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-Chain Rearrangement Is Followed by Selection for Shorter TCR ß-Chain Complementarity-Determining Region 31
The Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee WI 53201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains are selected has also been
identified using ß-chain transgenic mice. Here we identify two
additional selection stages in human thymocytes based on
characteristics of the TCR. selection is measured directly by
identification of in-frame rearrangements and is associated with the
appearance of CD3 on the DP thymocyte surface. The next stage has not
yet been described and involves selection of thymocytes that express
shorter TCR ß-chain complementarity-determining region 3 (CDR3). This
stage is associated with the acquisition of high levels of CDR3 by DP
cells and the transition to SP thymocytes. The extent of CDR3 length
selection observed is a function of the TCR V and J genes. We propose
that CDR3 length selection is based on recognition of the MHC.
Thus, there exist limitations on the allowable length of that portion
of the TCR most intimately in contact with MHC and peptide. This may be
a physical representation of positive selection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (1). This occurs in the
CD44- compartment of DN mouse thymocytes
(2, 3) and is referred to as ß selection
(4). The subset of DN cells in which the ß-chain is
selected in man is not known, although recent data have implicated the
CD4+CD3-CD8-
immature single-positive (ISP) cells (5). Later stages,
which would include pairing of the TCR ß-chain with a productively
rearranged TCR -chain and recognition of the peptide-MHC ligand by
TCRß, are less well understood. Thymic selection has been divided into two conceptual frameworks, referred to as positive selection and negative selection (6, 7). Negative selection is easily understood as elimination of T cells whose receptor/coreceptor affinity for self-peptide-MHC is too high (8). Positive selection can be defined quite broadly, ranging from the rescue of thymocytes from programmed cell death to the specific stimulation of a thymocyte by a peptide mimic of the future Ag. The most accepted definition of positive selection implicates only those events in which the thymocyte is interacting with self-MHC-peptide (see Ref. 9 for review). Historically, this form of selection has been closely linked to lineage selection, which was used as the readout. There have been recent studies indicating a division between positive selection of thymocytes and lineage selection (10, 11). There has been a large effort in determining the roles of peptides in the selection process (12, 13, 14).
Selection has also been assayed independent of the lineage markers
using TCR ß-chain Tg mice. It has been reported that in some cases
the ß-chain pairs preferentially with -chains similar to those
with which it was paired in the hybridoma of origin. By assaying the
stage at which this -chain selection is observed, this form of
positive selection has been mapped to the CD69+
subset of DP cells (15).
We have been investigating the rearrangement status of the TCR - and
ß-chain loci during human thymocyte maturation. Evidence for ß- and
-selection was obtained. As part of these studies we have identified
an additional stage in the maturation process that involves the
accumulation of SP cells that contain TCR ß-chains with shorter
lengths of complementarity-determining region 3 (CDR3). These results
are discussed in terms of our current understanding of thymic
selection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thymi were obtained as surgical tissue discards from The Children’s Hospital of Wisconsin. PBMC were obtained as discards after removal of indwelling catheters. All materials were obtained under an institution review board-approved protocol.
Fluorescent staining and sorting
Thymi were disaggregated by passing them through a wire mesh.
Cells were suspended in RPMI medium (Life Technologies, Gaithersburg,
MD), 0.1% sodium azide, and 2% FCS. To determine whether the thymi
were normal, 0.5 x 106 cells were stained
using mouse mAbs to human cell surface markers; CD3-FITC conjugate,
TCRß-FITC conjugate, CD4-Tri-color conjugate, and CD8-R-PE
conjugate (Caltag, San Francisco, CA). The stained cells were analyzed
using FACScan (Becton Dickinson, San Jose, CA). Thymi that had normal
CD3, CD4, and CD8 profiles were then stained for sorting. Three color
sorts were performed using FACStar (Becton Dickinson), and different
populations were collected. Primary gating was on the CD3 marker, which
resolved the thymocytes into three populations, referred to as
CD3neg, CD3low, and
CD3high (Fig. 1
A). These three populations
were then further divided on the basis of CD4 and CD8 expression (Fig. 1A). Cells were collected into 0.5 ml of FCS so that the
final concentration in the tube was 10% (5-ml final volume).
