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Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purpose of this paper was to report on four issues. First, we report details of the method to estimate the peptide content from extracts of I-Ak molecules and on our first results using HEL as an Ag. We generated a panel of mAbs that react with the core sequence of the peptides. These Abs were selected on the basis of their reactivity with free peptide in solution. Using these mAbs, we developed sensitive ELISA methods that allowed for quantitation of the HEL peptides from class II molecules of APC. Second, we indicate that there are marked differences (as much as 250-fold) in the presentation of various peptide families on APC. Third, we report studies in vivo in which the levels of peptide-MHC complexes can also be estimated and related to immunologic responses. Lastly, we comment on peptide selection and immunogenicity based on these results.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used the murine B cell lymphoma lines M12.C3.F6 (M12-Ak) (9) expressing class II I-Ak molecules and the M12.C3.F6 cell line transfected with a membrane form of HEL (M12-Ak mHEL) (10). Both cell lines were cultured in DMEM supplemented with 5% heat-inactivated normal calf serum.
Mice
To produce the mAbs, we used CAF.1/J or B10.BR mice (obtained
from The Jackson Laboratory, Bar Harbor, ME) or CBA/J mice (purchased
from the National Cancer Institute, Bethesda, MD). The transgenic mHEL
mice had been generated previously using a cDNA gene for a chimeric
HEL-Ld fusion protein under the I-E 
promoter
(7, 11). The mHEL mice express HEL tethered to the
membrane of class II-positive cells in both thymus and peripheral
lymphoid organs. The ML-5 HEL transgenic mice expressing HEL under the
metallothionine promoter were obtained from Christopher Goodnow (John
Curtain School of Medical Research, Canberra, Australia)
(12, 13). All mice were maintained at Washington
University small animal facility.
Peptides
The peptides used in this study were synthesized by F-moc chemistry (model 432A, Applied BioSystems, Foster City, CA) and purified by reverse phase HPLC. To synthesize the biotinylated peptides, biotinamidocaproate N-hydroxysuccinimide ester (Sigma, St. Louis, MO) was used to bind to the amino terminus. The sequences and purity of each synthetic peptide were checked by mass spectrometry.
Generation of anti-HEL peptide mAbs
We generated a panel of mAbs to the core sequences of the
different I-Ak-restricted HEL epitopes. The mAbs
used in this study were 15B7-1B5 mAb (IgG1,
anti-HEL18–33 peptide), VAL-3 (IgG1,
anti-HEL31–47 peptide) (1),
48.151.2 (IgG1, anti-HEL48–61 peptide), and
115.171 (IgG1, anti- HEL115–129 peptide)
(Table I
). These mAbs recognize the free HEL peptide in solution or
bound to an ELISA plate. The anti-HEL peptide mAbs were generated
by repeated immunizations of mice with synthetic HEL peptides coupled
to a carrier protein using bromoacetyl succinimide (Sigma).
Specifically, the synthetic peptide HEL18–33
(DNYRGYSLGNWVSAAK) and HEL118–127 (TDVQAWIRGC)
were coupled to OVA, the HEL34–45 peptide was
coupled to the carrier protein keyhole limpet hemocyanin
(1), and the HEL48–61 peptide was
coupled to BSA. (There was no particular reason for the use of
different carrier proteins or mice; the mAbs were developed at
different times, involving more than one investigator.) In all peptides
except 118–127, a cysteine was added at the carboxyl terminus to
facilitate coupling to the carrier. In the 18–33 peptide, a serine was
substituted for cysteine at residue 30. Mice were first injected s.c.
with 200–400 µg of HEL peptide-carrier protein in CFA (Sigma),
followed by three i.p. injections in IFA (200–400 µg of
peptide-carrier protein/immunization) at 2- to 4-wk intervals. Finally,
3 days before B cell fusion, the mice were boosted i.v. with 100 µg
of HEL peptide-carrier protein in sterile saline solution. Spleen cells
from immunized mice were fused with the myeloma fusion partner P3
x 63.Ag8 using polyethylene glycol 1500 (Roche, Indianapolis, IN) by
standard procedures. The B cell hybridomas were selected by testing
supernatants in ELISA to plates coated with the peptides. Positive
supernatants were immediately screened by the binding to free peptide
in the same ELISA procedure (see below).
