By
From the * Laboratoire de Biologie Moléculaire du Gène, Institut National de la Santé et de la
Recherche Médicale U277 and Institut Pasteur, 75724 Paris, France; the Department of Genetics,
Medical Institute of Bioregulation and Ministry of Education, Science, Sports and Culture, Japan
Science and Technology Corporation, Fukuoka 812, Japan; and the § Information Genetique et
Structurale, Centre National de la Recherche Scientifique, Institut de Biologie Structurale et
Microbiologie, 13402 Marseille, France
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Abstract |
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The positive selection of CD4+ T cells requires the expression of major histocompatibility
complex (MHC) class II molecules in the thymus, but the role of self-peptides complexed to
class II molecules is still a matter of debate. Recently, it was observed that transgenic mice expressing a single peptide-MHC class II complex positively select significant numbers of diverse
CD4+ T cells in the thymus. However, the number of selected T cell specificities has not been
evaluated so far. Here, we have sequenced 700 junctional complementarity determining regions 3 (CDR3) from T cell receptors (TCRs) carrying V11-J
1.1 or V
12-J
1.1 rearrangements. We found that a single peptide-MHC class II complex positively selects at least 105 different V
rearrangements. Our data yield a first evaluation of the size of the T cell repertoire.
In addition, they provide evidence that the single E
52-68-I-Ab complex skews the amino
acid frequency in the TCR CDR3 loop of positively selected T cells. A detailed analysis of
CDR3 sequences indicates that a fraction of the
chain repertoire bears the imprint of the selecting self-peptide.
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Introduction |
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During development, thymocytes undergo two steps of selection, each involving interaction with self-MHC molecules (1). Positive selection rescues thymocytes from programmed cell death and ensures that the mature T cell repertoire is directed against foreign peptides bound to self-MHC molecules (5). Subsequently, negative selection eliminates, through clonal deletion, T cells with potentially autoreactive receptors (1, 2, 9, 10).
Using in vitro fetal thymic organ culture system from 2
microglobulin- or transporters associated with antigen processing (TAP)-deficient mice, it was shown that peptides
were important for positive selection of CD8+ T cells (11,
12). Similar experiments with mice expressing rearranged
TCR genes of known specificity revealed a stringent requirement for peptide recognition during the positive selection process (13). Affinity measurements of TCR-peptide-MHC interaction indicated that peptide-MHC
complexes capable of positive selection were of low affinity
for the TCR, whereas high affinity ones were deleting
ligands (17). More recently, natural self-peptides, extracted
from MHC class I groove and capable of driving positive selection, were identified (18, 19). It was shown that different peptides could select thymocytes expressing different
TCRs, suggesting that weak but specific interactions with
self-peptide-MHC complexes promote positive selection
of CD8+ T cells. Hence, a particular TCR could be selected by different peptides.
Peptide involvement in positive selection of CD4+ T
cells was addressed using genetically engineered mice designed to express MHC class II molecules complexed to a
single peptide. In mice lacking the MHC-encoded H-2M
molecule and involved in the removal of the class II-associated invariant chain peptide (CLIP)1 during the MHC class
II maturation process (20), almost all MHC class II
molecules are occupied with the CLIP peptide (24). Transgenic mice (Tg) for the I-A chain connected to the
E
52-68 peptide (25, 26) backcrossed to MHC class II-
and invariant chain-deficient animals express the single
E
52-68 peptide-I-Ab complex. Studies conducted with
these two types of Tg suggested that a large and diverse
repertoire of CD4+ T cells is selected (22). Staining
with anti-V
and -V
antibodies showed that a close to
normal spectrum of V
and V
was used by mature CD4+
T cells. Sequencing studies in the H-2M
/
model, from
two V
s and one V
, confirmed the polyclonality (24). Finally, positively selected cells were capable of responding to
immunization with several peptide antigens (22, 27).
