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ß TCR Repertoire of Naive Mouse Splenocytes1
*
Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale, Unité 277, Institut Pasteur, Paris, France; and
Information Génétique et Structurale, EP 91, Centre National de la Recherche Scientifique, Institut de Biologie Structurale et Microbiologie, Marseille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß variable domains of
the ß TCR and by the rearrangements of various gene segments
encoding these domains. In the periphery, it results from competition
between various T cell subpopulations including recent thymic migrants
and long-lived T cells. Quantitative data on the actual size of the T
cell repertoire are lacking. Using PCR methods and extensive
sequencing, we have measured for the first time the size of the
TCR-ß repertoire of naive mouse T splenocytes. There are 5–8
x 105 different nucleotide sequences of BV chains in the
whole spleen of young adult mice. We have also determined the size of
the BV repertoire in a subpopulation of AV2+ T splenocytes,
which allows us to provide a minimum estimate of the ß repertoire.
We find that the mouse spleen harbors about 2 x 106
clones of about 10 cells each. This figure, although orders of
magnitude smaller than the maximum theoretical diversity (estimated up
to 1015), is still large enough to maintain a high
functional diversity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß TCR is composed of two polypeptide chains with variable (AV and
BV) and constant (AC and BC) domains. The variable regions are
generated by the somatic recombination of V and J gene segments for AV
and V, D, and J gene segments for BV (1, 2). The published
three-dimensional structure of the TCR has shown that the three
complementarity-determining regions
(CDR)3 of each AV or
BV chains form loops that interact with peptide/MHC class I complexes
(3, 4). The CDR1 and 2 loops are encoded within the V
genes, whereas the CDR3 results from the rearrangement of V and J
segments for AV and V, D, and J for BV. CDR3s are the most variable.
They are in a central position and are involved in peptide
recognition.
The diversity of the TCR repertoire results from
rearrangements of various gene segments, their imprecise joining,
addition of template-independent N nucleotides during this process, and
from the pairing of different - and ß-chains (2). In
addition, within the thymus, positive selection favors the emergence of
a highly diverse T cell repertoire and ensures the quality control of
TCRs, while negative selection eliminates self-reactive thymocytes
(5, 6). Both processes involve specific interactions with
self-MHC molecules (7, 8, 9, 10).
It has been established that the expansion and maintenance of peripheral T lymphocytes are dependent on interactions of their TCRs with MHC molecules (11). Furthermore, appropriate self-peptides bound to MHC class II molecules are required for the persistence of CD4+ T cells in the periphery (12). Nevertheless, little is known about how the peripheral T cell repertoire is established. Given the constant influx of recent thymic migrants and the persistence of long-lived T cells, its composition is likely to be the result of competition between different subpopulations of T lymphocytes (13).
The number of potentially different TCRs generated by AV and BV domain
pairing and by junctional diversity has been estimated to be up to
1015 (2). The total number of T
lymphocytes in a mouse is around 1 x 108,
including some 2 x 107 T splenocytes. Thus,
only a small fraction of the potential T cell repertoire can be used at
any given time in a mouse. However, its diversity has so far not been
investigated in detail at the molecular level. Using PCR methods and
extensive sequencing, we report here the first direct estimate of the
size of the TCR-ß repertoire from naive T splenocytes in young
adult mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice used in this study were 8-wk-old DBA/2 or C57BL/6 mice raised in specific pathogen-free conditions and obtained from IFFA-Credo (l’Arbresle, France).
Abs and sorting of AV2+/AV2- T splenocytes
FITC-conjugated anti-V2 and PE-labeled anti-TCRß
and anti-Vß10 Abs were purchased from PharMingen (San Diego, CA);
biotinylated anti-B220 was obtained from Caltag (South San
Francisco, CA).
Splenocytes from 8-wk-old DBA/2 male were depleted of
B220+ cells using biotinylated mAbs and
streptavidin beads (Dynals, Oslo, Norway). B220-negative splenocytes
were incubated with FITC-labeled anti-V2 and PE-labeled
anti-TCRß at 4°C for 1 h and washed. Single- and
double-positive cells were sorted on an Epics-Elite ESP (Coultronics,
France) at the Flow Cytometry Unit (Institut Jacques Monod, Paris,
France). Cell purity after sorting was analyzed by flow cytometry and
was above 98% in both samples.
