B6dom1 peptide mixed
with competitor peptides extracted from 8 × 106 C3H.SW
male cells, respectively. One representative experiment of two is
shown. Targets were T2Db cells. E:T ratio was 5:1 for
anti-B6dom1 effectors (SW10/B cell line) and 50:1 for
anti-H-Y effectors.
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Relative avidity of CTL target cell recognition of B6dom1
and H-Y epitopes.
The avidity of CTL/APC interaction is the product of the cell surface
density of the peptide/MHC complex on the APC and the intrinsic
affinity between these complexes and their specific TCR. To compare the
relative avidity of target cell recognition by specific CTLs,
polyclonal anti-B6dom and anti-H-Y CTLs were tested in
cytotoxicity assays with female C3H.SW targets sensitized with graded
concentrations of synthetic B6dom1 and H-Y peptides. The
concentration of H-Y peptide necessary to sensitize targets for 50%
lysis was very low (1012 mol/L;
Fig 4) when compared with other class
I-associated peptides,5 and was 100-fold lower than that
required for B6dom1 (2 × 1010
mol/L; Fig 4). These results were highly reproducible in four of four
experiments. This indicates that Db/H-Y complexes interact
with high-affinity TCRs, whereas TCRs that recognize
Db/B6dom1 show a lower intrinsic affinity for
their ligand. These results, together with the demonstration that the
cell surface density of B6dom1 is 100-fold higher than that
of H-Y (Fig 3), support the concept that interactions between TCRs and
peptide/MHC complexes obey the law of mass action, even though these
reactants are confined to their respective cell membranes. Thus, for a
designated level of lysis, the product of the required number of
complexes per target cell (epitope density) and TCR affinity for
peptide/MHC complexes is constant, low-density ligands usually interact
with high-affinityTCRs and vice versa.49
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| Fig 4.
Activity of B6dom1 and H-Y peptides in CTL
sensitization assays. 51Cr-labeled T2Db targets
that had been preincubated with various concentrations of H-Y or
B6dom1 synthetic peptides were incubated for 4 hours with
polyclonal anti-H-Y or anti-B6dom1 CTLs (E:T ratio 50:1).
One representative experiment of four is shown.
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In vivo expansion of anti-B6dom1 and anti-H-Y CTLs.
Epitope density and TCR affinity regulate the rate of expansion of
cognate CTLs.50-52 To evaluate the kinetics of the primary response, we monitored the frequencies of B6dom1- and
H-Y-specific CTLs at various time points after immunization using LDA.
This approach has been widely used to evaluate the kinetics of CTL
responses.53,54 Thus, C3H.SW female mice were primed with
C3H.SW male cells (H-Y+) or C57BL/6 female cells
(B6dom1+), their spleen cells were harvested on day 5, 10, 15, or 20, and cultured under limiting dilution conditions in
IL-2-supplemented medium with the same cell type as for priming. After
9 days, CTL activity was tested on T2Db targets coated with
optimal concentrations of synthetic B6dom1 or H-Y peptide.
Assays performed after culture in the presence of low concentrations of
IL-2 (2.5 U/mL) showed that the expansion of anti-B6dom1
CTLs was much more rapid than that of anti-H-Y CTLs
(Fig 5). Thus, on day 5, the mean frequency
of anti-B6dom1 CTL precursors was 20-fold greater than that
of anti-H-Y precursors. The difference was still significant on day 15 (7.5-fold) and disappeared on day 20. CTL precursor frequencies were
similar, however, when the culture medium was supplemented with high
concentrations of IL-2 (20 U/mL); in the latter case, anti-H-Y and
anti-B6dom1 precursor frequencies reached 2 × 10-5 on day 20 (data not shown). Thus, unless large amounts
of IL-2 were added, the proliferation rate of anti-B6dom1
CTLs was higher than that of anti-H-Y CTLs. The fact that under these
assay conditions H-Y-specific CTLs were more dependent on the supply
of exogenous IL-2 than B6dom1-specific CTLs is consistent
with previous evidence that in vivo expansion of anti-H-Y CTLs
requires CD4 help, whereas expansion of anti-B6dom1 CTLs
does not.33,55
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| Fig 5.