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For DNA, sorted cells were spun down and resuspended in nucleic lysis buffer, pH 8.2 (10 mM Tris, 0.4 M NaCl, and 2 mM EDTA), in the presence of SDS and proteinase K, then the cells were incubated overnight at 45 C to ensure the complete lysis. After the incubation, proteins were precipitated by adding 5.3 M NaCl, and DNA was isolated from the supernatant by ethanol precipitation (16). RNA was made using TRIzol reagent (Life Technologies, Gaithersburg, MD).
Rearrangement analysis
Rearrangement analysis was performed by PCR amplification of the
CDR3 using V and J region-specific primers. A description of the
methods has been published (17, 18, 19). The J region primer
was labeled with 5'-carboxyfluorescein (FAM), the PCR products were
analyzed on denaturing polyacrylamide gels, and the fluorescent PCR
products were quantitated using a FluorImager (Molecular Dynamics,
Sunnyvale, CA). Data were collected as a 16-bit Tiff file. Band
intensities could be further analyzed using ImageQuant and spreadsheet
software. For calculation of CDR3 length changes, band intensities
originally measured as relative fluorescence units by the FluorImager
were converted to the relative frequency (RF) of each band over the
total band intensity. The relative band intensities correct for minor
fluctuations in the data. The use of RF to calculate shortening is
shown in Fig. 5, and a general description is given in Ref.
19 .
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B. Volumes of the DNA preparations were chosen so that the
signals would be identical. The titration procedure was described in
greater detail previously (19). RT-PCR
Levels of pre-T and -chain mRNA were measured by RT-PCR
using primers specific for each cDNA. One microgram of total RNA was
converted to cDNA using Moloney murine leukemia virus reverse
transcriptase. The cDNA from a different population of thymocytes was
titrated to determine the amount needed to obtain an equivalent actin
ß-chain mRNA signal. Serial dilutions of cDNA were amplified for 24
cycles using two primers, one in exon 2 and the other in the exon 3 of
the actin ß-chain locus. Based on the ß-actin titration, levels of
pre-T and -chain mRNA were measured using primers specific for
each cDNA. Three concentrations of cDNA were used for the PCR to insure
a linear response of fluorescent signal to input. The sequences of the
primers used are as follows: ß-actin direct,
5'-CGTGTGGCTCCCGAGGAGCACC-3'; ß-actin anti-Fam labeled,
5'-CCCTGTACGCCTCTGGCCGTACCAC-3'; pre-T direct,
5'-GGCACACCCTTTCCTTCTCTG-3'; pre-T anti-Fam labeled,
5'-GCTTCTACAGCCAGGACCTGC-3'; C direct,
5'-GATATCCAGAACCCTGACCC-3'; and C anti-Fam labeled,
5'-ATGACGCTGCGGCTGTGGTCCAG-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have used the variations in intensity of CDR3 length of the TCR
ß-chain to assay changes in the T cell repertoire. It was of some
interest to extend these studies to thymocytes, as mature SP thymocytes
represent the immediate precursor of naive circulating T cells. The
recombination assay consists of generating a PCR product that amplifies
the CDR3 using V family-specific and J region-specific primers. The
length of the CDR3 thus amplified is resolved on denaturing acrylamide
gels. We have published an analysis of the thymic rearrangement
profiles of normal V genes compared with pseudogenes (18)
and have also used this method for analysis of the relationship of
thymocytes to ß thymocytes (17). The technique
and approach are similar to those described by Hayday and colleagues
(4, 20).
Analysis of TCR V ß-chain genes during thymocyte maturation
The thymic maturation series in man, as assayed by the
surface expression of CD3, CD4, and CD8, consists of the following
stages,
CD3negCD4negCD8neg
cells (triple negative) followed by two DP compartments,
CD3negCD4+CD8+
and
CDlowCD4+CD8+.