In the E
52-68-I-Ab model, Fukui et al. (26) have shown
that the level of expression of this complex in the thymus
affects the CD4+ T cell selection dramatically. Transgenic
lines with low expression of E
52-68-I-Ab complexes positively select CD4+ T cells, whereas such cells are eliminated in the thymus of another line with high expression
(26). Furthermore, two thirds of the selected CD4+ T cells
react with the syngeneic cells that express the same MHC
class II molecules complexed to the natural set of self-peptides (25). In wild-type mice, such lymphocytes are eliminated by negative selection on bone marrow-derived cells
expressing wild-type class II molecules in the thymus (23,
24). These results indicate that the Tg repertoire of CD4+
T cells is different from the wild type. Furthermore, it was shown using mice expressing various transgenic TCRs
(which are positively selected in mice expressing wild-type
class II molecules) that these TCRs are not selected in the
CLIP mice or the E
52-68-I-Ab Tg (22). These observations imply that the selecting peptide influences, to some
extent, the emerging repertoire. However, the diversity of
the repertoire selected by a single peptide-MHC class II
complex has not been quantitated so far. In addition, the
available evidence does not rule out that a few positively selected specific TCR rearrangements have not yet been
detected over a polyclonal background.
These studies were designed to quantitate the number of
positively selected T cells. We have analyzed the and
T
cell repertoire of CD4+ T lymphocytes selected by the
E
52-68-I-Ab complex extensively. We show that CD4+
CD8
NK1.1
HSA
thymocytes selected on this complex
bear TCRs that include all V
s and 10 V
s tested. The J
usage and CDR3 length distribution of V
and V
chain
rearrangements are indistinguishable from the repertoire of
CD4+CD8
NK1.1
HSA
thymocytes from normal C57Bl/6
mice. Extensive sequencing of particular V
-J
combinations with the same CDR3 length enabled us to calculate
that a minimum of 105 different V
rearrangements are selected by the single peptide-MHC class II complex. Careful analysis of these sequences revealed some differences in
the CDR3 amino acid composition of T cells selected by
the single peptide-MHC complex or by wild-type MHC class II molecules. Altogether, our results provide a lower
limit on the size of the selected CD4+ T cell repertoire in
vivo and indicate that part of the repertoire bears the imprint of the selecting peptide.
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Materials and Methods |
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Animals.
Mice used in this study have been described elsewhere (26) and were bred in the animal facility at the Medical Institute of Bioregulation. In brief, Tg were produced by injection of DNA encoding thePurification of CD4+ T Cells.
Thymocytes were prepared from 6-wk-old mice and counted. CD4+CD8mRNA Extraction and cDNA Synthesis.
Poly(A)+ mRNA from CD4+CD8Immunoscope Analysis of V and V
Repertoires.
Sequencing of Particular V-J
Rearrangements with a Given
Length.
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Results |
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Single positive CD4 thymocytes were purified from the thymus of B2L, which are transgenic for a single peptide-MHC class II complex (26). The
repertoire analysis did not include CD4+CD8 NK1.1+ cells,
which were eliminated by specific lysis with antibodies and
complement, because it is known that such cells are selected by nonclassical MHC class I molecules such as CD1 (38).
mRNA from 3-4 × 105 CD4+CD8NK1.1
HSA
thymocytes was extracted and reverse transcribed into
cDNA. 23 V
- and 12 V
-specific PCR reactions were
then carried out. The product of each PCR was visualized by performing a runoff extension with internal C
- (or
C
-) specific fluorescent primer. Such primers enable
elongation through the CDR3 region of each amplified
product, and therefore reveal peaks of different sizes for all
V-J combinations after separation on a sequencing gel. Previous studies from our laboratory, using unstimulated
mouse splenocytes, have shown that, in all functional rearranged V
or V
segments, the CDR3 size patterns display
six to eight peaks spaced by three nucleotides. These peaks
are distributed in Gaussian-like patterns that are characteristic of polyclonal T lymphocytes (34). The repertoire analysis performed on single positive CD4 cells selected on the
E
52-68 peptide-I-Ab complexes is shown in Fig. 1. 22 out of the 23 V
genes were amplified (Fig. 1 A). Amplification of V
6 gene failed for technical reasons. All V
genes tested were amplified (Fig. 1 B). The size distribution
of CDR3 from each V
or V
family gives a set of about
seven peaks, each spaced by three nucleotides and corresponding to in-frame transcripts. For V
17 and V
19
genes that are pseudogenes in C57Bl/6 mice (39), in-frame transcripts are difficult to detect above the background of out-of-frame transcripts. To detect more subtle
modifications of the V
repertoire, we performed runoff reactions with 12 fluorescent primers that recognize individual J
segments for the V
4, V
8.2, V
10, V
11,
V
12, and V
14 families. The CDR3 distribution of specific V
-J
rearrangements was then obtained for the six
different V
s of two individual Tg. Our results clearly
show that for each V
, all J
s are used. Furthermore, the
CDR3 distribution of these rearrangements has a Gaussian-like profile (V
11 in Fig. 2 B; otherwise, data not shown).