Anti-B6dom1 CTLs expand more rapidly than
anti-H-Y CTLs. Limiting numbers of responder spleen cells from C3H.SW
female mice primed either with C57BL/6 female (B6dom1+)
or C3H.SW male (H-Y+) splenocytes (20 × 106
cells injected IP) were restimulated in vitro on days 5, 10, 15, or 20 postimmunization with 3 × 105 irradiated stimulator cells
in culture medium supplemented with 2.5 U/mL of IL-2. After 9 days,
cultures were evaluated in a 4-hour cytotoxicity release assay. Targets
were T2Db cells coated with optimal concentrations of H-Y
or B6dom1 peptide. Mean ± SD of three mice per group.
*, Not detectable.
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Is B6dom1 expressed by cells with a different genetic
background always a dominant epitope?
The results presented above strongly suggest that the high cell surface
density of B6dom1 has a determining influence on the fact
that this antigen is a dominant MiHA recognized by C3H.SW mice when
primed against C57BL/6 cells. Thus, it is logical to assume that if the
expression of the B6dom1 epitope was decreased below a
certain threshold, B6dom1 would lose its dominant status.
The next series of experiments were designed to test this possibility
and to evaluate what could be the expression threshold required for
B6dom1 to be a dominant MiHA.
First, to assess the strain distribution of B6dom1, CML
assays were performed with SW10/B effectors and Con A blast targets
from six strains of H2b mice. Results were negative with
C3H.SW, A.BY, and BALB.B, but strongly positive with C57BL/6, 129, D1.LP, and LP targets (data not shown). To determine if
B6dom1 was a dominant MiHA, C3H.SW mice were primed IP and
their splenocytes were restimulated in MLC with cells from the six
strains of H2b mice mentioned above. Effectors from these
cultures were tested on T2Db cells coated with synthetic
B6dom1 peptide. T2Db cells coated with the
YSNENMDAM peptide were used as a negative control. The latter
nonapeptide is a variant of the dominant influenza nucleoprotein
epitope presented by H2Db and its ability to stabilize
Db molecules on the surface of T2Db cells is
similar to that of B6dom1.32 As expected,
effectors primed with B6dom1-negative cells (C3H.SW, A.BY,
and BALB.B) did not kill target cells coated with B6dom1
(data not shown). In contrast, C3H.SW-derived CTLs primed with C57BL/6,
D1.LP and LP cells (all H2b) killed T2Db
targets coated with B6dom1 peptide
(Fig 6A); thus, for these strains,
B6dom1 is not only present, but is also dominant. The fact
that LP-derived anti-C57BL/6 CTLs did not kill targets coated with
B6dom1 suggests that only B6dom1-negative mice
can generate CTLs that kill these targets and that CTLs specific for
other antigens present on B6dom1-expressing cells do not
cross-react with B6dom1 (Fig 6B). The most interesting
results were observed with C3H.SW-derived CTLs primed with 129 cells
(the latter are B6dom1-positive, Fig 6C), as these
effectors did not kill targets coated with B6dom1 (Fig 6D).
Thus, in the latter case, B6dom1 is expressed at the cell
surface, but is not immunodominant. The nature of the dominant MiHAs
recognized by C3H.SW CTLs on 129 APCs is currently unknown.
View larger version (32K):
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| Fig 6.
B6dom1 is a dominant epitope recognized by
C3H.SW-derived CTLs on C57BL/6, LP, and D1.LP, but not on 129 cells.
(A) B6dom1 is dominant on C57BL/6, LP, and D1.LP cells.