This division of DP thymocytes into almost equivalent numbers of
CD3neg and CD3low is
specific to man and is not found in nontransgenic laboratory mice. In
man, triple-negative cells proceed to the DP
CD3neg stage through an ISP
CD3-CD4+CD8-
compartment (21). The most mature cells are SP, showing
the following markers:
CD3highCD4+CD8-
and
CD3highCD4-CD8+
(reviewed in Ref. 22). The flow cytometric profile of a
typical human thymus is shown in Fig. 1A. The three levels
of CD3 expression are shown in the first panel, and the CD4 and CD8
profiles of the three CD3 gates are shown in the following panels.
An example of a typical recombination analysis is shown in Fig. 2. The amount of DNA used for each
amplification has been normalized by titration using a common set of
primers amplifying exon 1 of the ß constant region gene. The banding
pattern shows a 3-bp spacing indicative of in-frame selection. The
intensity of rearranged genes is the same throughout the maturation
series, indicating no further ß-chain rearrangements. We have
analyzed six human thymi, representing all age groups in which a
reasonable amount of thymic tissue is still found, and have observed
the same patterns of BV rearrangement in these five subsets.
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-chain genes during thymocyte maturation
Recent experiments using ß-chain TCR transgenic mice have
identified a stage occurring in DP cells in which the -chain is
selected (10). These took advantage of a propensity of the
ß-chain to pair with -chains similar to that with which it was
paired in the hybridoma of origin (15, 23). This
identifies a stage that can be referred to as selection. A
candidate for such a stage in man is the CD3low
DP subset. Expression of detectable CD3 on the surface implies that
both TCR chains are expressed on the surface, i.e., that pairing has
taken place.
We tested this supposition by using rearrangement analysis to determine
whether the TCR -chain was selected in the
CD3low DP cells. Fig. 3A shows analysis of the same
five thymocyte subsets as were analyzed for BV rearrangement in Fig. 2.
The data are for the AV8 family. It is obvious that there are very few
rearranged -chain genes in the CD4 ISP and
CD3neg DP compartments. There is a large jump in
the overall intensity of rearranged AV genes in the
CD3low DP population, which would indicate that
rearrangement is taking place at this stage. The increases in intensity
in the SP populations indicate further rearrangement. However, the
rearrangement profiles show a single base pair spacing throughout the
different compartments. This is probably due to the amplification of
rearranged genes that are on excised circles generated as part of the
continuing rearrangement process (24, 25). The
accumulation of circles with rearranged -chain genes, most of which
are out-of-frame, thwarts the ability of the analysis to define at
which point thymocytes with in-frame rearrangements are selected.
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B), whereas the CD3neg DP compartment
has accumulated thymocytes with out-of-frame rearrangements for the
same AV gene. The analysis was also performed with AJ4 primers. AJ4 is
the first AJ locus that is used to any great extent in AVJ joining and
thus must be maintained on the chromosome. In the analysis with AJ4 and
AV12S2 primers (Fig. 3C), the same in-frame selection
was observed between the two DP stages. The results in Fig. 3, B and C, are from two different thymi, and
similar results were obtained with three other thymi.
Further data supporting the observation the selection takes place
between the CD3neg and
CD3low DP compartments come from analysis of
pre-T and TCR -chain mRNA levels. Before pairing with the
-chain, the TCR ß-chain is paired with the pre-T. It would be
expected that at the point at which selection has occurred, the
levels of pre-T mRNA would decrease, whereas the levels of TCR
-chain mRNA would increase. We performed such an analysis on the RNA
from three thymocyte subsets, and the results are shown in Fig. 4. Quantitative RT-PCR was performed at a
number of dilutions of the cDNA for either pre-T (Fig. 4A) or TCR -chain (Fig. 4B). Before the
analysis, the cDNA was titrated to determine the amount needed to
obtain an equivalent ß-chain mRNA signal. The data are presented as a
dilution series to ensure that the response of the PCR is a linear
function of the input cDNA. It can be seen that there is a very
significant decrease in pre-T mRNA between the two DP stages.