RNA extraction and cDNA synthesis
Unfractionated splenocytes from mouse or purified AV2+ and AV2- T splenocytes were used for RNA preparation. Total RNA or poly(A)+ mRNA from splenocytes was extracted as previously described (14). Total RNAs or mRNAs were reverse-transcribed into cDNA using random primers (5 µM) for mRNA or oligo(dT) for total RNA (14, 15).
Immunoscope analysis
PCR were conducted in 50 µl on 1/50 of the cDNA with 2 U of Taq polymerase (Goldstar; Eurogentec, Brussels, Belgium) in the supplier’s buffer. cDNA was amplified using TCR BV-specific sense primers and an antisense primer hybridizing in the BJ segment (16).
Amplified products were then used as a template for an elongation reaction with fluorescent-tagged oligonucleotides (run-off reactions) as described elsewhere (16, 17).
Cloning of BV-TCR rearrangements
PCR were performed as above with 2 U of Taq polymerase (Goldstar; Eurogentec) or 5 U Pfu polymerase (Stratagene, La Jolla, CA) in the supplier’s buffer. PCR products were visualized by a DNA silver staining system (18) (Promega, Madison, WI). Bands corresponding to a given CDR3 length were cut from the gel and disrupted in 40 µl water. A second PCR was conducted using the same primers on 2 µl of the isolated PCR product with 2 U of Taq polymerase or 5 U Pfu polymerase for 20 cycles. Further purification was performed on a 15% nondenaturating acrylamide gel in 1x TBE buffer. PCR products were then cloned in pCR2.1 vector using the TA cloning kit or Topo TA cloning kit (Invitrogen, Carlsbad, CA).
In addition, the protocol (see Fig. 1
) was designed to minimize PCR and
cloning biases. The first round of PCR was always performed in a
separate area where no amplification products are allowed. Negative
controls were included in every experiment and at various steps to
ensure that reagents were clean. To control the second round of PCR,
empty fragments of acrylamide gel were processed in parallel to control
for spurious amplifications. As previously shown, cloning of the PCR
products does not introduce major biases because for each CDR3ß
studied, the length and the ends of the PCR fragments were identical
(15).
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For sequencing purposes, PCR was conducted directly on LacZ- colonies with Taq polymerase as described (14, 15). Sequencing reactions were conducted directly on these products using M13(-20) primer and with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit or the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystem, Foster City, CA). CDR3-corresponding sequences were extracted and analyzed using software designed for this purpose.
The BV segment sequences were taken from Arden et al. (19) and completed for the BV1 genomic segment with the sequence of BV locus submitted by Rowen et al. in the DDBJ/EMBL/GenBank databases under accession numbers AE000663 and AE000522.
Statistical calculations
Under the following assumptions—1, all rearrangements present
in the sample have an equal probability of being sequenced; 2, sequence
obtention can be considered as a random draw with replacement, i.e.,
the obtention of a given sequence does not alter the probabilities to
observe the following sequences; 3, the frequencies of all
rearrangements are identical, i.e., all rearrangements correspond to
the similar number of transcripts within the sample—the equation used
by Barth et al. (20) and Behlke et al. (21)
was used to estimate the maximum probable number of distinct CDR3
sequences found in the cDNA preparation. Namely, the maximum likelihood
estimate (MLE) of the number of distinct sequences is the value that
maximizes the equation:
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In these calculations, the percentages of erroneous nucleotide sequences were subtracted from every sequence numbers. We had previously evaluated at 0.3% the rate of errors made at each position of conserved residues of the frameworks adjacent to the CDR3 (14). For the MLE values of distinct amino acid sequences, the percentages of erroneous amino acid sequences were estimated from the percentage of erroneous nucleotide sequences. Thus, for a CDR3 of 6 aa, we have 5.4% erroneous nucleotide sequences corresponding to 5.4 x 75/100 = 4.05% erroneous amino acid sequences because in average 25% of all substitutions are synonymous and the remaining ones are nonsynonymous (22).
In Table II, the rate of errors made at each position of conserved
residues was evaluated at 0.1%. This lower error rate (0.1% instead
of 0.3%), due to the use of Pfu polymerase vs
Taq polymerase, was used for the calculations in Table II.
