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Introduction |
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
E52-68 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 Vs 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 E52-68-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 E52-68-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 E52-68-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
E52-68-I-Ab complex extensively. We show that CD4+
CD8NK1.1HSA thymocytes selected on this complex
bear TCRs that include all Vs and 10 Vs tested. The J
usage and CDR3 length distribution of V and V chain
rearrangements are indistinguishable from the repertoire of
CD4+CD8NK1.1HSA 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 |
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 the chain of I-Ab covalently linked to the
peptide derived from MHC class II E (E52-68) into fertilized
eggs of I-Ab gene knockout (I-A/) mice carrying H-2b haplotype (28). To avoid peptide replacement, Tg were backcrossed with mice deficient for the invariant chain (Ii/; reference 29).
The B2L mice that express ~10% of the level of I-Ab found in
C57Bl/6 animals were chosen for all studies.
Purification of CD4+ T Cells.
Thymocytes were prepared from
6-wk-old mice and counted. CD4+CD8NK1.1HSA T cells
were prepared using depletion and cell sorting as previously described (26). In brief, thymocytes were incubated with anti-CD8 and anti-HSA (J11D) monoclonal antibodies and killed by addition of rabbit complement. Remaining living cells were stained
with anti-CD4-PE, anti-CD8-FITC, and anti-NK1.1-FITC (PharMingen, San Diego, CA). CD4+CD8NK1.1HSA T cells were
analyzed and sorted on an Epics cell sorter (Coulter Corp., Miami, FL). For Immunoscope analysis of the T cell receptor repertoire, cells were washed with PBS and immediately frozen down
into liquid nitrogen until further manipulation.
mRNA Extraction and cDNA Synthesis.
Poly(A)+ mRNA from
CD4+CD8NK1.1HSA thymocytes was extracted (Quick
mRNA Micro Prep Kit; Pharmacia, Piscataway, NJ). In brief, cells were lysed in guanidium thiocyanate and mRNA samples
were purified by affinity chromatography on oligo-d(T) cellulose.
After ethanol precipitation, mRNAs were reverse transcribed
into cDNA using a cDNA synthesis kit (Boehringer Mannheim
GmbH, Mannheim, Germany). RNA were denaturated at 70°C
for 10 min and then incubated with random primers (5 µM),
dNTP (1 mM), Rnasin (40 U; Promega, Madison, WI) and 2 U
of reverse transcriptase from avian myeloblastosis virus (Boehringer
Mannheim GmbH) at 43°C for 1 h, followed by an incubation at
53°C for 10 min.
Immunoscope Analysis of V and V Repertoires.
PCR was carried
out in 50 µl on 1/50 of the cDNA with 2 U of Taq polymerase
(Goldstar, Eurogentec, Seraing, Belgium) in the buffer provided
by the supplier. Each V mRNA was amplified using one of a set
of 23 V-specific sense primers and an antisense primer designed
to hybridize in the C gene (30). Similarly, V mRNAs coding
for V 1, 2, 3, 4, 5, 6, 8, 10, 11, 17, 18, and 19 were amplified
with V-specific sense primers and an antisense primer from the
C region (31).
Each amplified product was then used as a template for an
elongation reaction with oligonucleotides labeled with a fluorescent tag (runoff reactions). The fluorescent runoff products, corresponding to the elongation of individual V or V PCR products with various CDR3 sizes, were loaded on polyacrylamide
gels and subjected to electrophoresis in an automated DNA sequencer. CDR3 size distribution and signal intensities were then
analyzed with the Immunoscope software (30, 32, 33). The patterns observed from unprimed lymph node cells or splenocytes
usually contain six to eight size peaks each spaced by three nucleotides, corresponding to the lengths of in-frame transcripts. The
area of each size peak is proportional to the quantity of the TCR
transcripts of the corresponding CDR3 length in the sample. It
should be noted that each peak corresponding to a given CDR3
length is likely to contain multiple distinct sequences. Increase in
the height and area of a size peak signals clonal expansion, occurring against polyclonal background. The fluorescent C (or C)
primers used in the runoff reactions reveal all V-J (V-J)
rearrangements. Runoff reactions using specific fluorescent J
primers reveal the CDR3 length distributions from particular V-J
combinations. In brief, 2 µl of each PCR product was subjected
to three to five cycles of elongation with dye labeled specific
primer (0.1 µM) in a final volume of 10 µl, containing 0.2 mM
dNTP, 3 mM MgCl2, and 0.2 U of Taq polymerase (Promega) in
buffer (Promega). The elongation starts with 1 min at 94°C, followed by three to five cycles each consisting of 45 s at 94°C, 45 s
at 60°C, 45 s at 72°C, and ending with a step at 72°C for 3 min.
