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Département d’Ingénierie et d’Etudes des Protéines, Commissariat à l’Energie Atomique-Saclay, Gif sur Yvette, France; and
Institut National de la Santé et de la Recherche Médicale U396, Hôpital St Louis, Paris, France
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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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T cell epitopes are indeed carried by peptides that are derived from
proteins of the allergenic extracts, and which complex to the molecules
of the MHC of class II (MHC II or HLA II in humans). These molecules
bind a large array of peptides by using few peptide residues as anchors
(11) and by interacting with the peptide backbone
(12, 13). Most of the polymorphic residues reside in the
peptide binding groove and evidently are responsible for MHC II binding
specificity (14). As a result, allergens as well as other
Ags are recognized by specific T lymphocytes through various regions,
depending on the expressed MHC II molecules (15). In
particular, these regions depend on the HLA-DRB1 molecules that are the
most abundant ones at the surface of APC. More than 200 different
alleles have been described for the HLA-DRB1 locus (16),
suggesting an impressive number of potential T cell epitopes within a
single allergen. This elevated number of alleles does not allow an
exhaustive description of the T cell epitopes diversity. However,
alleles are not equally distributed worldwide. In defined populations,
a limited number of alleles are preponderant and are present in the
majority of individuals. In Caucasian populations, seven alleles
(DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and
DRB1*1501) cover 
60% of the HLA-DRB1 allele frequency. Moreover,
peptides common to several HLA-DR alleles exist (17, 18, 19)
and illustrate similarities in the peptide binding modes
(20, 21, 22). By using binding assays specific to these HLA-DR
alleles, we may thus expect to delineate a reasonable number of peptide
binders from an allergen and to combine them in minimal peptide
sequences, suitable for immunotherapy of most of the patients.
In this paper, we have tentatively developed this approach to a major
allergen, the bee venom phospholipase A2 (API
m1).2 Bee venom
immunotherapy is a well-suited treatment for patients suffering from
severe reactions to bee stings, but it causes undesirable side effects
for 15% of them (23). A number of T cell epitopes from
API m1 have already been delineated by proliferative cellular assays,
performed with PBMC from allergic patients (9). Three
peptides, namely 45–62, 81–92, and 113–124, have been selected and
successfully used to desensitize some patients (7).
However, other studies have also revealed that human T cell response to
API m1 involves multiple epitopes (10, 24), thus
addressing the question of the peptide sequences to be used. We have
adapted peptide binding assays to the seven preponderant HLA-DRB1
alleles found in Caucasian populations and investigated the capacity of
overlapping peptides encompassing the whole API m1 sequence to bind to
each of them. We have delineated allele-specific and promiscuous
binding regions that are interesting candidates for specific
immunotherapy and illustrate the functional similarities between
HLA-DRB1 alleles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides were synthesized on an Advanced ChemTech model 357 MPS synthesizer (Advanced Chemtech Europe, Brussels, Belgium) as previously described (15). API m1 peptides sequences were based on the amino acid sequence deduced from the cDNA (25). HA306–318 (PKYVKQNTLKLAT), A3152–166 (EAEQLRAYLDGTGVE), MT2–16 (AKTIAYDEEARRGLE), YKL (AAYAAAKAAALAA), and B121–36 (TERVRLVTRHIYNREE) were biotinylated with biotinyl-6-aminocaproic acid (Fluka Chimie, St Quentin Fallavier, France) on the N terminus. Peptides were cleaved from the resin by 95% trifluoroacetic acid and purified by reversed-phase HPLC on a C18 Vydac (Interchim, Montluçon, France) column. Their quality was assessed by electrospray mass spectroscopy.