We have observed previously that in every situation characterized by the expansion of specific clone(s) (35, 36, 42)
or the presence of oligoclonal populations (43), the analysis
with the Immunoscope technique always revealed perturbations of the CDR3 size profile. Therefore, the Gaussian-like
profile obtained for CD4+CD8
NK1.1
HSA
thymocytes
selected by a single peptide-MHC class II complex indicates that the repertoire is polyclonal.
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Repertoire analysis
was performed on CD4+CD8NK1.1
HSA
thymocytes
from C57Bl/6 mice as described in previous sections.
CDR3 size distributions obtained with C
or C
primers
for each V
or V
chain were compared with those from
cells selected by the single peptide-MHC class II complex
(Fig. 2 A and data not shown). Our results show that for all
V
and V
, the profiles are superimposable. On Fig. 2 A
are shown representative results for V
4, V
8.2, V
10, V
11, V
12, and V
14.
A more detailed analysis of V-J
rearrangements was
performed for V
4, V
8.2, V
10, V
11, V
12, and
V
14. Representative results obtained with V
11 are
shown on Fig. 2 B. The repertoires were superimposable
and no significant modification was found with any J
primer.
The relative J usage in the V
11, V
12,
and V
14 T cell population measured from one individual
mouse of each strain (B2L and C57Bl/6) is shown in Fig. 3.
The ratio of the area of the peaks generated with a given J
primer to the areas of all the J
primers was calculated as a
measure of the relative frequency of J
usage. This analysis
does not reveal any significant evidence of selection for a
particular J
and shows that the J
2 segments are used
more frequently than the J
1 segments as previously described in the normal repertoire (44). Altogether, these results strongly suggest that the T cell repertoire selected by a
single peptide-MHC complex is indistinguishable in terms
of V
, J
, and V
usage and CDR3 size distribution from
the one selected by multiple peptide-MHC class II complexes.
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Each peak corresponding to a
given CDR3 length contains multiple distinct sequences.
To evaluate the sequence complexity of the repertoire
from CD4+ T cells selected by the E52-68 peptide-I-Ab
complex, we have chosen to focus arbitrarily on two particular rearrangements with a given CDR3 length: V
11-J
1.1 with a CDR3 length of six amino acids and V
12-J
1.1 with a CDR3 length of eight amino acids. These
two V
s are well selected in B2L mice, but reasonably
abundant in both B2L and C57Bl/6 animals so that the anticipated complexity would be more amenable to our analysis. The same consideration guided the choice of the
CDR3 lengths.
Each peak corresponding to these rearrangements was
isolated and purified. The sequence mixture contained in
these peaks was cloned and every positive clone was sequenced. More than 700 sequences were collected. We
found that, in the two rearrangements tested, the number
of different sequences collected from each sample is in the
same range of magnitude, with a mean of 30 different sequences in the V11-J
1.1 peak with a CDR3 length of
six amino acids and a mean of 70 different sequences in the
V
12-J
1.1 peak with a CDR3 length of eight amino acids (Table 1). Hence, sequence diversity contained in a
given V
rearrangement with a particular length is the
same between wild-type mice and Tg. Since we could estimate the number of different sequences contained in a single peak, we calculated the minimal number of rearrangements positively selected by the E
52-68-I-Ab complex.
The V
11-J
1.1 combination with a CDR3 length of six amino acids represents 13% of the rearrangements using the
V
11 and J
1.1 gene segments. The J
1.1 is used in 5% of
the rearrangements involving the V
11 gene segment.
Staining with specific monoclonal antibody shows that 5%
of the CD4+CD8
NK1.1
HSA
thymocytes from the Tg
bear the V
11 chain (26). We then estimated that a minimum of 105 TCR-
rearrangements (30 × 7.7 [to correct
for the CDR3 length] × 20 [to correct for all the J
s] × 20 [to correct for all the V
s]) are selected by the peptide-
MHC class II complex. The same calculation was applied
for the V
12-J
1.1 rearrangement and a total of 7 × 104
TCR-
rearrangements were found.
