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Cutting Edge: The Relative Distribution of T
Cells Responding to Chemically Dominant or
Minor Epitopes of Lysozyme Is Not Affected by
CD40-CD40 Ligand and B7-CD28-CTLA-4
Costimulatory Pathways
Richard J. DiPaolo and Emil R. Unanue
J Immunol 2002; 169:2832-2836; ;
doi: 10.4049/jimmunol.169.6.2832
http://www.jimmunol.org/content/169/6/2832
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Copyright © 2002 by The American Association of
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References
The Journal of Immunology
●
Cutting Edge: The Relative Distribution of
T Cells Responding to Chemically Dominant or
Minor Epitopes of Lysozyme Is Not Affected by
CD40-CD40 Ligand and B7-CD28-CTLA-4
Costimulatory Pathways1
Richard J. DiPaolo and Emil R. Unanue2
W
e recently reported on the clonal distribution of the
CD4 T cell response after immunization with hen egg
white lysozyme (HEL)3 in CFA (1). We had identified, chemically sequenced, and directly quantitated the abundance
of four HEL peptides presented by the murine MHC class II I-Ak
molecule (2– 4). Despite levels of presentation that differed by
⬎200-fold between the chemically dominant, or high abundant
epitopes and the chemically subdominant, or low abundant
epitopes, the proportion of T cells responding to each at different
doses of HEL was changed to a minor degree (1, 5). We concluded
that chemical dominance, or the levels of peptide presentation, was
not related to immunodominance, or the number of responding T
cells, and hypothesized that increased levels of costimulatory mole-
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
Received for publication June 19, 2002. Accepted for publication July 29, 2002.
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 National Institutes of Health.
2
Address correspondence and reprint requests to Dr. Emil R. Unanue, Department of
Pathology and Immunology, 660 South Euclid Avenue, Box 8118, Washington
University School of Medicine, St. Louis, MO 63110. E-mail address:
[email protected]
3
Abbreviations used in this paper: HEL, hen egg white lysozyme; CD40L, CD40
ligand; LDA, limiting dilution analysis.
Copyright © 2002 by The American Association of Immunologists, Inc.
●
cules induced by adjuvant may have been the condition that equalized
the T cell response to this disproportionate display of peptides.
Although the role of costimulatory molecules in T cell responses
to epitopes displayed at various levels by a single APC has not
been examined previously, there are many reports that would lead
us to conclude that costimulatory molecules may have a greater
effect on low abundance epitopes, or that CTLA-4 may preferentially influence the response to the chemically dominant epitope.
Indeed, B7-CD28 costimulation has been shown to augment T cell
proliferation to suboptimal levels of TCR stimulation through the
up-regulation of IL-2 and other cytokines and up-regulation of
anti-apoptotic proteins such as Bcl-xL (6). The CD40-CD40 ligand
(CD40L) costimulation was demonstrated to be important in B cell
class switching, APC activation, and enhancing T cell priming
(7–11). Concerning CTLA-4, it is expressed on activated T cells
and may negatively regulate T cell responses by restricting IL-2
production, inhibiting cell cycle progression, recruiting phosphatases to dampen TCR signaling, and/or sequestering B7 molecules
from interacting with CD28 (reviewed in Ref. 12). The role of
CTLA-4 in negatively regulating T cell activation is best exemplified by the massive lymphoproliferative disorder of mice genetically deficient in it (13, 14). It was recently suggested that
CTLA-4 may be preferentially inhibiting T cells that receive stronger signaling through the TCR, for example, T cells with higher
affinity receptors, or responding to high-density epitopes (15).
In these studies, we examine whether the expression of costimulatory molecules induced by immunization with CFA, or the regulation of T cell responses by CTLA-4, masks a relationship between levels of peptide presentation and the T cell response after
immunization. We describe the CD4⫹ T cell response to the four
epitopes of HEL under two conditions: first, using mice lacking
either CD40, or both B7-1 and B7-2; and second, in mice treated
with Abs to CTLA-4. Under both situations, we immunized with
HEL in CFA, followed by limiting dilution analysis (LDA) of the
CD4 T cell response.