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The size of the BV repertoire equals the number of distinct sequences found in a CDR3 peak (MLE value) divided by the product of the frequency of BV, the frequency of BJ segment, and the frequency of the CDR3 peak in the immunoscope profile of the BV-BJ rearrangements under study.
The number of cells present in a CDR3 peak of given length equals the total number of T cells in the analyzed sample times the frequency of BV times the frequency of BJ segment times the frequency of the CDR3 peak in the immunoscope profile of the BV-BJ rearrangements under study.
The number of cells bearing the same CDR3 sequence equals the calculated number of cells present in a CDR3 peak of given length divided by the number of distinct nucleotide sequences (MLE value) found in the CDR3 peak.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immunoscope method is a PCR-based technique that allows the
measurement of the CDR3 lengths of the TCR V regions. In naive mice, it
has been shown previously that the V-J profiles yield a typical
bell-shaped distribution of the CDR3 lengths resolved in six or eight
distinct peaks (16, 23) However, this method provides no
information on the number of distinct CDR3 sequences present in a given
BV-BJ rearrangement and size peak. We evaluated the diversity in a
single peak as summarized in Fig. 1
. In a
first set of experiments, we started from 7 x
106 T splenocytes and determined the number of
distinct BV nucleotide sequences among the BV10-BJ1.2 rearrangement. We
isolated the 10-aa-long CDR3 band from a BV10-BJ1.2 rearrangement and
cloned these PCR fragments into a plasmid vector. Plasmids derived from
individual bacterial clones were sequenced. In Fig. 2A, the number of distinct
CDR3 nucleotide sequences was plotted vs the number of determined
sequences. A total of 731 sequences were determined before we reached
or approached a plateau. Using the method described by Barth et al.
(20) and Behlke et al. (21), we calculated
the MLE of the number of distinct nucleotide sequences within the
sample. It was necessary to eliminate unique nucleotide sequences
generated by PCR and/or sequencing errors. We had previously evaluated
at 0.3% the rate of errors made at each position of conserved residues
such as Y, L, C, and A of the frameworks adjacent to CDR3
(14). This percentage was subtracted (see Materials
and Methods) to obtain the MLE values displayed in Fig. 2B.
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B) are 204 (189–223), 206
(196–219), and 207 (199–217) for 400, 500, and 600 accumulated
sequences, respectively. Thus, the MLE curve reaches a plateau. This
experimental result justifies the assumptions made in Materials
and Methods. The data show that a fairly good estimate of the
number of distinct sequences, because 96% were obtained after 370
sequences. In summary, the results show that exhaustive sequencing of
cloned CDR3 sequences allows one to determine the extent of diversity
in a single CDR3 peak. The diversity found in CDR3 ß of different sizes is proportional to the peak area from the immunoscope profile
The above results provided a measure of the density of
diversity in a given CDR3 size peak. The area of the analyzed size peak
represents 11% of the total area of the BV10-BJ1.2 combination with a
CDR3 length of 10 aa, and the BV10 chain is used in 7% of the
splenocytes, while BJ1.2 represents 5% of the rearrangements involving
the BV10 segment (24, 25) (cf footnotes of Table I). We then calculate that 5.4 x
105 distinct nucleotide BV rearrangements equals
210 x 100/11 (to correct for all CDR3 size peaks) x 100/7
(to correct for all BVs) x 100/5 (to correct for all BJs) and
should represent the size of the BV repertoire of naive T
splenocytes.
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displays the calculated sizes of the BV
repertoire: 6 x 105 and 5 x
105 for the 6- and 10-aa-long CDR3,
respectively.
To determine whether the density of diversity varied with the BV
usage, we analyzed the 11-aa-long CDR3 of the BV1-BJ1.2 rearrangement.
The MLE value (71 ± 3 distinct nucleotide sequences for 278
sequences), the area of the size peak (11%), and the usage of this
particular BV-BJ combination (0.1%) yielded an estimated size of the
BV repertoire of 4.7 x 105 (Table I), in
excellent agreement with the two previous estimates. Thus, the density
of diversity does not depend upon BV and BJ usage, nor on CDR3 length.
We conclude that the spleen of DBA/2 mice harbors in the order of
4.7–6 x 105 distinct BV chain nucleotide
sequences, each being shared, on the average, by 39–45 T
splenocytes.