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Among the
700 sequences collected, we found some recurrent sequences between animals. The results obtained with the
V11-J
1.1 and V
12-J
1.1 rearrangements are shown in
Fig. 4. For the V
11-J
1.1 rearrangement, only one recurrent amino acid sequence was found in all animals. This
CDR3 is generated by the same nucleotide sequence and is
encoded by the germline segments without trimming or N
nucleotide addition (data not shown). For the V
12-J
1.1, four recurrent sequences were found between the C57Bl/6
mouse and one Tg, and seven between the same C57Bl/6
mouse and another Tg. Between the two Tg, nine amino
acid sequences of the CDR3 were recurrent. Among these,
two CDR3 amino acid sequences were common to all animals tested: SLGANTEV and SLTANTEV (see Fig. 4).
The SLGANTEV sequence was found one time in the
C57Bl/6 animal, four times in one Tg, and two times in
the other Tg tested (Table 2). Nucleotide sequence analysis
of these rearrangements revealed that in each mouse, the
sequence was generated differently by the recombination
machinery (see Table 2). From the C57Bl/6 mice, the coding sequence was obtained with the trimming of three nucleotides from the V
12 germline segment, the usage of
four nucleotides from the D
1 segment, 1 P nucleotide addition between the D and the J segments, and the germline
sequence of the J
1.1 gene segment. This specific rearrangement was never found in the two Tg tested. In these
two animals, recombination events (V
trimming, D
usage, N nucleotide addition) were different (see Table 2).
However, one should notice that recurrent nucleotide sequences were also found between Tg. Comparable conclusions were reached when we analyzed the nucleotide sequences encoding the recurrent SLTANTEV (Table 2).
Altogether, these results strongly suggest that these two
CDR3 regions are not preferentially generated by the recombination machinery, but are positively selected at the
amino acid level.
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We
compared the amino acid usage in the CDR3 region of the
two sequenced rearrangements V11-J
1.1 (CDR3 length
of six amino acids) and V
12-J
1.1 (CDR3 length of eight
amino acids) of the CD4+CD8
NK1.1
HSA
thymocytes
from Tg and wild-type mice. No difference was detected between mice when the individual percentage of amino acids in the CDR3 was plotted (data not shown). However,
interesting differences became apparent when we plotted
the frequency of individual amino acids at each position in
the CDR3 loop (Figs. 5 and 6). Frequencies were calculated with nonredundant sequences in order to eliminate bias due to overrepresented sequences. Positions 2 and 3 for the V
11-J
1.1 rearrangement and 2, 3, and 4 for the
V
12-J
1.1 rearrangement are more variable due to N or
P nucleotide additions and reading frame usage of the D
gene segment. One can notice that for each variable position, a limited number of amino acid residues are found
and that they are similar in all mice. Furthermore, the percentage of amino acid residues found at each variable position (2, 3, and 4) is strikingly similar between C57Bl/6
mice and Tg. However, for the V
11-J
1.1 rearrangement, it appears that at position three of the CDR3 from
Tg, there is an increase in the frequency of the asparagine
residue as compared with wild-type mice (24 versus 8%; see
Fig 5). Similar skewing is also found with the V
12-J
1.1
rearrangement. When comparing amino acid frequency between wild-type mice and Tg, we found an increase in
leucine at position 2, threonine and glutamine at position
3, and glycine at position 4 of the CDR3 loop of transgenic
animals (Fig. 6). In contrast, in C57Bl/6 mice, glycine is
preferentially found in position 3 of the CDR3. Analysis of
nucleotide sequences encoding glycine or glutamine at this
position shows that both amino acids are encoded by the
D
1.1 gene segment in all CDR3s. Glycine can be encoded by all three D
1.1 reading frames. There are six possibilities to code for glycine. In Tg and wild-type mice, all
encoding possibilities are used (Fig. 7), indicating that there
is no bias due to the recombination machinery between
these mice. Strikingly, in Tg animals, glutamine is found
more frequently at this position (Fig. 6) even though this
residue is only encoded by frame 2 of the D
1.1 gene segment. Altogether these results suggest that this residue has
been selected for at the amino acid level. Furthermore, the
preferential usage of the D
1.1 frame 2 in the transgenic animals should favor the appearance of a glycine residue at
position 4. This increase in glycine frequency at position 4 is observed in transgenic animals and not in C57Bl/6 (Fig.