Purification of HLA-DR molecules
EBV homozygous cell lines were used as sources of human HLA class II molecules. HOM2 (DRB1*0101), SCHU (DRB1*1501, DRB5*0101), STEILIN (DRB1*0301, DRB3*0101), PITOUT (DRB1*0701, DRB4*0101), and SWEIG (DRB1*1101, DRB3*0202) were from Prof. H. Grosse-Wilde (European Collection for Biomedical Research, Essen, Germany). BOLETH (DRB1*0401, DRB4*0103) and 0206AD (DRB1*1301, DRB3*0101) were kindly provided by Dr. J. Choppin (Hôpital Cochin, Paris) and Prof. J. Dausset (Centre d’Étude du Polymorphisme Humain, Paris), respectively. HLA-DR molecules were purified by affinity chromatography using the monomorphic mAb L243 (American Type Culture Collection, Manassas, VA) coupled to protein A-Sepharose CL 4B gel (Amersham Pharmacia Biotech, Orsay, France) (26). Briefly, cells were lysed on ice at 5 x 108 cells/ml in 150 mM NaCl, 10 mM Tris-HCl (pH 8.3) buffer containing 1% Nonidet P40, 10 mg/L aprotinin, 5 mM EDTA, and 10 µM PMSF. After centrifugation at 100,000 x g for 1 h, the supernatant was applied to Sepharose 4B and protein A-Sepharose 4B columns and then to the specific affinity column. HLA-DR molecules were eluted with 1.1 mM n-dodecyl ß-D-maltoside (DM), 500 mM NaCl and 500 mM Na2CO3 (pH 11.5). Fractions were immediately neutralized to pH 7 with 2 M Tris-HCl (pH 6.8) buffer and extensively dialyzed against 1 mM DM, 150 mM NaCl, 10 mM phosphate (pH 7) buffer.
HLA DR peptide binding assays
HLA-DR molecules were diluted in 10 mM phosphate, 150 mM NaCl, 1 mM DM, 10 mM citrate, and 0.003% thimerosal buffer with an appropriate biotinylated peptide and serial dilutions of competitor peptides. More precisely, HA306–318 was used at pH 6 for the 101 and 401 alleles at 10 nM and 30 nM concentration, respectively, and at pH 5 for the 1101 allele at 20 nM concentration. YKL (10 nM) was used for the 701 allele at pH 5. Incubation was done at pH 4.5 for the 1501, 1301, and 301 alleles in the presence of A3152–166 (10 nM), B121–36 (200 nM), and MT2–16 (50 nM), respectively. Samples (100 µl per well) were incubated in 96-well polypropylene plates (Nunc, Roskilde, Denmark) at 37°C for 24 h, except for the 1301 and 301 alleles which were incubated 72 h. After neutralization with 50 µl of 450 mM Tris-HCl (pH 7.5), 0.003% thimerosal, 0.3% BSA, and 1 mM DM buffer, samples were applied to 96-well Maxisorp ELISA plates (Nunc) previously coated with 10 µg/ml L243 mAb and saturated with 100 mM Tris-HCl (pH 7.5), 0.3% BSA, and 0.003% thimerosal buffer. They were allowed to bind to the Ab-coated plates for 2 h at room temperature. Bound biotinylated peptide was detected by incubating streptavidin-alkaline phosphatase conjugate (Amersham, Little Chalfont, U.K.), and after washings, by adding 4-methylumbelliferyl phosphate substrate (Sigma, St Quentin-Fallavier, France). Emitted fluorescence was measured at 450 nm upon excitation at 365 nm on a Fluorolite 1000 fluorometer (Dynex, Issy les moulineaux, France). Maximal binding was determined by incubating the biotinylated peptide with the MHC II molecule in the absence of competitor. Binding specificity was assessed by adding an excess of nonbiotinylated peptide. Background did not significantly differ from that obtained by incubating the biotinylated peptide without MHC II molecules. Data were expressed as the peptide concentration that prevented binding of 50% of the labeled peptide (IC50). Average and SE values were deduced from at least three independent experiments. Validity of each experiment was assessed by reference peptides. Their IC50 variation did not exceed a factor of three.
Statistical analysis
The factor analysis was performed using SPSS 8.0 software (SPSS France, Paris). Maximum likelihood method was applied to extract the principal components. The varimax method of rotation was used to facilitate the three-dimensional graph interpretation. Validity of the model was assessed by Kaiser-Mayer-Olkin (KMO) (KMO = 0.76) and Barlett’s tests (p = 10-6). Variance analyses (ANOVA) were performed using STATVIEW 5.0 software (Abacus Concepts, Berkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To delineate interesting peptides for specific immunotherapy, we
established peptide binding conditions for seven HLA-DRB1 preponderant
alleles (Table I
). Each allele was
present in >5% of the HLA-DRB1 allele frequency in the French
population and altogether reach the HLA-DRB1 allele frequency of 63%
and 58% in France and in the United States, respectively
(27). Since we immunopurified these alleles from
homozygous EBV cell lines using the monomorphic L243 mAb, the HLA-DR
preparations also contained the second gene product (see
Materials and Methods). To overcome this contaminating
activity, we cautiously selected biotinylated peptides for each assay.