Analysis of the CDR3 ß diversity in splenocytes from a different strain of mice
As an alternative mouse strain, we chose C57BL/6 mice that lack
both the MHC class I Lb and the class II
I-Eb molecules. Thus, we could ask whether their
absence had any effect on the BV diversity. We analyzed the 10-aa-long
BV10-BJ1.2 rearrangement, which corresponds to 11% of all
rearrangements present in this combination. Among 633 sequences, we
found a MLE of 125 (±2) distinct ones. As shown in Table I, the size
of the BV repertoire is in the order of 5.7 x
105 distinct nucleotide BV rearrangements for
8.4 x 106 T lymphocytes. Thus, the absence
of two restricting elements did not affect the repertoire
significantly.
For the sake of completion, all CDR3 nucleotide sequences were
translated into amino acid sequences, and MLE values were calculated
with the appropriate corrections for erroneous amino acid sequences (cf
Materials and Methods). These MLE values and the resulting
estimate of diversity were about 10% lower than with nucleotide
sequences (Table I). This was expected because identical amino acid
sequences can be generated by different nucleotide sequences. Thus,
some T cell clones sharing the same BV chain generated by different
nucleotide sequences originate from different precursors and are likely
to be associated with different AV chains.
Size estimate of the TCR ß repertoire of T splenocytes
Because, on average, the number of T lymphocytes bearing the same
BV nucleotide sequence is about 39–45 per spleen (Table I), the
theoretical maximum ß diversity would be (6 x
105) BVs x (39 or 45) = 2.3 or
2.7 x 107 distinct TCR ß, assuming
every BV chain is associated with a different AV. To verify this
assumption and to gain insight into the combinatorial diversity
generated by the - and ß-chain pairing, we isolated the
AV2+ and AV2- T
lymphocytes from one DBA/2 spleen and analyzed the diversity of the
CDR3 sequences in BV10-BJ1.2 rearrangements within each sample. We
estimated the size of the BV repertoire of AV2+
cells. Knowing the percentage of AV2+ lymphocytes
in total splenocytes, we could estimate the minimal size of the ß
T cell repertoire in the spleen.
B220-negative splenocytes were incubated with FITC anti-V2 and
PE anti-TCRß mAbs, and the AV2- cells and
the double-positive lymphocytes were sorted. Total RNAs from
AV2+ and AV2- cells were
reverse transcribed into cDNAs. PCRs were performed with BV10 and BJ1.2
primers, and the amplified DNA fragments corresponding to the 6-aa-long
CDR3 were isolated, cloned, and sequenced as above.
The results are summarized in Table II.
Eleven of 41 distinct nucleotide sequences in the
AV2+ subset were found in the 118 sequences from
AV2- lymphocytes of this mouse. The fact that
identical CDR3 sequences are detected in both
AV2+ and AV2- lymphocytes
implies that a single BV10-BJ1.2 rearrangement can associate with at
least two distinct AV chains.
We calculated the size of the BV repertoire using the number of
distinct nucleotide sequences found in the AV2+
population (MLE = 41 ± 1). Because the frequency of
AV2+ lymphocytes is 13%, the total diversity of
the ß repertoire is at least 1.85 x
106 (Table II). It is worth noting that the
increase in diversity brought by AV pairing is at least 2.4 because the
BV repertoire size is about 7.7 x 105 (see
footnotes of Table II).
Thus, the observed ß diversity (1.85 x
106) appears to be 10 times lower than the
theoretical value (2.3–2.7 x 107). This is
most probably the result of a limited number of cell divisions after
the appearance of a BV chain on a given thymocyte.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß repertoire size.
Our approach relied on two assumptions. First, the number of distinct
sequences found in a BV-BJ rearrangement of a given CDR3 length had to
reach a plateau that, as we showed, is best defined by an index known
as MLE (Fig. 2). In calculations of the MLE, we eliminated the
percentage of nucleotide sequences potentially generated by errors that
turned out to be close to that of unique sequences. Nevertheless, a few
percent of these could correspond to T cell clones of lower abundance.
Second, and most importantly, we had to demonstrate that in naive mice,
the number of distinct sequences, comprised in a BV-BJ rearrangement of
a given CDR3 length is directly proportional to its peak area on the
immunoscope profile, irrespective of the BV, BJ usage and CDR3 length.