6). These differences in amino acid usage at different positions of the
chain CDR3 junctions reveal the imprint of
the E
52-68 peptide-I-Ab complex on the selected T cell
repertoire.
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Discussion |
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The involvement of peptides presented by MHC
molecules in the process of thymic positive selection of T
cells has been investigated in vivo (25, 26, 45) and in
vitro (11, 12, 14, 50). Recent studies (23, 24, 27) have
shown that positive selection is a promiscuous process enabling the selection of many different TCR rearrangements
by a single peptide-MHC class II complex, but their diversity has not been quantified so far. Here, we have estimated
the number of TCR rearrangements in mature thymocytes positively selected in Tg B2L mice (26) designed to express a single peptide-MHC class II complex (the E52-68 peptide hooked to I-Ab). Their Ii
/
genetic background guarantees that the physiological route of peptide loading is
blocked (29). Similar mice, engineered by Ignatowicz et al.
(25), have been shown (contrary to H-2M
/
mice) to
present no other detectable self-peptides (22).
B2L mice express low levels of the E52-68-I-Ab complex in their thymus and on ~5-10% of their splenocytes
and positively select a number of CD4+ T cells, which is
~20-50% of C57Bl/6 (26). We analyzed RNA from
CD4+CD8
NK1.1
HSA
T cells isolated from C57Bl/6
and from B2L mice by a PCR-based approach designed to
determine the CDR3 lengths of the
and
chains. All
V
-J
combinations were used to visualize an overall picture of the
repertoire based upon some 2,000 measurements. Given the larger number of J
segments, the
repertoire was not totally analyzed but sampled with 12 V
families. The normal mouse repertoire is characterized
by bell-shaped, Gaussian-like, CDR3 length distributions
(30). Distortions are observed upon antigenic stimulations
that cause sufficient clonal expansion (34, 42). Oligoclonal distributions in pathological infiltrates are easily depicted (51). B2L CD4+ T cell profiles were remarkably
Gaussian-like and quasisuperimposable to wild type. The
J
usage for V
11, V
12, and V
14 closely matched that
observed in C57Bl/6. Moreover, B2L and C57Bl/6 displayed overlapping CDR3 size distributions, even though
the average CDR3 length is different for distinct V
segments (30). No spikes suggesting clonal or oligoclonal expansions were detected. Therefore, the single MHC-peptide complex in B2L mice did not preferentially select a
small number of clones over an otherwise polyclonal background.
We cloned and sequenced from B2L and C57Bl/6 mice
the V11-J
1.1 and V
12-J
1.1 size peaks with CDR3
lengths of six and eight amino acids, respectively. We could
thus (Table 1) establish that the Tg and wild-type mice
repertoires both include a minimum of 105
rearrangements, suggesting that the Tg repertoire is about as diverse
as the wild-type one. In addition, the number of distinct
chains capable of pairing with a given rearranged
chain
has not been determined in physiological conditions. The
work of Sant'Angelo et al. (56) showed that 20-30 different
chains can associate with a unique transgenic rearranged
chain. If this figure can be extrapolated to physiological situations, the number of different TCRs would be
much higher than 105, leaving open the possibility that each
positively selected CD4+ thymocyte displays a unique TCR.
The above data show
that the preferential selection of certain Vs, observed in
B2L mice by Fukui et al. (26), is not reflected in a few
clonal expansions but in a larger number of rearrangements as revealed by the Gaussian distributions. This suggests that the E
52-68 peptide prevents proper association with the
subset of V
segments that are not efficiently selected, that
it is instrumental in binding those V
segments that are selected (57), or both. The amino acid usage, compiled in
Figs. 5 and 6, shows a second level of skewing now involving the CDR3 regions. We are confident that these differences in the amino acid composition at some CDR3 positions are significant. First, in conserved positions we find no variation of the amino acid usage between Tg and wild-type mice. Second, we evaluated the percentage of sequencing errors at conserved positions such as the C92
,
A93
,S94
-encoded positions of the CDR3. The figure of
0.3% at each position cannot explain the results. Third,
amino acid usage calculations were done with nonredundant sequences in order to avoid skewing due to overrepresentation. Fourth, such percentage variations are consistent
from one mouse to another of the same lineage (Tg versus
wild-type mice).