Likewise, the most significant increase in TCR -chain mRNA was
observed between the two DP stages. This fits with selection taking
place at the CD3neg to
CD3low DP boundary.
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One of the more interesting data to come from the rearrangement
analyses is the observation that the average length of the CDR3
shortens between the DP CD3low and SP stages.
This can be observed to some extent in the data presented in Fig. 2, although it is not overtly apparent. For BV2-J2.7 rearrangements (Fig. 2), visual inspection of the data is difficult, so we will use these
data to introduce a more quantitative approach for analysis of the band
fluorescence intensities. The steps involved in this are shown in Fig. 5. The FluorImager data are converted to
relative fluorescence units using ImageQuant software (Fig. 5A). This is shown for the CD3low DP
and the CD4 SP lanes. The intensity of each band is converted into the
RF of the band with respect to the total band intensity (Fig. 5B). The difference of the RF of equivalent bands in any two
samples compared yields the 
RF. If two band distributions are
similar, the RF will hover around zero. If there is shortening, then
the RF will be positive for the higher m.w. bands (right)
and negative for the lower mw bands (left). The data
will show a well-defined shift from negative to positive values. This
form of analysis shows that for BV2-BJ2.7 rearrangements there is
indeed shortening between the CD3low DP and CD4
SP populations (Fig. 5C).
Shortening is observed between the CD3low DP and SP stages
To determine whether the selection of thymocytes with shorter CDR3
is a continuing process or whether there is a particular stage at which
this occurs, we analyzed the CDR3 length distributions in the two DP
compartments and the SP compartments of the thymus. An example of such
an analysis is shown for the BV7 and BV5.1 families (Fig. 6). The shortening for these families
between the CD3low DP and CD4 SP is readily
discernible by visual inspection of the gel data (Fig. 6A).
The RF analysis clearly shows that there is shortening that occurs
when the CD3low DP is compared with the CD4 SP
(Fig. 6B). The same is not seen when the
CD3neg DP is compared with the
CD3low DP (Fig. 6C). Thus, a
characteristic of SP thymocytes is that they tend to have shorter TCR
BV CDR3.
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. As a control for
the RF analysis, the two CD3neg DP populations
from two thymi were compared. Formally these profiles should be similar
and represent the CDR3 size distribution of the initial rearrangement
process if no selection has taken place. Comparison of the
CD3neg DP from the two thymi gave very similar
profiles (Fig. 6D), as evidenced by the low RF. In all,
we have clearly observed the phenomenon in five different thymi and for
10 different BV genes.
Thus far, the data have shown the selection for CD4 SP thymocytes. The
same can be observed for CD8 SP thymocytes. A representative gel
analysis of a recombination analysis (Fig. 7A) shows that the average
CDR3 length of CD4 SP and CD8 SP are very similar. The RF data for a
number of V gene rearrangements from two different thymi (Fig. 7, B and C) show that this is a general phenomenon.
Thus, there does not appear to be a difference between the two major SP
thymocyte lineages with respect to this phenotype.
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RF data show evidence of shortening, although the
overall pattern of shortening differed by the J gene used. The average CDR3 length of CD3high DP thymocytes is shorter than that of the other DP subsets
While a majority of DP thymocytes show either no or low expression
of CD3 on the surface, there is a small, but distinct, population of DP
thymocytes that express higher levels of CDR3. It is postulated that
CD3high DP cells may the precursors of
CD3high SP cells (28, 29). The
generation of SP thymocytes from transferred
CD3low DP thymocytes has been reported
(30), but a direct precursor relationship of
CD4high DP and SP thymocytes has not yet been
shown. The rearrangement status of the CD3high DP
population was investigated to determine whether the selection was
already taking place at this stage. The CD3high
thymocytes (Fig. 8A) were
fractionated by their CD4 and CD8 expression (Fig. 8B), and
the DP and the CD4 SP cells were collected.