Table I shows that, for the BV10-BJ1.2 rearrangement, there is a good
agreement between the numbers of distinct nucleotide CDR3 sequences
found in the 6- vs 10-aa-long CDR3 peaks, 84 for an area of 4% vs 210
for an area of 11% (84/0.04 = 2100 vs 189/0.11 = 1909),
respectively. The sequences in the latter could, in principle, be many
orders of magnitude more diverse (644), and it
will be interesting to assess whether their diversity is limited to the
same level as that of the 6-aa peak by functional selection or by the
recombination machinery.
We were cautious to avoid technical biases in PCR and cloning and to use naive mice bred in specific pathogen-free conditions to minimize the risk that infections would trigger oligoclonal expansions, which could hide the diversity of other BV chains. We analyzed one-third or one-half of total splenocytes to ensure an adequate representation of splenic T cell clones. We knew from Bousso et al. (15) that when the two halves of the same spleen are processed independently, 82% of the CDR3 nucleotide sequences overlap, showing that most T splenocytes are present at least twice (82% is a minimum figure because the CDR3 size peaks were not sequenced as exhaustively as done here). We find each BV rearrangement in 10–15 copies in one-third of a spleen, implying that it is present in 30–50 cells in the total spleen. Moreover, because there are 200–400 copies of BV TCR mRNA per T lymphocyte (26), we could safely work with aliquots of the RNA preparation without distorting the representation of the various TCR. It follows that figures obtained for one-third or one-half spleen do represent the entire spleen, probably with a minor underestimate due to sampling effects. Another element that may result in an underestimation of the repertoire size stems from memory T cells that were not separated from the splenocytes. However, because we carefully checked that the BV-BJ rearrangements under study gives gaussian profiles and that exhaustive sequencing were performed, the underestimate remains within the error margin of the technique. We conclude that the TCR ß-chain diversity, estimated from nucleotide sequences, is in the order of 6–8 x 105, each BV sequence being shared by about 30–40 T splenocytes. The figures obtained for two different mouse strains (DBA/2 and C57BL/6) are strikingly similar, showing that the lack of two major MHC restriction elements does not affect the BV repertoire size significantly.
Immunoscope profiles (14) and limited sequencing
studies suggest that the TCR -chain diversity may be similar to BV
chains. However, the large number of AV and AJ segments, and the lack
of information about their usage, complicate the analysis. Rather than
measuring the -chain diversity per se, we evaluated the increase in
diversity that is introduced by the AV chains. We determined the size
of the BV repertoire in isolated AV2+ T
splenocytes. We found an increase in diversity of 2.4, yielding a size
of the ß repertoire of 1.85 x 106
(Table II). This value would be underestimated if several BV chains
could pair to the same AV chain. This is unlikely to occur frequently
because, in thymocytes, the BV chain rearrangement is fixed before the
AV chain is rearranged. The pairing of several AV chains with a given
BV sequence reflects the major pathway of thymic T cell production,
while the pairing of several BV chains to a given AV sequence is a
matter of chance. Thus, assuming an AV diversity in the order of
105, as many as (105)
AVs x (6 x 105) BVs = 6 x
1010 ß combinations could be produced but
only a small fraction actually shows up at any given time. Note that
the number of T splenocytes (2 x 107)
imposes a limit resulting in, at most, 30–40 AV chains per BV
rearrangement. Structural constraints may prevent the emergence of a
significant fraction of potential ß combinations, but the
ontogenic limitation is probably the major one. Penit et al. estimated
that thymocytes with a rearranged BV chain divide six to seven times,
generating 64 to 128 CD4+
CD8+ thymocytes sharing the same BV chain
(27). Of these, 95% will die by neglect or as the result
of negative selection (28, 29), leaving about three to
seven thymocytes undergoing full maturation. The increase in diversity
of 2.4 due to AV pairing, which we observed is quite consistent with
this figure.