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10-fold better) or than the
HEL48–63 peptide (300-fold better). The same
pattern of peptide recognition was observed with other mAbs, suggesting
that these HEL peptides in solution adopt different conformations.
Based on this observation, we used a mix of the HEL peptides 48–61,
48–62, and 48–63 as standard peptide in the
HEL48–61 ELISA. The molar ratio of these
synthetic peptides in the mix was equal to the molar ratio observed
among the naturally processed forms of these peptides on the B lymphoma
cell line (M12-Ak mHEL) and other APC (4, 5, 10). The ratios established are 48–61/62/63 = 10/45/45.
The 15B7-1B5 (anti-HEL18–33 peptide) and the
VAL-3 (anti-HEL31–47 peptide) mAbs did not
show any preferential recognition for any member of the
HEL18–33 or HEL31–47
peptide families, respectively. Our chemical studies of the 115–129
epitope are in progress. Thus, we used the synthetic
HEL18–33, HEL31–47, and
HEL115–129 peptides as standard peptides to
generate calibration curves in the ELISAs. Isolation of I-Ak-bound HEL peptides
Class II I-Ak molecules were isolated from
4 x 109 to 1010 B
cells (M12-Ak mHEL) or control B cells
(M12-AK; 20 l of cell culture) and from the
spleens and thymi of 132 mHEL or ML-5 transgenic mice (7)
by immunoaffinity chromatography as previously described
(1), but with some modifications to improve the peptide
isolation method. (Although we present results using 6 x
109 cells, the method for quantitation can easily
detect peptides in 107 cells.)
The APCs were lysed with 40 mM MEGA8/MEGA9 detergent (Sigma) in PBS with enzyme inhibitors (1 mM PMSF, 10 mM iodoacetamide, and 20 µg/ml leupeptin). The suspension was centrifuged at 8000 rpm for 30 min at 4°C. The I-Ak molecules were isolated from the supernatant using anti-I-Ak mAb 40 F (14). Specifically, the supernatant was incubated with mAb 40 F coupled to cyanogen bromide-activated Sepharose beads (Sigma) overnight at 4°C. Then the Sepharose beads were loaded into a BSA-precoated chromatography column (Bio-Rad, Hercules, CA). The column was precoated to minimize loss of peptides by incubation with 1% BSA in PBS for 1 h at room temperature and then washing with PBS. The Sepharose beads were washed with 5 column volumes of 10 mM MEGA 8/MEGA9 in PBS, 5 column volumes of 2.5 mM MEGA 8/MEGA9, at least 25 column volumes of PBS, and 5 column volumes of Milli-Q water for the removal of excess salt. The I-Ak-peptide complexes were subsequently eluted with 0.1% trifluoroacetic acid. Peptides were separated from I-Ak molecules by filtration through a Centriprep (YM-10) centrifugal filter device (Amicon, Beverly, MA); the peptide extract was dried, reconstituted in a total volume of 100–200 µl of 1% BSA in PBS, and subsequently analyzed in the different HEL peptide ELISAs.