These peptides derived from the most efficient binders to the first
gene product and did not bind at low concentration to the second one.
HA306–318 was used for the 101, 401, and
1101 alleles in agreement with previous data (20, 28, 29)
and in particular with those obtained with transfected cells (28, 30). However, it was not suited to perform binding assays with
the 1501 allele because it efficiently bound to the DRB5*0101 molecule
(28, 30, 31). We thus used the naturally eluted
A3152–166 peptide, which was unambiguously
restricted to the 1501 allele (31). Because of the
moderate affinity of the HA306–318 peptide, we
used other biotinylated peptides for investigating the 701, 1301, and
301 alleles. These are, respectively, the synthetic YKL peptide
(28), the naturally eluted B121–36
peptide (32), and the MT2–16 T cell
epitope (33). Competitive experiments with YKL on
DRB4*0101 and with B121–36 and
MT2–16 on DRB3*0101 alleles confirmed their weak
reactivity for these molecules (data not shown). The binding assays
were therefore specific to the HLA-DRB1 products. They were also
sensitive because the IC50 values of the
nonbiotinylated peptides ranged between 14 and 330 nM (Table I).
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The competitive ELISA allowed us to evaluate the relative binding
affinities of an extensive number of peptides from the major bee venom
allergen (API m1) to each allele. We first tested a set of 30 peptides
that encompassed the whole API m1 sequence (25) and that
we previously used for characterizing the T cell response to API m1 in
BALB/c mice (15). These peptides of 18 aa contained all
possible peptides of 15 residues present in API m1 sequence. They were
expected to cover most, if not all, binding determinants
because 15 aa was the length most frequently found in naturally
processed peptides (34). As proposed by others (22, 35), we discriminated the active and inactive peptides on the
basis of an upper 1000 nM threshold. As shown in Table II, the 101, 401, and 1101 alleles
displayed a similar binding pattern. In particular, four peptides
(P81–98, P85–102, P89–106, and P93–110) bound to them with good
efficiency. One peptide (P105–122) exhibited a good binding activity
to the 401 allele only, whereas two others (P77–94 and P117–134)
bound to the 1101 allele. The 701 allele was characterized by seven
peptides with a substantial binding ability (P13–30, P17–34, P21–38,
P45–62, P77–94, P81–98, and P85–102), whereas four peptides
(P53–70, P57–74, P81–98, and P85–102) were active toward the 301
allele. The P81–98 and P85–102 peptides also bound efficiently to the
1301 allele as did the P117–134 peptide. Finally, two regions of high
affinity were found for the 1501 allele and comprised on one side the
P65–82, P69–86, P73–90, P77–94, P81–98, and P85–102 peptides and
on the other one the P113–130 and P117–134 peptides. Clearly, each
allele displayed a unique binding pattern of API m1 peptides, but
interestingly a number of peptides were common to several alleles. In
particular, the P81–98 peptide bound efficiently to all the alleles,
whereas the P85–102 peptide bound to six of them. The common peptides
defined three distinct regions: a N-terminal region (P13–30 to P21–38
peptides, most of them being of moderate activity), a central one
(P77–90 to P93–110 peptides), and a C-terminal one (P105–122 to
P117–134 peptides). We also observed that 13 peptides of the 30 ones
tested displayed few, if any, binding activity irrespective of the
allele used in the assay.
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To compare the binding areas of API m1 determinants, we used three
sets of 13-aa-long peptides that encompassed the three identified
common regions in an exhaustive manner. The length of 13 residues was
expected to be small enough to discriminate between two HLA-DR contact
areas in an 18-mer peptide, as we previously did with MHC II molecules
from BALB/c mice (15). The N-terminal part of API m1 was
analyzed with 11 peptides for the 101, 401, 701, and 1101 alleles (Fig. 1). The P18–30 to P22–34 peptides bound
efficiently to the 701 allele; in particular, the P18–30 peptide was
almost as active as the corresponding 18-mer peptide. The 101, 401, and
1101 alleles bound approximately the same peptides as the 701 allele,
but the binding activities were weaker in agreement with the
IC50 values of the corresponding 18-mer peptides.
The central part of API m1 was investigated for the seven alleles using
24 peptides of 13 residues (Table III).
For the 101 allele, the active peptides (P85–97 and P91–103 to
P95–107) defined two distinct binding regions. These regions were also
found for the 401 and 1101 alleles, but presented slight variations.