Materials and Methods
Mice and Reagents
B10.BR and CD40⫺/⫺ mice were obtained from The Jackson Laboratory (Bar
Harbor, ME) and B7-1/B7-2⫺/⫺ mice were a generous gift from Dr. A. Sharpe
(Harvard University, Boston, MA) (16). Knockout mice were originally obtained on the B6 background, and were outcrossed to B10.BR for one generation. Progeny from this mating were intercrossed to obtain knockout genotypes on the H-2k haplotype.
The hybridoma-producing Abs to CTLA-4 (UC10-4F10) were obtained
from American Type Culture Collection (Manassas, VA), and produced
0022-1767/02/$02.00
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We examined the frequencies and specificities of the CD4ⴙ T
cell responses to the protein hen egg white lysozyme in mice
deficient in the CD40-CD40 ligand or B7-CD28 costimulatory
pathways. The frequency of T cells was decreased by between
3- and 4-fold in CD40ⴚ/ⴚ mice, and 12-fold in B7-1/B7-2ⴚ/ⴚ
mice, but surprisingly, the relative distribution of T cells responding to peptides that were presented at levels that differed
by >250-fold was similar. We also examined the CD4 response
after blocking the regulatory molecule CTLA-4 during immunization. We observed no difference in either the frequency or
specificity of the CD4ⴙ T cell response if CTLA-4 was blocking
during priming. Thus, the T cell response was generated toward the constellation of chemically dominant and subdominant epitopes as a whole, and did not discriminate among them
based on their relative abundance. The Journal of Immunology, 2002, 169: 2832–2836.
The Journal of Immunology
and purified in our laboratory. The control hamster IgG was a generous gift
from Dr. R. Schreiber (Washington University, St. Louis, MO). One hundred micrograms of each was injected i.p., daily, starting 1 day before
immunization. Lymph nodes were harvested 7 days after immunization.
Assays
from the draining lymph nodes (Fig. 1). The hybridomas used in
these assay, which are less dependent on the expression of costimulatory molecules, responded similarly to APCs from draining
lymph nodes of B10.BR, CD40⫺/⫺, and B7-1/B7-2⫺/⫺ mice. Thus,
there are similar levels of HEL presented in the draining lymph node
of the wild-type and costimulatory-deficient mice (Fig. 1).
Ab responses, frequencies of HEL-reactive CD4⫹ T cells,
and the specificity of the CD4⫹ T cell response in B10.BR,
CD40⫺/⫺, and B7-1/B7-2 ⫺/⫺ mice
Mice were immunized with 10 nmol of HEL in CFA, a dose that
is known to generate a strong response 7 days after immunization.
B10.BR mice produced a strong Ab titer to HEL protein, as expected, CD40⫺/⫺ mice made significantly less, and B7-1/B7-2⫺/⫺
mice did not produce detectable levels of Abs (Fig. 2A).
The frequencies of responding T cells was determined by LDA
7 days after immunization (5). The frequency of HEL-reactive
CD4⫹ T cells in B10.BR mice was 1 in 5000 lymph node cells
(Fig. 2B). These are the same results reported in our previous study
(1) and which represented the pool from six different experiments
comprising 162 clones (both the present and the previous experiments were done at about the same time). To summarize the results
in B10.BR mice, the frequency of T cells responding to the family
of peptides 48-63, 20-35, and 115-129 were similar, 30, 17, and
25%, respectively, when immunizing with 10 nmol (Fig. 2C). To
note, 48-63 is expressed 200-fold higher than 18-33 and ⬎250-fold
Results
Similar levels of HEL presentation in the draining lymph nodes
of B10.BR, CD40⫺/⫺, and B7-1/B7-2⫺/⫺ mice
The relative levels of presentation of two HEL epitopes was examined in cells from the draining lymph nodes 24 h after immunization with HEL protein in CFA. T cell hybridomas to the 48-63
and 18-33 epitopes were cocultured with various numbers of cells
FIGURE 1. Similar levels of Ag presentation in the draining lymph
nodes of costimulation-deficient mice. B10.BR mice (f), CD40⫺/⫺ mice
(䡺), and B7-1/B7-2⫺/⫺ mice (E) were immunized with 10 nmol of HEL
in CFA. Twenty-four hours later, the draining lymph nodes were removed
and cocultured with HEL-specific hybridomas 3A9 which is specific for
48-63 (A) and 3F12 which is specific for 18-33 (B).
FIGURE 2. Abs, frequencies, and specificities in B10.BR and costimulatory-deficient mice. A, Serum Ab titers after immunization with 10 nmol of
HEL in CFA. Shown is the average of between four and six mice per group.