The elegant studies by Sant'Angelo et al. (56) have provided strong evidence for an imprint of the peptide in the
process of positive selection. Here we had no direct way to
evaluate the respective impact of positive and negative selection in shaping the B2L repertoire of CD4+ thymocytes.
However, the recurrent chain rearrangements that we
have observed (Fig. 4) are likely to reflect positive selection events, since they could hardly be the result of chance, or
of a negative process that would delete all sequences but
these ones. Remarkably, among these recurrent
chains,
two were found in both B2L and C57Bl/6. They were encoded by nonidentical nucleotide sequences and generated
by distinct junctional events (Figs. 4 and 7), implying that
they were selected at the amino acid level in a positive
fashion. It is worth noting that C57Bl/6 mice do not express nor present the E
52-68 peptide (58, 59). Therefore, in this case, the event that positively selected the
chain
involved no peptide or self-peptides sharing amino acid
residues with E
52-68, or was mediated by the
chain.
In the peripheral response to several defined antigens,
one or a few specific CDR3 sequences, in given V-J
and/or V
-J
combinations, have been found to be highly
reproducible and shared by individual animals. They were
named "public" (35) by analogy with recurrent idiotypes
found in immunoglobulins (60). Whether these public rearrangements are selected in the periphery or in the thymus
has not been determined so far. Since, as shown above, at
least some recurrent rearrangements are positively selected in the thymus, it may be proposed that peripheral public
rearrangements, in general, bear the imprint of thymic positive selection.
Altogether, our results also show that the impact of a
peptide in positive selection may not be readily detected.
First, our data indicate that the peptide may not be directly
involved in all selection events regarding the chain since
the
chain may also be involved in the selection process.
This raises the possibility that not all TCR-
rearrangements bear the imprint of the peptide. Second, in order to
detect the latter, we had to focus on a specific V
-J
combination with a fixed CDR3 length, whereas Sant'Angelo et al. studied
chain rearrangements associated with a
chain that had been fixed by transgenesis (56). This may
explain some apparently conflicting results about peptide
specificity in positive selection.
The physiological relevance of observations made in transgenic animals is questionable. Among the few reports on
positive selection in nontransgenic animals (for review see
reference 61), the comparison of positive selection of antiovalbumin TCR in Kb and Kbm8 mutant mice has provided
a clear indication for the involvement of presented peptides
(45). The single peptide-MHC complex of B2L mice
makes up ~10% of physiological complexes in the thymic cortex of B10-A(5R) mice. According to Grubin et al. (22),
rare peptides (expressed, in their case, in the H-2M-deficient background) appear functional in positive selection
whether the diversity of the T cell repertoire is built up
only by the most abundant self-peptides like E52-68, or
by rarer self-peptides as well. In the latter case, it remains to
be determined to which extent the diversity of thymocytes
is increased by rarer peptides in a cumulative fashion. We
are currently performing an extensive repertoire analysis
of B10-A(5R) mice in order to evaluate whether and to
which degree it includes or overlaps with that of B2L
animals.
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Footnotes |
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Address correspondence to Philippe Kourilsky, Laboratoire de Biologie Moléculaire du Gène, INSERM U277 and Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris cedex 15, France. Phone: 33-1-45-68-85-45; Fax: 33-1-45-68-85-48; E-mail: kourilsk{at}pasteur.fr
Received for publication 15 January 1998 and in revised form 20 March 1998.
L. Gapin is supported by l'Association pour la Recherche contre le Cancer and Pasteur-Weizmann fellowships. This work was supported by grants from Institut National de la Santé et de la Recherche Médicale and Institut Pasteur.We would like to thank Drs. Anna Cumano, Mitchell Kronenberg, François Lemonnier, Jean-Pierre Levraud, and Alfred Singer for discussion and critical review of the manuscript.
1Abbreviations used in this paper CLIP, class II-associated invariant chain peptide; Tg, transgenic mice.
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References |
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