Each runoff product was then diluted vol/vol in 30 mM EDTA-formamide solution. This mix was heat-denatured during 10 min
at 80°C and 2 µl aliquot was loaded on a 6% (or 4.25%) polyacrylamide 8 M urea gel. Gel electrophoreses were performed on a
DNA sequencer (373A or 377; PE Applied Biosystems, Foster
City, CA). Peak size and fluorescent intensity were determined
with the Immunoscope software (32, 34). Because the technique is semiquantitative, we could evaluate the relative frequency of J usage for a given V population, as described in reference 37. The ratio of the area of the peak 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 in the V+ cell
population under study.
Sequencing of Particular V-J Rearrangements with a Given
Length.
Two PCR reactions were performed using specific
V11-J1.1 or V12-J1.1 primers in 50 µl on 1/50 of the
cDNA with 2 U of Taq polymerase (Goldstar, Eurogentec) in the
supplier's buffer. The elongation starts with 1 min at 94°C, followed by 40 cycles each consisting of 45 s at 94°C, 45 s at 60°C,
45 s at 72°C, and ending with a step at 72°C for 3 min. The PCR
product was ethanol precipitated and resuspended in 10 µl formamide containing 0.05% bromophenol blue and 0.05% xylene cyanol. This mix was heat denatured for 10 min at 80°C and then
loaded on a 8% polyacrylamide/7 M urea gel. After migration,
PCR products were visualized by a silver coloration (DNA Silver
Staining System; Promega) following manufacturer's instructions.
We usually obtained six to eight bands each spaced by three nucleotides, corresponding to in-frame transcripts of the V-J combinations. Bands of interest, corresponding to a given CDR3
length, were cut from the gel and disrupted in 40 µl of water. A
second PCR was carried out using the same primers on 2 µl of the isolated PCR product with 2 U of Taq polymerase (Goldstar, Eurogentec) in the supplier buffer for 20 cycles. Further purification was realized on a 15% nondenaturating acrylamide gel in
TBE. Staining of this gel was obtained with a 30 min bath in a
0.5 mg/ml ethidium bromide solution in TBE (Tris-borate-EDTA). PCR product was electroeluted in TBE and purity of
the sample was estimated by a runoff reaction with the fluorescent
J primer. We usually obtained 90-95% purity of the final product with the expected size. PCR products were then cloned in
pCR®2.1 vector from the TA cloning kit (Invitrogen, Carlsbad,
CA). 10-20 ng of DNA were ligated with an equimolar concentration of the vector. The ligation was carried out in 10 µl with 4 U
of T4 DNA ligase during 12 h at 14°C, following the manufacturer's instructions. Competent INVF' bacteria (Invitrogen)
were then transformed by heat shock at 42°C for 30 s and plated
on Luria-Bertani medium containing 50 µg/ml ampicillin and 60 µg/ml X-Gal (ICN Biomedicals Inc., Aurora, OH).
For the purpose of sequencing, PCR was carried out directly
on LacZ colonies in a final volume of 30 µl using universal
primers RP and M13(-40) designed to hybridize on each side of
the polylinker where the V-J PCR product was cloned. 5 µl
of this PCR product was treated during 40 min at 37°C with 1 U
of shrimp alkaline phosphatase (Nycomed Amersham, Buckinghamshire, UK) and 10 U of exonuclease (Nycomed Amersham)
in a final volume of 10 µl. Both enzymes were heat denatured at
80°C for 20 min. Sequencing reactions were carried out directly
on these products using M13(-20) primer and the ABI PRISMTM
Dye Terminator Cycle Sequencing Ready Reaction Kit following the manufacturer's instructions (PE Applied Biosystems). Reaction products were loaded on a 4.25% polyacrylamide 8 M urea gel. Gel electrophoreses were performed on a DNA sequencer
(377; PE Applied Biosystems), and CDR3 region corresponding
sequences were extracted and analyzed using software designed
for this purpose (Casrouge, A., E. Beaudoing, J.P. Levraud, L. Gapin, N. Garguier, D. Gautheret, J.M. Claverie, J. Kanellopoulos,
P. Kourilisky, manuscript in preparation).
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Results |
CDR3 Size Distribution of TCR-/ Repertoire from
CD4+CD8NK1.1HSA Thymocytes Positively Selected by a
Single Peptide-MHC Complex.