C3H.SW mice were primed IP and their splenocytes were restimulated in
MLC with cells from C57BL/6, LP, or D1.LP mice. Effectors were tested
at various E:T ratios on C3H.SW Con A blasts that had been incubated
with graded concentrations of B6dom1 peptide ( ), control
YSNENMDAM peptide ( ), or culture medium alone ( ). (B) LP-derived
anti-C57BL/6 CTLs do not kill T2Db targets coated with
B6dom1 peptide. E:T ratio was 50:1 (closed bar) or 25:1
(open bar). (C) B6dom1-specific CTLs (SW10/B cell line)
kill 129 Con A blasts. (D) C3H.SW-derived anti-129 CTLs do not kill
C3H.SW Con A blast targets coated with optimal concentration of
B6dom1 peptide (AAPDNRETF; 1010 mol/L).
|
|
Why do C3H.SW CTLs recognize B6dom1 as a dominant MiHA when
it is presented on C57BL/6, D1.LP, or LP, but not when presented on 129 cells? One possibility would be that the epitope density of B6dom1 is identical on C57BL/6 and 129 cells, but that the
immunodominant MiHA recognized by C3H.SW-derived anti-129 CTLs
(hereafter referred to as 129dom) dominates
B6dom1 expressed at 103 copies per cell.
This possibility cannot be directly evaluated because
129dom has not been defined. An alternative explanation
would be that the expression of B6dom1 at the cell surface
is lower in 129 mice than in C57BL/6 mice. To evaluate this
possibility, we assessed the abundance of B6dom1 at the
surface of 129 cells. B6dom1 was recovered in the same
fraction (ie, no. 18) after HPLC fractionation of eluates from C57BL/6
and 129 splenocytes (data not shown), but the number of epitopes per
cell was only 107 to 126 in 129 splenocytes (12 experiments), ie,
sevenfold less than for C57BL/6 cells (Fig 3B). This suggests that an
88% decrease in the cell surface expression of B6dom1 is
sufficient for this antigen to lose its dominant status. Theoretically, quantitative assessment of B6dom1 expression could be
biased if there were a variation in the structure of the
B6dom1 peptide expressed in 129 versus C57BL/6 cells.
However, this possibility is very unlikely because both peptides had
exactly the same elution profile when extracts from B6 and 129 cells
were fractionated with three different previously described
high-resolution HPLC gradient systems (data not shown).32
| |
DISCUSSION |
B6dom1/Db complexes are optimal CTL ligands.
The results presented herein (summarized in
Table 1) disclose major differences between
the nondominant and the dominant antigens recognized by C3H.SW-derived
CTLs on C57BL/6 cells: H-Y/Db complexes show a low cell
surface density, but are recognized by TCRs with very high relative
affinity, whereas B6dom1/Db complexes are more
abundant and interact with lower affinity TCRs.
Efficient T-cell triggering requires sustained signaling, is correlated
with the number of TCR engaged, and is dependent on optimal kinetics of
TCR interaction with peptide/MHC complexes.50,52,56-58 A
high epitope density, such as found for B6dom1 when
compared with H-Y, is an advantage because the number of triggered TCRs
is a function of the logarithm of the number of complexes
offered.52,56 Indeed, antigen dose has a determinant influence on the level of proliferation of cognate T cells, and high
epitope density can even overcome the requirement for costimulation ("signal two").50,51,59 The fact that
B6dom1 is not dominant on 129 cells, where its expression
is only 117 copies/cell (v 1,012 for C57BL/6 cells)
strongly suggests that epitope density plays a pivotal role in
dominance. The relation between TCR affinity and T-cell triggering is,
however, more complex because optimal affinity does not correspond to
maximal affinity, but rather to "intermediate" affinity. Indeed,
acceleration of TCR-ligand complex dissociation allows serial TCR
triggering and increases the efficiency of antigen recognition by CTL
up to a critical threshold, beyond which a too rapid dissociation
results in aberrant TCR signaling and TCR antagonism.60
Whereas epitopes with an optimal off-rate can trigger several TCRs
serially, high-affinity ligands, such as anti-CD3 antibodies, are
inefficient because their incapacity to dissociate does not allow
serial triggering.58 According to this model, the
intermediate affinity of B6dom1/Db complexes
for their cognate TCRs should be more advantageous for T-cell
activation than the high-affinity of Db/H-Y complexes.