If the size of the TCR repertoire of the mouse spleen is about 2 x 106, the thymocyte repertoire before negative selection, which removes about 70–80% of positively selected thymocytes (30), may be five times larger, i.e., 1 x 107. This value is many orders of magnitude smaller than the potential diversity estimated at 1015 (2). The total number of recent thymic migrants has been found to be 2 x 106 cells per day (31). The fraction that actually seeds the periphery is not known yet. Even if peripheral T cells are fully replaced by these thymic migrants and if each of these expresses a distinct TCR, it would take up to 109 days (1015 - 107 TCRs/2 x 106 cells per day) to exploit the "potential" repertoire. Thus, in any individual only a small fraction of the potential repertoire is ever used and two individuals may not share many similar sequences. When the immune and naive T cell repertoires of individual DBA/2 mice were compared, a large individual variability was found (15). Strikingly, 75% of the naive CD8+ T lymphocytes from two mice had different BV chains. When we compared the percentages of recurrent BV sequences in two different animals and for various BV-BJ combinations, the percentages of recurrent CDR3 sequences were comprised between 17% and 6% for BV10-BJ1.2 of 6- and 10-aa CDR3, respectively. These observations confirm that CDR3 sequence variability is extensive from one individual to the other. Thus, the size of the potential repertoire may be useful to allow maximal variability among individuals in a given species.
Several years ago, Langman and Cohn (32) proposed
the notion of "protecton," which they defined as a functional unit
of 107 B lymphocytes/ml of body fluid with a
repertoire of 105 different combining sites
capable of interacting with 104 Ags. They
hypothesized that "in animals with more than
107 total B cells, this unit is simply
repeated" (32). Our estimate of the T cell repertoire in
mice is about 1–2 x 106 TCRs/2 x
107 cells, a value that is about 10–20 times
larger than what was hypothesized for B cell protecton. In humans,
Wagner et al. (33) found a diversity of 2 x
107 for ß-chains of CD4+
blood lymphocytes. More recently, Arstila et al. (34)
found a lower diversity for the ß-chains of blood T lymphocytes
(about 1 x 106), with a total TCR-ß
diversity of 2.5 x 107 (34).
Thus, even though there are 250 times more T lymphocytes in human blood
than in a mouse spleen, the total diversity in blood may be only 10–20
times higher. Therefore, the T cell diversity in humans is not
increased proportionally to the total number of T lymphocytes. This is
suggestive of a putative T cell protecton, envisioned as a functional
unit comprising a few million T cells with distinct TCRs and not
varying much in size in different species.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean Kanellopoulos, Laboratoire de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale, Unité 277, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris cedex 15, France.
3 Abbreviations used in this paper: CDR, complementarity-determining region; MLE, maximum likelihood estimate.
Received for publication December 7, 1999. Accepted for publication March 10, 2000.
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and ß chain gene families. Immunol. Rev. 101:149.[Medline]
ß T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex. Science 274:209.ß T-cell receptor with major histocompatibility antigens. Nature 338:591.[Medline]
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ß T cell receptor diversity. Science 286:958.This article has been cited by other articles:
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  G. A. Rizzuto, T. Merghoub, D. Hirschhorn-Cymerman, C. Liu, A. M. Lesokhin, D. Sahawneh, H. Zhong, K. S. Panageas, M.-A. Perales, G. Altan-Bonnet, et al. Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response J. Exp. Med., April 13, 2009; 206(4): 849 - 866. [Abstract] [Full Text] [PDF]
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E. J. Yager, M. Ahmed, K. Lanzer, T. D. Randall, D. L. Woodland, and M. A. Blackman Age-associated decline in T cell repertoire diversity leads to holes in the repertoire and impaired immunity to influenza virus J. Exp. Med., March 17, 2008; 205(3): 711 - 723. [Abstract] [Full Text] [PDF]