The first region also extended to the P82–94 and P83–95 peptides for
the 401 allele and to the P83–95 peptide for the 1101 allele. The
second region was reduced to only one peptide for the 1101 allele
whereas it was strictly identical between the 101 and 401 alleles. None
of the 13-mer peptides reproduced the activity level of the P77–94
peptide for both the 1101 and 0701 alleles. The latter was
characterized by three active peptides (P85–97 to P87–99) in
agreement with the IC50 values of the P81–98 and
P85–102 18-mer peptides. The P85–97 peptide efficiently bound to both
the 301 and 1301 alleles, whereas the P86–98 peptide was active toward
the 1301 allele only. Seven peptides (P73–85 to P78–90 peptides and
P81–93 peptide) were as efficient binding to the 1501 allele as the
corresponding 18-mer peptides (P73–90, P77–94, and P81–98). Finally,
the C-terminal part of API m1 was analyzed for the 401, 1501, 1101, and
1301 alleles using sixteen 13-mer peptides (Fig. 2). The 401 allele was the only one for
which the six P109–121 to P114–126 peptides were active. Seven other
peptides (P116–128 to P122–124) bound to the 1501 allele, whereas the
P121–133 and P122–134 peptides exhibited a high binding activity to
the 1101 and 1301 alleles.
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. This list is predominantly composed
of 13-mer peptides; however, 18-mer peptides have been sometimes
introduced if the 13-mer peptides did not display a correct activity.
Note that the peptide P85–97 binds to the 101, 401, 1101, 701, 301,
and 1301 alleles and reaches an impact level in Caucasian populations
of 55% (Table IV). This sequence is surrounded by various
determinants, namely P76–88, P77–94, P81–93, and P94–106 peptides.
Even though they have a moderate frequency, their combination with the
P85–97 peptide provides at least one determinant to each allele. Then,
the recognition level of the 76–106 region reaches the maximal
frequency of about 63%. In particular, the sequence 81–97, which is a
combination of the P81–93 and P85–97 peptides, is active for all the
alleles tested. In the C-terminal extremity of API m1, the P122–134
peptide has a nonnegligible frequency of 23%. Other peptides (P18–30,
P45–62, P57–74, P65–82, and P111–123) are allele-specific and have
lower importance in the Caucasian populations.
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Taking advantage of our extensive binding data, we
applied statistical analyses that could account for functional
similarities and differences of these representative HLA-DR molecules
toward the API m1 peptides. We first applied a factorial analysis and
then represented graphically the functional similarity of the HLA-DRB1
alleles (Fig. 3). In this approach, we
only used data from 18-mer peptides because they entirely cover the API
m1 sequence. By this method, the 101 and 401 alleles were nearly
indistinguishable and in proximity to the 701 and 1101 alleles. The
301, 1301, and 1501 alleles constituted an heterogeneous group in which
the 301 allele was clearly apart from the others. The seven HLA-DRB1
alleles display 14 polymorphic residues in the peptide binding groove,
which are responsible for their binding specificity (14).
It is likely that some of them only affect the peptide activities. We
therefore evaluated the influence of each HLA-DRB1 polymorphic position
on the API m1 binding pattern by a variance analysis (Table V). More precisely, for each position, we
measured the interaction effect between the two factors that
contributed to the IC50, namely, amino acid at
that position and peptides. By this method, we found that the
well-known ß86 position of HLA-DR molecules exerted a very
significant effect in complete agreement with the factor analysis (Fig. 3). The main component separated the 0301, 1301, and 1501 alleles from
the other ones (101, 401, 1101 and 701), which precisely differed from
the former ones by position ß86 (Table V). The other positions have
lower influence, except perhaps the position ß71
(p = 0.07). We could not exclude that the lack
of apparent influence is hidden by the effect of the position
ß86.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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illustrate
the main different regions of API m1 interacting with these
preponderant alleles. The 13-mer P85–97 peptide of API m1 binds to six
of the seven alleles, and when it is combined with the peptide P81–93,
all alleles are bound. Therefore, the 81–97 region appears as a
minimal sequence for binding all these alleles. Universal
HLA-DR-restricted peptides have been previously reported for other
proteins (17, 18). However, few of them, especially from
natural sequences (19), also bind to the 301 allele
(36). In addition, we found that elongation of the N- and
C-terminal parts of the P81–97 peptide to the P76–106 peptide
advantageously encompasses other active regions. Thus, the P76–106
peptide contains one determinant of the 0301 and 1301 alleles; two of
the 101, 401, 701, and 1501 alleles; and three of the 1101 allele.