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+CD8NK1.1HSA
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
E52-68 peptide-I-Ab complexes is shown in Fig. 1. 22 out of the 23 V genes were amplified (Fig. 1 A). Amplification of V6 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 V17 and V19
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 V4, V8.2, V10, V11,
V12, and V14 families. The CDR3 distribution of specific V-J rearrangements was then obtained for the six
different Vs of two individual Tg. Our results clearly
show that for each V, all Js are used. Furthermore, the
CDR3 distribution of these rearrangements has a Gaussian-like profile (V11 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+CD8NK1.1HSA thymocytes
selected by a single peptide-MHC class II complex indicates that the repertoire is polyclonal.
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Fig. 1.
Profiles of the fluorescent V-C (A) and V-C (B) runoff products obtained with CD4+CD8NK1.1HSA thymocytes from single
chain peptide-MHC class II complex Tg. Results are representative of two mice tested independently. The intensity of fluorescence is represented in arbitrary units as a function of the size of the single DNA fragments.
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Fig. 2.
Comparison of the fluorescent runoff products profiles obtained from CD4+ mature thymocytes from single chain peptide-MHC class II
complex Tg (solid lines) and C57Bl/6 mice (dotted lines). Runoff extensions were performed for V4, V8.2, V10, V11, V12, and V14 with internal
C-specific fluorescent primer (A) and for V11 with 12 J-specific primers (B). The intensity of fluorescence is represented in arbitrary units as a function of CDR3 size in amino acids. Two mice were tested independently for each repertoire. Results were superimposable for the two mice tested in each
combination.
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Comparison of the / Repertoire of CD4+CD8NK1.1
HSA Thymocytes from Transgenic Mice for the E52-68 Peptide-I-Ab Complex and C57Bl/6 Mice.
Repertoire analysis
was performed on CD4+CD8NK1.1HSA 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 V4, V8.2, V10, V11, V12, and V14.
A more detailed analysis of V-J rearrangements was
performed for V4, V8.2, V10, V11, V12, and
V14. Representative results obtained with V11 are
shown on Fig. 2 B. The repertoires were superimposable
and no significant modification was found with any J
primer.
Similar Usage of J Gene Segments by TCR from CD4+
CD8NK1.1HSA Thymocytes Selected in Wild-type Mice
or in Transgenic Animals Expressing a Single Peptide-MHC Class
II Complex.
The relative J usage in the V11, V12,
and V14 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 J2 segments are used
more frequently than the J1 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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Fig. 3.
J usage of V11, V12, and V14 of CD4+CD8
NK1.1HSA thymocytes from B2L Tg (black bars) and C57Bl/6 mice
(white bars). In the abscissa are represented all members of the J family. In
the ordinate is plotted the relative frequency of J usage, calculated as described in Materials and Methods. The results were generated from one
mouse of each strain.
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Sequence Complexity in a Given V-J Rearrangement with
a Defined CDR3 Length.
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: V11-J1.1 with a CDR3 length of six amino acids and V12-J1.1 with a CDR3 length of eight amino acids. These
two Vs 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-J1.1 peak with a CDR3 length of
six amino acids and a mean of 70 different sequences in the
V12-J1.1 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 E52-68-I-Ab complex.
The V11-J1.1 combination with a CDR3 length of six amino acids represents 13% of the rearrangements using the
V11 and J1.1 gene segments. The J1.1 is used in 5% of
the rearrangements involving the V11 gene segment.
Staining with specific monoclonal antibody shows that 5%
of the CD4+CD8NK1.1HSA thymocytes from the Tg
bear the V11 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 Js] × 20 [to correct for all the Vs]) are selected by the peptide-
MHC class II complex. The same calculation was applied
for the V12-J1.1 rearrangement and a total of 7 × 104
TCR- rearrangements were found.