B, The frequencies of HEL-reactive T cells were determined by LDA. The
average frequency in B10.BR mice was 1 in 5,000 lymph node cells, in
CD40⫺/⫺ was 1 in 12,600 lymph node cells, and in B7-1/B7-2⫺/⫺ mice was
1 in 64,500 lymph node cells. C, The graph shows the relative proportion of
HEL-specific T cells that were specific for each HEL epitope. These data
represent the specificity analysis of 162 individual clones from B10.BR, 123
individual clones from CD40⫺/⫺, and 56 clones from B7-1/B7-2⫺/⫺ mice.
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Mice were immunized in the hind footpads with 10 nmol of HEL (SigmaAldrich, St. Louis, MO) emulsified in CFA H37Ra (Difco, Detroit, MI).
Lymph nodes were removed after 24 h and cocultured with either 3A9 T
cell hybridoma to detect presentation of the 48-63 epitope, or the 3F12
hybridoma to detect presentation of the 18-33 epitope. After 24 h, supernatants were removed from the cultures and the presence of IL-2 was tested
using a standard CTLL-2 assay.
The presence of Abs to HEL protein in immunized mice was detected by
ELISA (1, 4) using Nunc Maxisorp ELISA plates (Roskilde, Denmark)
precoated with 10 ␮g/ml of HEL protein. Serial dilutions of immune serum
were added and detected with goat anti-mouse IgG-peroxidase (1/1500).
The LDA was performed as reported (1, 5). Mice were immunized in the
hind footpads with 10 nmol (144 ␮g) of HEL protein emulsified in CFA.
Seven days later, the draining lymph nodes were removed and placed into
the LDA, using as APCs 5 ⫻ 105 splenocytes from mice expressing a
membrane form of HEL as a transgene, and 50 U/ml of IL-2 (5). The
growth positive wells were expanded in two wells under the same conditions, and tested 7–10 days later for proliferation to HEL protein in the
absence of exogenous IL-2. HEL-specific T cell clones were further expanded for 7–10 days, and their specificity was determined in a proliferation assay using 5 ␮M of each HEL peptide (18-33, 31-48, 48-63, and
115-129) (5, 17). Ag-specific T cells were those showing thymidine incorporation ⬎2-fold above background. The LDA detects between one in two
and one in three reactive clones, reflects the spectrum of T cells that proliferate in response to the various epitopes at the time of harvest, after
cloning on an APC line presenting the naturally processed epitopes from
HEL protein, and the synthetic peptides that reflect the naturally presented
peptides were used to test specificity.
2833
2834
CUTTING EDGE: COSTIMULATION AND CD4 RESPONSE TO LYSOZYME EPITOPES
Blocking CTLA-4 has no measurable effect on the primary
CD4⫹ response.
FIGURE 3. Frequencies, specificities, and functional avidities in 4F10treated mice. A, Frequencies of HEL-reactive T cells in mice treated with
control Ab or an Ab to CTLA-4, and immunized with 1 nmol of HEL in
CFA. The average frequencies of HEL-reactive T cells were 1 in 27,500
and 1 in 32,000 in control and anti-CTLA-4-treated mice, respectively. B,
The graph shows the relative proportion of the HEL-reactive T cells that
were specific for the HEL peptides. The data represent the specificity of 56
clones from control mice and 55 clones from anti-CTLA-4-treated mice. C,
The average proliferation shown as the percent of maximal response for six
individual clones that were 48-63-specific from control-treated (Œ) and
anti-CTLA-4-treated (f) mice.
higher than 115-129. The percentage of cells reactive to 31-47 was
9%; this epitope happens to be the second highest, ⬃60-fold ⬍4863, but 3- to severalfold higher than 18-33 and 115-129, respectively. Immunizing with lower doses of HEL resulted in a decrease
in the total number of HEL-responding cells, i.e., the response was
proportional to the immunizing dose, but nevertheless, it affected the
relative proportion of the reactive clones to a small degree (1). Similar
distributions were found at 7 or 30 days after immunization (1).
We immunized the B7-1/B7-2⫺/⫺ and CD40⫺/⫺ mice with the
10 nmol dose expecting to find a reduction in the total number of
positive T cells, and examining whether a preferential reduction
was observed in T cells responding to low abundance epitopes.
