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Cutting Edge |
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 8, 9, 10, 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.
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Materials and Methods |
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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 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
Mice were immunized in the hind footpads with 10 nmol of HEL (Sigma-Aldrich, 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 µg/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 µg) of HEL protein emulsified in CFA. Seven days later, the draining lymph nodes were removed and placed into the LDA, using as APCs 5 x 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 µM 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 from
the draining lymph nodes (Fig. 1
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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).
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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).
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B). 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 higher than 115-129. The percentage of cells
reactive to 31-47 was 9%; this epitope happens to be the second
highest, 
60-fold <48-63, 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.
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.
Blocking CTLA-4 has no measurable effect on the primary CD4+ response.
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 µg of this Ab into BDC2.5 x nonobese
diabetic mice days 9, 12, and 15 after birth, led to rapid onset
of diabetes (18).
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B). These
data and the previously discussed results are summarized in Table I.
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C). 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 21, 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 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Emil R. Unanue, Department of Pathology and Immunology, 660 South Euclid Avenue, Box 8118, WashingtonUniversity School of Medicine, St. Louis, MO 63110. E-mail address: Unanue{at}pathology.wustl.edu
3 Abbreviations used in this paper: HEL, hen egg white lysozyme; CD40L, CD40 ligand; LDA, limiting dilution analysis.
Received for publication June 19, 2002. Accepted for publication July 29, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), CD40-/-
mice (
), and B7-1/B7-2-/- mice (
) 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).
)
and anti-CTLA-4-treated (