Addition of the P57–74 and P122–134 peptides also increases the
number of determinants for the 0301, 1101, 1301, and 1501 alleles.
Other peptides described here are of lower incidence. As demonstrated
previously (15), peptides that bind to MHC II molecules
are not necessarily T cell stimulating, but previous papers confirmed
the in vivo potency of some of them. Three of 10 patients
respond to the P81–99 peptide (10), whereas the P81–98
peptide elicits a significant T cell proliferation for 46% of the
allergic patients (9). Both peptides encompass the 81–97
sequence that we identified and that is contained in the 76–106
sequence. Other fragments such as the P45–62, P72–90, and P113–124
peptides have also been reported to stimulate T cells (9)
and may also correspond to particular HLA-DRB1 alleles such as 701,
1501, and 401, respectively. As inferred from T cell clone
investigations (9, 24), we cannot exclude that these T
cell epitopes are restricted to other MHC II molecules such as DQ
(9, 24) and DP (24). Nevertheless,
HLA-DR-restricted T cells have been also isolated but their peptide
specificity remains unknown (9, 37). Further
investigations are required to tackle this question, and they may be
facilitated by our binding data. Our results are also in agreement with
previous observations (10), showing that T cell response
to API m1 involves multiple epitopes. It was even suggested that long
fragments might be more appropriate than short peptides for
immunotherapy to encompass the maximum of T cell determinants. We
propose that such a sequence contains the 76–106 fragment at
least.
By these data, we document the capacities of the Caucasian preponderant
HLA-DRB1 alleles to recognize a natural protein, i.e., API m1. To
appreciate the differences of binding patterns, we applied statistical
analyses on values obtained with the peptides encompassing the whole
API m1 sequence. Clearly, each HLA-DR molecule possesses its own
binding profile, but two main subsets emerge as a result of a
dimorphism at position ß86 of HLA-DR molecules. Obviously, this
position is not the only one important polymorphic positions
contributing to binding data. Several peptide differences exist between
HLA-DR molecules that share identical amino acid at position ß86
(Table II). This position is occupied by either a glycine for alleles
101, 401, 701, and 1101 or a valine for the alleles 301, 1301, and
1501. This dimorphism valine/glycine is known to control the P1 anchor
residue (11, 28, 32, 38) and to contribute to the dimer
stability of HLA II molecules (39). Our binding data
suggest that position ß86 segregates the preponderant alleles upon
different binding modes. The alleles 101, 401, 701, and 1101 have in
common the use of the P1 position as primary anchor and hence have an
overlapping peptide repertoire (21, 22). For example, both
101 and 401 binding motifs of the alleles 101 and 401 accept Y96 as a
potential P1 anchor in the P94–106 peptide and Y87 and F88 in the
P85–97 peptide (11, 40, 41). Differences are also
observed in Fig. 3 between these alleles and the alleles 701 and 1101.
They may result from the size of the P6 pocket that is large enough in
the latter to accommodate bulky side chains (21). In sharp
contrast, the P1 position may not necessarily constitute the main
anchor residue in valine-possessing alleles. Interactions are also
ensured by other anchoring positions and in a different way as compared
with that of glycine-possessing alleles. For instance, the P85–97
peptide is likely to bind to the 0301 allele by using D94 and K96 as P4
and P6 anchors and thus are different from the 101 and 401 alleles
(36, 42). L59 in P1 and D62 in P4 may participate to the
binding of the P57–74 peptide to the 301 allele only (36, 42). In the P122–134 peptide, a 1501 motif exists with amino
acids V125 at P1, W128 at P4, and L131 at P7 (31), and it
does not fit with the binding specificity of the 1101 allele, to which
this peptide also binds (11). Suitable 1501 anchors
(31) are also found in the P76–88 peptide (Y68 in P4 and
L71 in P7) and in the P81–93 peptide (Y87 in P4 and L90 in P7); both
of which exclusively bind to the 1501 allele. Therefore, our results
provide an overview on the functionality of HLA-DR molecules, which is
consistent with their known specificity. This view may be useful to
address the basis of genetic linkage between disease and HLA typing. In
bee allergic patients, HLA-DR7 was found in higher frequency than in
control population (43), while it was the opposite for DR4
(44). Interestingly, we found that peptides P13–30,
P17–34, P21–38, and P45–62 bind with good efficiency exclusively to
the 701 allele. Only one peptide (P105–122) is exclusive to the 401
allele. Such differences may support previously described genetic
association. However, we have to also mention that nonassociated
HLA-DR, as exemplified by alleles 1501 and 301, also display their own
peptides. Particularities in the binding pattern could not therefore
furnish sufficient arguments to account for the genetic link between
bee venom allergy and HLA II molecules.