To determine whether CTLA-4 was affecting the frequency or the
distribution of T cells recognizing high and/or low abundance
epitopes, we immunized mice with an intermediate dose while
administering blocking Abs to CTLA-4. The average frequency of
CD4⫹ T cells responding with 1 nmol of HEL in CFA in two
independent experiments was 1 in 27,500 cells when administering
a hamster IgG control Ab, and 1 in 32,000 when administering Abs
to block CTLA-4 (Fig. 3A). No differences were observed in the
bulk lymph node proliferation assay, or in Ab titers at day 7 either
(data not shown). The anti-CTLA-4 Ab used in these studies
stained intracellular CTLA-4 on activated T cells, and as a proof
that the anti-CTLA-4 Ab was effective in vivo, we repeated previously reported studies demonstrating that injecting 100 ␮g of
this Ab into BDC2.5 ⫻ nonobese diabetic mice days 9, 12, and 15
after birth, led to rapid onset of diabetes (18).
The specificity of 56 individual clones treated with control Ab,
and 55 individual clones treated with anti-CTLA-4 was determined. The percent of the HEL-reactive T cells responding to the
most abundant epitope (48-63) in control vs CTLA-4 treated mice
were 32 vs 25%, respectively. Five percent of HEL-reactive T cells
were specific for 31-47 both in control and anti-CTLA-4-treated
mice. Little change was observed in the proportion of T cells responding to the lowest abundant epitopes as well, with 17 and 11%
specific for 18-33, and 25 and 20% specific for the least abundant
Table I. Similar distributions of T cells to high and low abundance epitopesa
Specificity (%)
Mouse
B10.BR
CD40⫺/⫺
B7–1/B7–2⫺/⫺
Anti-CTLA-4
Control IgG
Dose
(nmol)
Frequency
48–63
31–47
18–33
115–129
10
10
10
1
1
1/5,000
1/12,600
1/64,500
1/32,000
1/27,500
30
45
37
25
32
9
7
16
5
5
17
21
18
11
17
25
27
8
20
25
a
Summary of the frequencies of T cells responding to immunization with HEL in CFA and the distribution of the responses
to four epitopes. The 48 – 63 is the chemically dominant epitope in that it is presented at levels 60-fold higher than 31– 47,
200-fold higher than 18 –33, and greater than 200-fold higher than 115–129. Despite these large differences in the relative
abundance of each epitope, there is little correlation between levels of peptide presentation and T cell responses.
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Three independent experiments were performed in CD40⫺/⫺
mice, and the specificity of 123 individual HEL-reactive CD4⫹ T
cells was analyzed. In CD40⫺/⫺ mice, the frequency of HEL-reactive T cells was modestly reduced by ⬃2- to 3-fold, from 1 in
5,000 to 1 in 12,600 lymph node cells (Fig. 2B). The proportion of
T cells reacting to the four epitopes was not changed to a major
extent; there was a slight increase in the percentage of those clones
directed to 48-63 (Fig. 2C).
The frequencies in B7-1/B7-2⫺/⫺ mice were determined in four
independent experiments, and the specificity of 56 HEL-specific
clones was examined. There was a marked reduction in the frequency of HEL-reactive T cells, to 1 in 64,500 lymph node cells
(Fig. 2B). There was some shift in the percentage of T cells reacting to the three minor epitopes: a 3-fold reduction in the least
represented peptide, 115-129, which went from 25% of clones in
the normal mice, to 8% in the B7-1/B7-2⫺/⫺ mice; the number
reactive to the 31-47 epitope increased from 9 to 16% while the
percentage reacting to 20-35 was unchanged (Fig. 2C). The proportion of clones seeing the minor epitopes relative to those seeing
the chemically dominant epitope was not changed to any major
degree, 30% in the normal to 37% in B7-1/B7-2⫺/⫺ mice.
The Journal of Immunology
115-129 peptide in control and anti-CTLA-4-treated mice, respectively (Fig. 3B). These data and the previously discussed results
are summarized in Table I.
The functional avidity, or amount of Ag required to produce
50% maximal proliferation, was determined in six and seven individual T cell clones reactive to 48-63 after administering either
control or anti-CTLA-4 Abs (Fig. 3C). Fig. 3C shows that the
average functional avidity was very similar in the two groups,
although the average T cell from CTLA-4 blockade responded to
slightly lower amounts of Ag.