Recurrent Sequences of the V Chain CDR3 Region from CD4+
CD8NK1.1HSA Thymocytes of Wild-type Mice or Transgenic Animals Expressing E52-68-I-Ab Complex.
Among the
700 sequences collected, we found some recurrent sequences between animals. The results obtained with the
V11-J1.1 and V12-J1.1 rearrangements are shown in
Fig. 4. For the V11-J1.1 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 V12-J1.1, 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 V12 germline segment, the usage of
four nucleotides from the D1 segment, 1 P nucleotide addition between the D and the J segments, and the germline
sequence of the J1.1 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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Fig. 4.
Recurrent sequences of the V chain CDR3 region from
CD4+CD8NK1.1HSA thymocytes of wild-type mice or transgenic
animals expressing E52-68-I-Ab complex are displayed. Recurrent sequences between individual animals are shown and the sequences common to all animals are in bold.
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Table 2
Nucleotide and Amino Acid Translated Sequences of V12-J1.1 Recurrent Rearrangements with a CDR3 Length of Eight
Amino Acids
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Comparison of the Amino Acid Frequency at Different Positions in the CDR3 Region of V-J Rearrangements from
CD4+CD8NK1.1HSA Thymocytes of Wild-type Mice or
Transgenic Animals Expressing E52-68-I-Ab Complex.
We
compared the amino acid usage in the CDR3 region of the
two sequenced rearrangements V11-J1.1 (CDR3 length
of six amino acids) and V12-J1.1 (CDR3 length of eight
amino acids) of the CD4+CD8NK1.1HSA 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 V11-J1.1 rearrangement and 2, 3, and 4 for the
V12-J1.1 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 V11-J1.1 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 V12-J1.1
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
D1.1 gene segment in all CDR3s. Glycine can be encoded by all three D1.1 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 D1.1 gene segment. Altogether these results suggest that this residue has
been selected for at the amino acid level. Furthermore, the
preferential usage of the D1.1 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 E52-68 peptide-I-Ab complex on the selected T cell
repertoire.
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Fig. 5.
Amino acid frequency at the different positions in the CDR3 region of V11-J1.1 (length: six amino acids) rearrangements from
CD4+CD8NK1.1HSA thymocytes. Top, the results obtained from two independent C57Bl/6 mice; bottom, the results obtained from two independent Tg. Amino acid frequencies were calculated with nonredundant sequences to avoid skewing due to overrepresented sequences (34 and 27 sequences
and 25 and 34 sequences were used for C57Bl/6 mice and Tg, respectively).
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Fig. 6.
Amino acid frequency at the different positions in the CDR3 region of V12-J1.1 (length: eight amino acids) rearrangements from
CD4+CD8NK1.1HSA thymocytes. Top, the results obtained from one C57Bl/6 mouse; bottom, the percentage of usage obtained from two independent Tg. Amino acid frequencies were calculated with nonredundant sequences to avoid skewing due to overrepresented sequences (73 sequences and 81 and 60 sequences were used for C57Bl/6 mice and Tg, respectively).
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Fig. 7.
D reading frame
usage coding for glycine or
glutamine residues at position 3 of eight amino acids-long
CDR3 region of V12-J1.1 rearrangements. (A) The nucleotide sequences (bold) encoding a
glycine residue at position 3 of
the CDR3 region from C57Bl/6
(top) and Tg (bottom) thymocytes.
(B) The nucleotide sequences
(bold) encoding a glutamine residue at position 3 of the CDR3
region from C57Bl/6 (top) and
Tg (bottom) thymocytes. All the
sequences were aligned following the D reading frame usage,
regardless of the V, J gene
segment trimming, and N addition.
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Discussion |
At Least 105 Distinct TCR- Rearrangements Are Selected
by the Single MHC-Peptide Complex Displayed in B2L
Mice.
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+CD8NK1.1HSA 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 V11, V12, and V14 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-J1.1 and V12-J1.1 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 Transgenic Repertoire Is Skewed in at least Two Ways
with Respect to the Wild-type Repertoire.
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 E52-68 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 E52-68 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 E52-68, 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.
How is the T Cell Repertoire Shaped in Wild-type Mice?
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.
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
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.
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Abstract
Introduction