HEL peptide quantitation
We developed four ELISAs to estimate the HEL peptide contents
extracted from I-Ak molecules. The Nunc Maxisorp
ELISA plate (Nunc, Roskilde, Denmark) was precoated with 100 µl of 1
µg/ml streptavidin (Sigma) in 0.1 M sodium bicarbonate buffer (pH
8.8) overnight at 4°C. The plate wells were washed with PBS-0.05%
Tween 20 and incubated with 100 µl of 0.1–0.4 µM biotinylated HEL
peptide in 0.1 M sodium bicarbonate buffer, pH 8.8, for 75 min at room
temperature. The plate was blocked with PBS-1% BSA for 1 h at
room temperature and washed. In an additional 96-well U-bottom plate
(Costar, Corning, NY), 100 µl of 0.025 µg/ml anti-HEL peptide
mAb in PBS-1% BSA solution was added per well and incubated for 75 min
at room temperature with known quantities of synthetic HEL peptide or
several dilutions of the peptide extract from the APC (the total volume
in the well was 110 µl). After this incubation time, 100 µl from
each well was transferred into an ELISA plate well, and the incubation
was continued 75 min longer. The ELISA plate was washed, and 100 µl
of secondary Ab (goat anti-mouse IgG-peroxidase; Roche) in PBS-1%
BSA was added per well. The ELISAs were developed with 1 mM
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in citrate buffer
with 0.03% H2O2 (Roche),
and the absorbency was measured at 415 nm. The total amount of HEL
peptides contained in the samples was determined by referring to the
calibration curves made with synthetic peptides. The recovery of
peptides was determined using radiolabeled peptides, quantitating their
amounts through the different steps of the procedure (Fig. 3).
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Binding to I-Ak molecules was performed as
described by Latek et al. (15, 16), but using a
baculovirus-produced preparation of I-Ak
molecules. Indicated in Table III is the concentration of unlabeled
peptide required to inhibit by 50% the binding of a standard peptide
(sequence YEDYGILQINSR). Binding was performed at the optimal pH of 5.5
for each peptide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An important aspect in the analysis of peptides selected and bound to class II MHC molecules during protein processing by APC is their quantitation. The standard procedure consists of fractionating the peptide extract by reverse phase HPLC, analyzing every peptide fraction in a T cell assay, and then sequencing the peptides that stimulate the T cell using mass spectrometry analysis (1, 2, 3, 17, 18, 19, 20, 21). For the class II MHC-bound peptides, this strategy introduces serious problems, particularly with regard to those peptides displayed in low abundance on APC. There is peptide loss during all the steps of peptide isolation and also during the chemical analysis. Another limitation is that the T cell assay skews against low affinity binding peptides, which are required to be at a high molar concentration and are competed by other peptides present in the same HPLC fraction. These problems can be resolved by the immunochemical approach described in this paper, which does not depend on a T cell readout and which avoids losses during the peptide isolation protocols and HPLC analysis.
We developed a sensitive and specific ELISA to quantitate the HEL
peptides isolated from I-Ak molecules using mAbs
specific for the core sequences of the main
I-Ak-restricted HEL epitopes,
HEL18–33, HEL31–47,
HEL48–61, and HEL115–129
(Table I
) (7). These Abs
were selected for their ability to recognize peptides both in solution
and bound to an ELISA plate. The Abs remove most of the T cell-reactive
peptides from solution (1, 22) (our unpublished
observations). Comparing the amount of inhibition by peptide extract
from I-Ak to the standard inhibition with
synthetic peptides allowed quantitation of the epitopes. Fig. 1 shows the calibration curves for the
four peptides in a competitive ELISA. The amount of the standard
synthetic peptide required to inhibit 30–50% of the maximum OD
reading was 0.3 pmol for both HEL18–33 and
HEL31–47, 1.0 pmol for
HEL48–61, and about 25 pmol for
HEL115–125 assay (Table I). The contents of HEL
peptides in the extract isolated from APCs were calculated using these
calibration curves.
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shows a representative result that
illustrates the procedure used to detect
HEL31–47. We previously reported the isolation
and quantitation of 31–47 peptide using electrospray tandem mass
spectrometry and the same Ab as a device to capture the peptide for
mass spectrometry analysis (1, 22). Quantitating the
peptides with both approaches gave roughly comparable results. For
example, we estimated using the current ELISA method in which the
amount of peptide eluted from I-Ak was 10.2
pmol/109 cells, while by mass spectrometry we
first reported 11.9 pmol/109 cells
(1). (The amounts recovered were corrected for losses,
which were estimated to be 69% for the ELISA approach and 35% for the
mass spectrometry approach.) However, quantitation by mass spectrometry
has varied as much as 100% in subsequent trials (22),
while the ELISA method described here is highly reproducible (Fig. 3).