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A. L. Furmanski, C. Ferreira, I. Bartok, S. Dimakou, J. Rice, F. K. Stevenson, M. M. Millrain, E. Simpson, and J. Dyson Public T Cell Receptor -Chains Are Not Advantaged during Positive Selection J. Immunol., January 15, 2008; 180(2): 1029 - 1039. [Abstract] [Full Text] [PDF]
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N. Fazilleau, H. Bachelez, M.-L. Gougeon, and M. Viguier Cutting Edge: Size and Diversity of CD4+CD25high Foxp3+ Regulatory T Cell Repertoire in Humans: Evidence for Similarities and Partial Overlapping with CD4+CD25 T Cells J. Immunol., September 15, 2007; 179(6): 3412 - 3416. [Abstract] [Full Text] [PDF]
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J. Wong, R. Obst, M. Correia-Neves, G. Losyev, D. Mathis, and C. Benoist Adaptation of TCR Repertoires to Self-Peptides in Regulatory and Nonregulatory CD4+ T Cells J. Immunol., June 1, 2007; 178(11): 7032 - 7041. [Abstract] [Full Text] [PDF]
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K. Kedzierska, E. B. Day, J. Pi, S. B. Heard, P. C. Doherty, S. J. Turner, and S. Perlman Quantification of Repertoire Diversity of Influenza-Specific Epitopes with Predominant Public or Private TCR Usage J. Immunol., November 15, 2006; 177(10): 6705 - 6712. [Abstract] [Full Text] [PDF]
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 J. Hataye, J. J. Moon, A. Khoruts, C. Reilly, and M. K. Jenkins Naive and Memory CD4+ T Cell Survival Controlled by Clonal Abundance Science, April 7, 2006; 312(5770): 114 - 116. [Abstract] [Full Text] [PDF]
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J. K. Whitmire, N. Benning, and J. L. Whitton Precursor Frequency, Nonlinear Proliferation, and Functional Maturation of Virus-Specific CD4+ T Cells. J. Immunol., March 1, 2006; 176(5): 3028 - 3036. [Abstract] [Full Text] [PDF]
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S.-K. Kim, M. Cornberg, X. Z. Wang, H. D. Chen, L. K. Selin, and R. M. Welsh Private specificities of CD8 T cell responses control patterns of heterologous immunity J. Exp. Med., February 22, 2005; 201(4): 523 - 533. [Abstract] [Full Text] [PDF]
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N. Fazilleau, J.-P. Cabaniols, F. Lemaitre, I. Motta, P. Kourilsky, and J. M. Kanellopoulos V{alpha} and V{beta} Public Repertoires Are Highly Conserved in Terminal Deoxynucleotidyl Transferase-Deficient Mice J. Immunol., January 1, 2005; 174(1): 345 - 355. [Abstract] [Full Text] [PDF]
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I. Messaoudi, J. LeMaoult, J. A. Guevara-Patino, B. M. Metzner, and J. Nikolich-Zugich Age-related CD8 T Cell Clonal Expansions Constrict CD8 T Cell Repertoire and Have the Potential to Impair Immune Defense J. Exp. Med., November 15, 2004; 200(10): 1347 - 1358. [Abstract] [Full Text] [PDF]
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 M. J. Anderson and K. L. Fritsche Dietary Polyunsaturated Fatty Acids Modulate In Vivo, Antigen-Driven CD4+ T-Cell Proliferation in Mice J. Nutr., August 1, 2004; 134(8): 1978 - 1983. [Abstract] [Full Text] [PDF]
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A. Srinivasan, J. Foley, and S. J. McSorley Massive Number of Antigen-Specific CD4 T Cells during Vaccination with Live Attenuated Salmonella Causes Interclonal Competition J. Immunol., June 1, 2004; 172(11): 6884 - 6893. [Abstract] [Full Text] [PDF]
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C. Joao, B. M. Ogle, C. Gay-Rabinstein, J. L. Platt, and M. Cascalho B Cell-Dependent TCR Diversification J. Immunol., April 15, 2004; 172(8): 4709 - 4716. [Abstract] [Full Text] [PDF]
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K. Kedzierska, S. J. Turner, and P. C. Doherty Conserved T cell receptor usage in primary and recall responses to an immunodominant influenza virus nucleoprotein epitope PNAS, April 6, 2004; 101(14): 4942 - 4947. [Abstract] [Full Text] [PDF]
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L. L. Pewe, J. M. Netland, S. B. Heard, and S. Perlman Very Diverse CD8 T Cell Clonotypic Responses after Virus Infections J. Immunol., March 1, 2004; 172(5): 3151 - 3156. [Abstract] [Full Text] [PDF]