In this paper, we describe the regions from the major bee venom allergen that efficiently bind to the predominant HLA-DRB1 molecules of Caucasian populations. Considering the potentiality of T cell epitopes as therapeutic leads for specific immunotherapy, our results provide a sound molecular basis for developing this approach to bee venom allergy. They also give information on functional similarities and differences within the main Caucasian HLA-DRB1 alleles.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: API m1, bee venom phospholipase A2; DM, n-dodecyl ß-D-maltoside
Received for publication August 24, 1999. Accepted for publication January 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion in specific allergen-stimulated T cell cultures. J. Immunol. 154:4187.[Abstract]
ß dimer stability. Eur. J. Immunol. 23:1346.[Medline]
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M. Mandic, F. Castelli, B. Janjic, C. Almunia, P. Andrade, D. Gillet, V. Brusic, J. M. Kirkwood, B. Maillere, and H. M. Zarour One NY-ESO-1-Derived Epitope That Promiscuously Binds to Multiple HLA-DR and HLA-DP4 Molecules and Stimulates Autologous CD4+ T Cells from Patients with NY-ESO-1-Expressing Melanoma J. Immunol., February 1, 2005; 174(3): 1751 - 1759. [Abstract] [Full Text] [PDF]
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 C. Buhot, A. Chenal, A. Sanson, S. Pouvelle-Moratille, M. H. Gelb, A. Menez, D. Gillet, and B. Maillere Alteration of the tertiary structure of the major bee venom allergen Api m 1 by multiple mutations is concomitant with low IgE reactivity Protein Sci., November 1, 2004; 13(11): 2970 - 2978. [Abstract] [Full Text] [PDF]
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 M. Mandic, C. Almunia, S. Vicel, D. Gillet, B. Janjic, K. Coval, B. Maillere, J. M. Kirkwood, and H. M. Zarour The Alternative Open Reading Frame of LAGE-1 Gives Rise to Multiple Promiscuous HLA-DR-restricted Epitopes Recognized by T-helper 1-type Tumor-reactive CD4+ T Cells Cancer Res., October 1, 2003; 63(19): 6506 - 6515. [Abstract] [Full Text] [PDF]
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 M. J. Holtzman Drug Development for Asthma Am. J. Respir. Cell Mol. Biol., August 1, 2003; 29(2): 163 - 171. [Abstract] [Full Text] [PDF]
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S. Fournel, S. Neichel, H. Dali, S. Farci, B. Maillere, J.-P. Briand, and S. Muller CD4+ T Cells from (New Zealand Black x New Zealand White)F1 Lupus Mice and Normal Mice Immunized Against Apoptotic Nucleosomes Recognize Similar Th Cell Epitopes in the C Terminus of Histone H3 J. Immunol., July 15, 2003; 171(2): 636 - 644. [Abstract] [Full Text] [PDF]
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Y. Zhang, P. Chaux, V. Stroobant, A. M. M. Eggermont, J. Corthals, B. Maillere, K. Thielemans, M. Marchand, T. Boon, and P. van der Bruggen A MAGE-3 Peptide Presented by HLA-DR1 to CD4+ T Cells That Were Isolated from a Melanoma Patient Vaccinated with a MAGE-3 Protein J. Immunol., July 1, 2003; 171(1): 219 - 225. [Abstract] [Full Text] [PDF]
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 M. Jacquemin, V. Vantomme, C. Buhot, R. Lavend'homme, W. Burny, N. Demotte, P. Chaux, K. Peerlinck, J. Vermylen, B. Maillere, et al. CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A Blood, February 15, 2003; 101(4): 1351 - 1358. [Abstract] [Full Text] [PDF]
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F. A. Castelli, C. Buhot, A. Sanson, H. Zarour, S. Pouvelle-Moratille, C. Nonn, H. Gahery-Segard, J.-G. Guillet, A. Menez, B. |
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