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HEL peptides from B lymphoma cells line expressing HEL
We conducted five experiments quantitating the four families of
HEL peptides from a B lymphoma line (M12-Ak.mHEL)
that expresses a membrane form of HEL. We found very marked differences
in the amounts of HEL peptides selected by the
I-Ak class II molecule (Fig. 3). From five
different experiments a mean of 2260 pmol of the 48–61 family of
peptides was isolated from 5.6 x 109 APCs
(Table II). The 31–47 set of
peptides was the second most abundant peptide represented in
I-Ak, with a mean of 38.8 pmol, amounts
58-fold less than that for 48–61. Peptide 18–33 was represented
251-fold less than 48–61 at 9 pmol. The actual characterization of
this family of peptides is now in progress; the natural epitope is
actually 20–35, in which the core sequence encompasses residues 24–32
(C. Velazquez, I. Vidavsky, K. van der Drift, M. Gross, and E. R.
Unanue, manuscript in preparation). Finally, peptide 115–129 could not
be detected, suggesting that the abundance of this epitope is below the
limit of detection of the ELISA, which is 25 pmol.
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. The 48–61 peptides bind with the highest
affinity (4, 5, 10, 15). The 20–35 peptide and 31–47
bind 7- and 13-fold less, respectively, than 48–61. Our studies with
115–129 are not completed, but this epitope appears to be the weakest
binding peptide for I-Ak molecules.
We compared the results of M12-Ak.mHEL with those
of M12-Ak cultured with exogenous HEL. More HEL
peptides were recovered, but the relative proportions were identical
with the line expressing the mHEL (Fig. 3B and Tables II and III). As expected, analysis of peptide
extract from control APCs (M12-Ak) did not detect
peptide in the different ELISAs (Fig. 2). Thus, the processing of the
vesicular bound protein and that of the exogenous protein are
identical.
HEL epitope quantitation on APCs from spleens and thymi of HEL transgenic mice
To determine whether the large differential in peptide
distribution also applies to physiological APC, particularly in an in
vivo situation, we examined the class II-bound peptide from the spleens
and thymi of 132 mHEL transgenic mice. These mice express a
membrane-bound form of HEL on all APCs (under a class II promoter)
(7). The relative abundance among HEL peptides on both
tissues was comparable to that observed in the B cell line, i.e.,
HEL48–61 peptide >
HEL31–47 peptide >
HEL18–33 peptide >
HEL115–129, which was not detected (Table III
and Fig. 3B). The molar ratio between the most abundant
peptide (HEL48–61) and the least abundant
(HEL18–33) detectable peptide on APCs from
spleen was 217-fold (Fig. 3B). The amount of HEL peptides
isolated from the spleens was about 10-fold higher than the amount of
HEL peptides recovered from thymi (Table II).
We also examined the content of HEL peptides from the ML-5 HEL
transgenic mice developed in the Goodnow laboratory: these mice
released soluble HEL at 10–20 ng/ml of blood (12, 13).
Although the tissue APC from these mice expressed considerably less
HEL, 48–61 was the chemically dominant peptide family.