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 S. O. Schonland, J. K. Zimmer, C. M. Lopez-Benitez, T. Widmann, K. D. Ramin, J. J. Goronzy, and C. M. Weyand Homeostatic control of T-cell generation in neonates Blood, August 15, 2003; 102(4): 1428 - 1434. [Abstract] [Full Text] [PDF]
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V. P. Badovinac, K. A. Nordyke Messingham, S. E. Hamilton, and J. T. Harty Regulation of CD8+ T Cells Undergoing Primary and Secondary Responses to Infection in the Same Host J. Immunol., May 15, 2003; 170(10): 4933 - 4942. [Abstract] [Full Text] [PDF]
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A. Hamrouni, A. Aublin, P. Guillaume, and J. L. Maryanski T Cell Receptor Gene Rearrangement Lineage Analysis Reveals Clues for the Origin of Highly Restricted Antigen-specific Repertoires J. Exp. Med., March 3, 2003; 197(5): 601 - 614. [Abstract] [Full Text] [PDF]
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N. Pasqual, M. Gallagher, C. Aude-Garcia, M. Loiodice, F. Thuderoz, J. Demongeot, R. Ceredig, P. N. Marche, and E. Jouvin-Marche Quantitative and Qualitative Changes in V-J {alpha} Rearrangements During Mouse Thymocytes Differentiation: Implication For a Limited T Cell Receptor {alpha} Chain Repertoire J. Exp. Med., November 4, 2002; 196(9): 1163 - 1174. [Abstract] [Full Text] [PDF]
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J. N. Blattman, R. Antia, D. J.D. Sourdive, X. Wang, S. M. Kaech, K. Murali-Krishna, J. D. Altman, and R. Ahmed Estimating the Precursor Frequency of Naive Antigen-specific CD8 T Cells J. Exp. Med., March 4, 2002; 195(5): 657 - 664. [Abstract] [Full Text] [PDF]
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J.-P. Cabaniols, N. Fazilleau, A. Casrouge, P. Kourilsky, and J. M. Kanellopoulos Most {alpha}/{beta} T Cell Receptor Diversity Is Due to Terminal Deoxynucleotidyl Transferase J. Exp. Med., November 5, 2001; 194(9): 1385 - 1390. [Abstract] [Full Text] [PDF]
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C. Baron, I. McMorrow, D. H Sachs, and C. LeGuern Persistence of Dominant T Cell Clones in Accepted Solid Organ Transplants J. Immunol., October 15, 2001; 167(8): 4154 - 4160. [Abstract] [Full Text] [PDF]
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J. L. Matsuda, L. Gapin, N. Fazilleau, K. Warren, O. V. Naidenko, and M. Kronenberg Natural killer T cells reactive to a single glycolipid exhibit a highly diverse T cell receptor beta repertoire and small clone size PNAS, October 5, 2001; (2001) 221445298. [Abstract] [Full Text] [PDF]
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C. Ronet, M. Mempel, N. Thieblemont, A. Lehuen, P. Kourilsky, and G. Gachelin Role of the Complementarity-Determining Region 3 (CDR3) of the TCR-{{beta}} Chains Associated with the V{{alpha}}14 Semi-Invariant TCR {{alpha}}-Chain in the Selection of CD4+ NK T Cells J. Immunol., February 1, 2001; 166(3): 1755 - 1762. [Abstract] [Full Text] [PDF]
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D. Laouini, A. Casrouge, S. Dalle, F. Lemonnier, P. Kourilsky, and J. Kanellopoulos V{beta} T Cell Repertoire of CD8+ Splenocytes Selected on Nonpolymorphic MHC Class I Molecules J. Immunol., December 1, 2000; 165(11): 6381 - 6386. [Abstract] [Full Text] [PDF]
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J. L. Matsuda, L. Gapin, N. Fazilleau, K. Warren, O. V. Naidenko, and M. Kronenberg Natural killer T cells reactive to a single glycolipid exhibit a highly diverse T cell receptor beta repertoire and small clone size PNAS, October 23, 2001; 98(22): 12636 - 12641. [Abstract] [Full Text] [PDF]
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T. Yokosuka, K. Takase, M. Suzuki, Y. Nakagawa, S. Taki, H. Takahashi, T. Fujisawa, H. Arase, and T. Saito Predominant Role of T Cell Receptor (TCR)-{alpha} Chain in Forming Preimmune TCR Repertoire Revealed by Clonal TCR Reconstitution System J. Exp. Med., April 15, 2002; 195(8): 991 - 1001. [Abstract] [Full Text] [PDF]
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) and lower (
) limit values of MLE at a 95%
confidence level are shown. The number of distinct nucleotide sequences
vs the number of determined sequences (
) are shown. Every value has
been reduced by 9% to take into account the contribution of sequencing
errors.