CD3neg DP and CD3low DP
were collected from the respective populations as described previously
(see Fig. 1). The four thymocyte populations were analyzed, and an
example is shown in Fig. 8C. The results show that the
selection for shorter CDR3 begins to be observed in the
CD3high DP population. The difference between the
CD3low DP and CD3high DP is
not obvious by visual inspection (Fig. 8C), but RF
analysis shows that there is a selection step between the two stages
(Fig. 8E) that continues between the
CD3high DP and CD4 SP stages (Fig. 8F). As shown before, there was no evidence for selection
between the CD3neg DP and
CD3low stages (Fig. 8D). These data
are compatible with the three stages, CD3low DP,
CD3high DP, and CD4 SP, constituting a sequential
maturation pathway characterized by increasing selection for short
CDR3.
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There is a simple explanation for the accumulation of thymocytes
with shorter CDR3 in the SP subset. This is that the SP thymocytes with
longer CDR3 rapidly exit the thymus, and thymocytes with shorter CDR3
are retained. While it is has been impossible for us to obtain both
thymus tissue discards and peripheral blood cells from the same
individual, the general nature of this phenomenon should insure that
comparison of thymus and peripheral T cells between different
individuals is sufficient. We compared two pairs of age-matched
samples, one from thymus and one from PBMC. Rearrangement analysis of
total thymocytes (predominantly DP cells) and peripheral T cells showed
easily visualized evidence of CDR3 shortening between the two
compartments (Fig. 9). The mean length in
the periphery was similar to that observed in SP thymocytes. Thus, it
is thymocytes with short CDR3 that are exported and accumulate in the
periphery.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain
gene rearrangement and pairing of the -chain with the ß-chain.
This takes place within the DP thymocyte population and results in DP
cells that are CD3low, as they now express
TCRß on the surface. Commensurate with this division of DP
thymocytes is the decrease in levels of pre-T mRNA and the increase
in AV mRNA in the CD3low DP population. A similar
observation was reported previously, although the DP populations were
not resolved (31). Direct evidence for selection is
obtained from rearrangement analyses of AV-AJ rearrangements that must
be maintained on the chromosome. For such rearrangements, the first
thymocyte population in which in-frame rearrangements are observed is
the CD3low DP population. The third stage of maturation is characterized by accumulation of SP thymocytes that have shorter CDR3. This is an unexpected characteristic of thymocyte maturation. The shorter CDR3 observed in the CD4 SP and CD8 SP subsets is not a result of rapid exit of thymocytes with longer CDR3, as peripheral T cells also have short CDR3. The short length of peripheral TCR BV CDR3 had been noted previously (32), but it was not clear whether this was a function of the rearrangement mechanism itself or of a shortening postrearrangement. The selection for thymocytes with shorter CDR3 can be observed at the CD3high DP stage, strongly supporting the precursor relationship between CD3low and CD3high DP cells (10, 15, 28, 29).
The shortening phenomenon is a general one, having been observed for a
number of individuals. However, it is not clear whether it will be
observed for the same V-J combination in all individuals. Within any
thymus, the phenomenon is characterized by a dependence on the BV-BJ
combination used for the analysis. For example, it is less evident for
the BV2 family used to obtain the data in Fig. 2, whereas it is much
more evident in the data for the other families shown. The same
sensitivity to the recombined J gene has also been observed. While our
studies have not been exhaustive, the data have always shown some
evidence of shortening, no matter which V or J gene was studied. In
contrast, comparison of the CD3neg DP subset from
different thymi does not show any evidence for shortening. This would
be expected if this less mature subset had not undergone any selection
and the components of the rearrangement machinery responsible for
determining CDR3 length were not polymorphic.
We propose that the observed accumulation of thymocytes with shorter CDR3 in the transitions between the two TCR-expressing DP populations, CD3low and CD3high, as well as that leading to SP cells is a direct result of selection on the TCR ligand, i.e., peptide-MHC molecules. This is the most reasonable interpretation of the observed dependence of the selection on the V-J combination being analyzed. Direct evidence for the role of peptide-MHC in the shortening process will have to come from work in the mouse, where inbred strains and mutants are available. We have observed selection of thymocytes with shorter CDR3 in CD4 SP thymocytes in the mouse. Results using inbred mouse strains show that there is an effect on the extent of shortening observed for a particular V-J combination if MHC disparate or recombinant strains are examined (our manuscript in preparation). The extent of shortening is much higher in 129 and B10 (H2b) than in B10.PL and PL (H2u) mice, indicating that the MHC plays a role in the process. These mouse data also speak to the generality of the CDR3 length selection.