The concept that B6dom1 triggers stronger signal 1 in CTL
than H-Y could well explain the observation that
B6dom1-specific CTLs expanded more rapidly and were less
dependent on exogenous IL-2 than H-Y-specific CTLs (Fig 5). Indeed,
potent helper-independent T-cell responses can only be triggered by
ligands that, because of their abundance and optimal TCR affinity,
induce strong TCR signals.51,61 This concept would also
provide a plausible explanation for the fact that, whereas CTL
responses toward B6dom1 and H-Y appear to be of similar
magnitude when evaluated in standard in vitro CTL assays (percent lysis
at various E:T ratios), the in vivo consequences of responses elicited
by these MiHAs are quite different. Indeed, in vivo responses toward
H-Y/Db are biologically attenuated; they cannot elicit
graft-versus-host disease and are not sufficient to cause skin graft
rejection (in H2b mice, male skin graft rejection is
elicited mainly by class II, as opposed to class I-associated H-Y
epitopes).30,62 In contrast, anti-B6dom1 CTLs
are sufficient to provoke graft-versus-host disease.33 However, it must be remembered that the dominance effect
results from competition between dominant and nondominant epitopes and is, therefore, defined in relative terms. Thus, the fact that other
MiHAs are neglected when presented with B6dom1 does not
imply that, when presented alone, they cannot elicit biologically
significant CTL responses. Nevertheless, other reports confirm the
differential ability of dominant versus nondominant epitopes to
generate protective responses against other types of antigens, ie,
viruses or cancer cells.63-65 Collectively, these observations provide compelling evidence that dominant antigens elicit
more vigorous CTL responses than nondominant epitopes. Furthermore, the
fact that H-Y/Db is an adequate CTL target in vitro, but
that in vivo, it elicits only a slow expansion of cognate CTLs (Fig 5),
which is of little biological significance30,62 illustrates
that the ability to kill target cells in vitro is not predictive of in
vivo efficacy.66,67 This can be explained by the existence
of more stringent requirements for the afferent arm of CTL responses
(proliferation) than for the effector phase of the killing
process.57
How can expression of B6dom1 inhibit anti-H-Y CTL
responses?
Previous analyses of CTL responses toward B6dom1 and H-Y
showed that the most critical characteristic of the immunodominance
effect is that "suppression" of T-cell responses to nondominant
epitopes by dominant epitopes is observed only when both types of
determinants are presented on the same APC.32,34 This
observation indicates that differential generation of T-cell help
cannot be responsible for B6dom1 immunodominance over
H-Y/Db. Indeed, help from other T cells, particularly from
the CD4+ subset, is available to all CD8+ CTLs
that recognize epitopes presented on the same APC, irrespective of the
specificity of helper cells and CTLs.55,68-70 Thus, after immunization with C57BL/6 male cells, anti-B6dom1 and
anti-H-Y CTLs receive the same help because both MiHAs are presented
on the same APC. Therefore, the fact that dominance takes place only
when the dominant and the nondominant epitopes are presented on the
same APC suggests that the immunodominance effect results from a
competition for the APC surface.