Discussion
activation (15). This model predicts that CTLA-4 should have a
greater effect on limiting the proliferation of T cells responding to
strong TCR signaling, such as higher affinity T cells or T cells
responding to higher abundant epitopes. Regardless, we found no
evidence that CTLA-4 was affecting either the frequency, specificity, or the average functional avidity of CD4⫹ T cells 7 days
after immunization. Although it remains a possibility that CTLA-4
was not completely blocked in vivo, we demonstrated its efficacy
in vivo in control experiments. It is possible that CTLA-4 acts later
in the response, as naive T cells are known to express low levels
of CTLA-4 (27, 28, 29). Perhaps other regulatory molecules have
greater effects on primary T cell responses.
In summary, the repertoire of T cells responding after immunization appears to be very much set and to respond “en bloc”, dictated by the total set of peptide-MHC molecules rather than by
their relative levels of presentation. The specificity of this repertoire is relatively independent of dose of immunization and the
presence or absence of costimulatory molecules, or of regulatory
molecules. Whether there is positive cooperativity among the high
and low epitopes presented by the same APC is an important issue
to consider next.
Acknowledgments
We thank Kathy Frederick for her help with generating and maintaining the
mice used in these studies, as well as in the Ab injections into BDC2.5 ⫻
nonobese diabetic mice, Kevin Clark for synthesizing and purifying the
peptides, and Craig Byersdorfer and Drs. Osami Kanagawa and Paul Allen
for helpful discussions.
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Our recent report indicated that the number of T cells responding
was proportional to the amount of HEL used to immunize, however, the number of clones reacting to each of the epitopes was not
proportional to their relative level of presentation. We described
this response as a “go-no go” response, where in the presence of an
inflammatory environment, the number of peptide-MHC complexes required to activate a T cell is low, and more TCR engagement by more peptide-MHC may not translate into better T cell
activation.
One hypothesis to explain these results was that the threshold
for activation may be lowered by high levels of costimulatory molecules induced by CFA, to the point that T cells responded equally
to high and low abundance epitopes, and that in the absence of
important costimulatory molecules, T cell responses would be focused on chemically dominant, or higher abundance epitopes. Previous studies demonstrated a role for CD40-CD40L costimulation
in CD4⫹ T cell priming in vivo. The importance of B7-CD28
costimulation on in vivo CD4⫹ priming was also demonstrated,
and at least in vitro, has been shown to have a greater effect at
suboptimal levels of TCR ligation (19). What has not been addressed previously is whether the requirement for these costimulatory pathways is different for T cell responses to high or low
abundance epitopes.
Our results indicate clearly that the frequencies of CD4⫹ T cells
responding to HEL was decreased in the costimulatory-deficient
mice, an indication of the importance of these costimulatory pathways in affecting the magnitude of CD4⫹ T cell responses. The
importance of the B7/CD28 pathway is illustrated by the lack of
Ab response and ⬎10-fold decrease in the number of T cells
primed. Yet, in parallel to our previous findings, the decrease in
CD4 priming was similar among T cells responding to epitopes
presented at high and low abundance (Table I). In other words, the
threshold for T cell priming was achieved for high and low abundance epitopes in the costimulation-deficient mice, as T cells were
primed to both similarly. We argue that these costimulatory pathways, in the inflammatory setting of adjuvants, may be more involved in T cell expansion, for example, through increased cytokine production or protecting activated cells from death, than in
decreasing the threshold for TCR signaling needed for priming.
We are currently investigating whether the decreased CD4⫹ response is at the level of priming fewer cells or less expansion by
primed cells.
Blocking CTLA-4 during immunization had no effect on the
frequency or specificity of the CD4⫹ T cell response under the
conditions tested in this study. Although there is abundant evidence showing Abs that block CTLA-4-enhanced T cell responses
to antigenic challenge (15, 20 –22) to some infections (23, 24), and
in some tumor models (25, 26), relatively little is known about
whether CTLA-4 was acting at the time of initial T cell priming,
or during subsequent interactions with Ag. Recently, a model was
proposed that implicates CTLA-4 in setting a threshold for the
potency or frequency of TCR ligation necessary for CD4⫹ T cell
2835
2836
CUTTING EDGE: COSTIMULATION AND CD4 RESPONSE TO LYSOZYME EPITOPES
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