TCR and Ig employ the same machinery for generating recombinational diversity, whereas the recognition events for these two classes of immune receptors are different. Therefore, it is not surprising that the recognition of peptide-MHC may require differences in the length of the contact specificity portion of the molecule. Shorter CDR3 could more easily form the flatter recognition surface characteristic of those observed in TCR-MHC crystal structures (33, 34, 35).
In addition to the role of the interaction of TCR with MHC:peptide, another molecule that may dictate a need for shorter CDR3 is the coreceptor, CD4 or CD8. The coreceptors are present at the time of selection and may also impose structural limitations on the preferred length of the CDR3.
Because the stage at which TCR -chains are selected is different
from that at which the shortening takes place, we do not think that the
shortening is related to pairing of the two chains. Our data imply that
selection is a distinct phenomenon from the selection for
thymocytes with shorter ß-chain CDR3. If CDR3 shortening represents
the first selection on peptide-MHC molecules, then the observation that
selection precedes CDR3 shortening would imply that selection
could be solely based on pairing and not on peptide-MHC
recognition.
While the data presented here provide a novel measure of thymocyte maturation, there remain a number of interesting issues that will require further investigation. For example, it would be of great interest to determine how the short CDR3 phenotype fits with current models of positive selection at the DP to SP boundary (10, 15, 29). If there is coreceptor involvement, the possible role of CDR3 length selection in lineage commitment could be explored. In the context of our current understanding of thymic maturation, two opposing explanations for the selection process could be envisaged, falling under the rubric of either positive or negative selection. If only short CDR3 are compatible with the interaction of the TCR with peptide-MHC (coreceptor) complexes needed to maintain viability, this could be considered positive selection, with elimination of unselected thymocytes by the "neglect" mechanism. While perhaps less likely, it is possible that a longer CDR3 demonstrates a high affinity interaction with the peptide-MHC ligand due to the generation of deeper, more complex, Ig-like contacts. This would result in elimination of these thymocytes by negative selection mechanisms. It will be interesting to determine which mechanism is at work. The data presented here provide a physical basis for considering the issues involved in thymocyte selection and open up further avenues for studying the process.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jack Gorski, The Blood Research Institute, The Blood Center of Southeastern Wisconsin, POB 2178, Milwaukee, WI 53201-2178.
3 Abbreviations used in this paper: DN, double negative; DP, double positive; ISP, immature single positive; CDR, complementarity-determining region; RF, relative frequency; FAM, 5'-carboxyfluorescein.
Received for publication March 13, 2000. Accepted for publication July 17, 2000.
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References |
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  A. C. Carpenter, K. S. Yang-Iott, L. H. Chao, B. Nuskey, S. Whitlow, F. W. Alt, and C. H. Bassing Assembled DJ{beta} Complexes Influence TCR{beta} Chain Selection and Peripheral V{beta} Repertoire J. Immunol., May 1, 2009; 182(9): 5586 - 5595. [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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 J. Nishio, M. Suzuki, T. Nanki, N. Miyasaka, and H. Kohsaka Development of TCRB CDR3 length repertoire of human T lymphocytes Int. Immunol., March 1, 2004; 16(3): 423 - 431. [Abstract] [Full Text] [PDF]
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M. Yassai, K. Ammon, J. Goverman, P. Marrack, Y. Naumov, and J. Gorski A Molecular Marker for Thymocyte-Positive Selection: Selection of CD4 Single-Positive Thymocytes with Shorter TCRB CDR3 During T Cell Development J. Immunol., April 15, 2002; 168(8): 3801 - 3807. [Abstract] [Full Text] [PDF]
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