The observation that unless excessive amounts of exogenous IL-2 are
added, B6dom1-specific CTLs expand more rapidly than
anti-H-Y CTLs could well explain the immunodominance effect. Indeed, a
direct consequence is that early in the course of the immune response,
anti-B6dom1 CTLs become activated more efficiently and are
likely to swiftly outnumber anti-H-Y CTLs. This should not influence
CTL responses when both MiHAs are presented on separate APCs. However,
when both MiHAs are presented on the same APC,
B6dom1-specific CTLs could prevent triggering of
H-Y-specific CTLs in two ways. First, the activation status and the
numerical supremacy of anti-B6dom1 CTLs should enable them
to compete more successfully for occupancy of the APC surface and for
cytokines generated at the T-cell/APC interface. Competition for the
APC surface and locally produced cytokines (mainly IL-2) may be a
recurring theme in the regulation of immune responses because these are
precisely the mechanisms whereby anergic T cells inhibit responses to
linked epitopes, ie, epitopes presented on the same APC as the
tolerizing determinant.71-73 Second,
B6dom1-specific CTLs may simply kill the APCs before
anti-H-Y CTLs have a chance to find them. Killing of APCs by cognate
CTLs has been documented both in vitro and in vivo and is likely
instrumental in limiting the duration of immune
responses.74-77
One limitation to the interpretation of our model is that we could not
ascertain whether the more rapid expansion of anti-B6dom1
versus anti-H-Y CTLs in vivo depended solely on the differential strength of signal 1 delivered to CTLs by the dominant versus nondominant epitope. Although differential generation of help cannot
explain B6dom1 dominance over H-Y when both MiHAs are
presented on the same APC (see above), expansion of MiHA-specific CTLs
could only be evaluated under experimental conditions where the two
MiHAs were presented on separate APCs (the presence of
B6dom1 inhibits the generation of H-Y-specific CTLs when
both epitopes are on the same APC). We cannot exclude the possibility
that, under these conditions, CTLs specific for B6dom1 and
H-Y could receive unequal amounts of help from T cells that recognize
other epitopes, such as class II-associated MiHAs. We believe that the
best strategy to address this issue would be to measure in vivo
expansion of anti-B6dom1 and anti-H-Y CTLs after priming
with APCs presenting only B6dom1/Db or
H-Y/Db, preferably on a class II-deficient background.
This will be possible only when B6dom1 congenic mice are available.
What is the raison d'être of the immunodominance effect?
Is it a good strategy for the immune system to restrict the repertoire
of CTL responses to a few dominant epitopes and hence to suppress
responses to other nondominant epitopes expressed by APCs? Analysis of
CTL responses to B6dom1 and H-Y shows that the
immunodominance effect is not a stochastic process. Indeed, based on
the characteristics of B6dom1, we conclude that dominant
epitopes are those, which have optimal ability to trigger TCR signals
in CTLs. Thus, whether the critical final event is competition for the
APC surface + cytokines or APC killing, the dominance effect can serve
two important purposes. First, considering that naive T cells require
approximately 20 hours of sustained signaling to be committed to
proliferation78 and that APCs disappear less than 48 hours
after interaction with cognate T cells in vivo,79 the
dominance effect may help to minimize the time required to mount a
protective immune response by devoting the APC surface to CTLs specific
for the best TCR ligands during the brief period of priming. This could
be extremely important considering how critical time limitations are
for CTL to mediate early control of a microbial infection in
vivo.80,81 Secondly, taking into account that TCRs are
degenerate receptors and that T lymphocytes responding to
nonself-antigens can cross-react with self-peptides,4,82,83
restricting the repertoire of T-cell responses may decrease the risk of
autoimmunity. Thus, the immunodominance phenomenon may, in fact,
represent an astute "low risk high efficiency" strategy for the
immune system: restricting the diversity of the immune response limits
the potential for autoimmune recognition, while focusing on the best
epitopes confers good chances to rapidly eliminate pathogens.
| |
FOOTNOTES |
Submitted April 30, 1998; accepted September 24, 1998.
Supported by the National Cancer Institute of Canada (C.P.) and the
National Institute of Allergy and Infectious Diseases (D.C.R.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Claude Perreault, MD, Research
Center, Maisonneuve-Rosemont Hospital, 5415 de l'Assomption Blvd,
Montreal, Quebec, Canada H1T 2M4; e-mail: c.perreault{at}videotron.ca.
| |
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Abstract
Introduction