Experiments in progress with isolated dendritic cells or macrophages indicate the same ratios in selected peptides. Thus, our conclusion is that although APC vary in their efficiency in yielding total peptides, the processing of HEL and the selection of peptides result in the same relative quantitative display, with some peptides in higher abundance than others.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides of the 48–61 family are clearly chemically dominant and, likewise, have a higher binding strength for the I-Ak molecule. The differences in the binding strength of the 48–61 family compared with those of the other HEL peptides is quite notable. In experiments in progress we have found that reducing the binding strength of the 48–61 segment by changing the Asp52 residue, the main residue that accounts for binding (23, 24), results in a marked loss in the amount selected and presented (experiments to be published with Robert Latek). Likewise, the 115–129 peptide has the weakest binding affinity for I-Ak and is the least represented (biochemical experiments on this epitope are in progress with Ravi Veraswamy). Thus, for these two families of peptides a relationship exists between degree of binding and selection. However, when examining and comparing the other two peptides, a strict one-to-one relationship between binding and display is not found. The 20–35 peptide family binds to I-Ak slightly better than 31–47, yet the 31–47 peptide is expressed in severalfold higher amounts. Other factors need to be taken into account in explaining the presence of a peptide bound to MHC molecules from the vesicles in which processing takes place, to the roles of auxiliary molecules in peptide editing, to their protease sensitivity and half-life in the APC of the peptide-MHC complex (5, 25, 26, 27, 28, 29).
The second issue is the lack of relationship between the relative abundance of peptide presentation and the number of T cells responding to each epitope after immunization. We previously reported that the number of T cells responding to each of these four epitopes was essentially the same after immunizing mice with 10 nmol of HEL protein in CFA (7). Although the HEL18–33 peptides are presented at close to 250-fold lower amounts than the HEL48–62 peptides, similar numbers of T cells were generated after immunizing mice with HEL protein. That is, there is not a direct correlation between the levels of presentation among these four epitopes and the numbers of T cells that are primed after immunizing with 10 nmol of HEL in CFA. We are currently investigating factors that may explain how similar numbers of T cells can be primed to epitopes that are presented at such different levels. Clearly the conditions used to activate T cells in the previous studies led to high levels of presentation of all epitopes under optimal priming conditions (CFA). We speculate that under such conditions the response becomes independent of the vast differences in the levels of presentation shown here. The dependence of the T cell response to these different epitopes on the amount of Ag and on the expression of costimulatory molecules is currently under investigation by testing lesser amounts of HEL, including costimulator-deficient mice.
Finally, we had established that tolerance in mHEL or ML-5 mice
was profound. When transgenic mice expressing a membrane form of HEL in
all their APC were immunized with 10 nmol of HEL in CFA, we could not
detect any T cells to HEL, including the minor epitopes now quantitated
in this analysis. Transgenic mice with soluble HEL at 10–20 mg/ml of
blood (ML-5) responded with 1/10th the number of clones. Yet all
epitopes were affected. Direct analysis of the amounts of peptides from
thymi of these mice allows us to further interpret these published
results (Table IV). In the mHEL we
calculate for the thymus that there are about 2600 molecules of the
48–61 family peptides/106 molecules of
I-Ak, while the 31–47 and 18–33 peptides have
45 and 13, respectively. In ML-5 mice the figures for the three
peptides are 284, 5, and 1 molecule of
peptide/106 I-A molecules. In neither of these
strains did Peterson et al. find a differential effect on tolerance to
peptides, that is to say, all four epitopes were affected to the same
degree when immunizing with 10 nmol of HEL in CFA (7). The
inevitable conclusion is that central tolerance is taking place with
single digits of peptide-MHC complex. (We are assuming that APC
have from 105 to 106 I-A
molecules/cell.) These figures match our own results ex vivo, where the
amounts of peptide-MHC complex needed to tolerize double-positive
thymocytes was two or three per APC (30).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Emil R. Unanue, Washington University School of Medicine, Department of Pathology and Immunology, 660 South Euclid Avenue, St. Louis, MO 63110.
3 Abbreviations used in this paper: HEL, hen egg-white lysozyme; mHEL, membrane form of HEL.
Received for publication December 7, 2000. Accepted for publication February 23, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), M12-Ak mHEL (
), and APCs from spleen (
)
and thymi (•) of mHEL transgenic mice). The relative abundances of
the HEL peptides were similar in the different APC analyzed.
, OD
reading in the presence of irrelevant peptide.