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Usage in Immature Thymocytes Is Independent of DJ Proximity and pT
Pairing1
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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locus rearranges before the
TCR 
locus. Pairing of productively rearranged TCR -chains with
an invariant pT chain leads to the formation of a pre-TCR and
subsequent expansion of immature pre-T cells. Essentially nothing is
known about the TCR V repertoire in pre-T cells before or after the
expression of a pre-TCR. Using intracellular staining, we show here
that the TCR V repertoire is significantly biased at the earliest
developmental stage in which VDJ rearrangement has occurred.
Moreover (and in contrast to the VH repertoire in immature
B cells), V repertoire biases in immature T cells do not reflect
proximity of V gene segments to the DJ cluster, nor do they
depend upon preferential V pairing with the pT chain. We conclude
that V gene repertoires in developing T and B cells are controlled by
partially distinct mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Ig signaling
complex (1), the TCR is a heterodimer of - and
-chains in association with the CD3 complex (CD3
, 
, 
, and
) (1). During development the BCR and TCR are assembled
from independent rearranging gene segments at the Ig and TCR loci,
respectively. In both instances variable (V), diversity (D), and
joining (J) gene segments are recombined in a combinatorial fashion to
produce a diverse BCR or TCR repertoire (2, 3, 4).
There are many striking parallels in B and T cell development. In both
cases, one Ag receptor chain (IgH for the BCR and TCR for the TCR)
rearranges early in development. Productive rearrangement of this chain
leads to the formation of a pre-BCR or a pre-TCR complex in which IgH
or TCR is paired with an invariant surrogate L chain or pT
chain, respectively (5, 6). Signaling through the pre-BCR
or pre-TCR leads to clonal expansion of immature B or T cells (7, 8), ultimately resulting in rearrangement of the IgL or TCR
locus and subsequently expression of a BCR or TCR. Maturing B and T
cells are then positively and negatively selected on the basis of their
Ag receptor specificity to yield the peripheral BCR and TCR repertoires
(9, 10, 11, 12, 13).
Based on these developmental parallels, there are several different
ways in which biases in the BCR or TCR repertoires can arise. At early
developmental stages, nonrandom VDJ recombination at the IgH or TCR
locus may lead to intrinsic biases in VH or V
gene usage in immature B or T cells. Moreover, the obligate formation
of a pre-BCR or pre-TCR for subsequent developmental progression may
further select for IgH or TCR -chains that can pair effectively with
the corresponding surrogate L or pT chains. Finally, positive and
negative selection events would be expected to select for mature BCR
and TCR based on the appropriate pairing and specificity of the IgH/IgL
and TCR /TCR heterodimers.
Although considerable data are available concerning the mature BCR and
TCR repertoires, less is known about the factors that shape the pre-BCR
and pre-TCR repertoires before positive and negative selection events.
Nevertheless, it has been shown that the pre-BCR repertoire is subject
to bias in at least two respects (7). First, a preference
for rearrangement of VH gene segments that are
proximal to the DJH locus results in a nonrandom
use of IgH chains in immature B cells. Second, the failure of a large
proportion (
50%) of IgH chains to pair effectively with the
surrogate L chain introduces further bias in the
VH repertoire that can successfully undergo
pre-BCR-mediated selection.
In contrast to B cells, essentially nothing is known about factors that
may influence the pre-TCR repertoire during early T cell development.
In particular, it is not clear whether the recombination machinery that
directs VDJ rearrangements acts at random or alternatively
introduces an intrinsic V repertoire bias. Moreover, the possible
influence of preferential TCR /pT pairing on the pre-TCR-selected
V repertoire has not been directly evaluated.
In this report, we have developed a novel two-color intracellular (ic)
immunofluorescence protocol that, when combined with two-color surface
staining, allows accurate determination of the TCR V repertoire in
immature thymocyte subsets. Our data indicate that significant biases
in the V repertoire are already apparent at the earliest
developmental stage in which VDJ rearrangement has occurred.
However, in contrast to the VH repertoire in
immature B cells, V repertoire biases in immature T cells do not
reflect proximity of V gene segments to the DJ cluster, nor do
they depend upon preferential pairing with pT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 female mice (Vb allotype) were
purchased from Harlan Olac (Bicester, U.K.). Congenic
Va allotype mice (backcrossed 15 generations
to the C57BL/6 background) were a kind gift from Alexandra Livingstone
(Basel Institute for Immunology, Basel, Switzerland). They were bred
and maintained at the Institut Suisse de Recherche Experimentale sur le
Cancer animal facility and have been described in more detail elsewhere
(14). pT-deficient mice on a C57BL/6 background
(15) were a kind gift from Hans Jorg Fehling (Basel
Institute for Immunology). All mice were used at 6–8 wk of age.
CD4-CD8- (double-negative
(DN)) thymocytes were prepared as previously described (16, 17). Contaminating mature or T cells and immature
CD44+ thymocytes were eliminated during subsequent FACS
analysis by gating out all cells stained with a mixture of
FITC-conjugated Abs to CD4, CD8, CD44, CD3, TCR , and
TCR (either prepared in this laboratory or purchased from
PharMingen, San Diego, CA). For the analysis of immature single
positive (ISP)
(CD4-CD8+CD3-)
thymocytes, depletion was performed using mAbs to CD4, CD25, and
CD44.
ic staining, FACS analysis, and Abs
Thymocyte subsets were analyzed by four-color simultaneous
surface and ic flow cytometry on a FACScalibur flow cytometer (Becton
Dickinson, San Jose, CA) as previously described (17, 18, 19).
Briefly, purified DN thymocytes were surface stained with the FITC
mixture described above and CD25-Cy5 (prepared in this laboratory) and
fixed in 2% paraformaldehyde before permeabilization with 0.5%
saponin (Sigma, St. Louis, MO). Two-color ic staining of TCR V vs
TCR C was performed by staining with PE-conjugated anti-V
mAbs followed by anti-TCR C-CyChrome (PharMingen). mAbs to TCR
Vs 2, 6, 8.2, 8 total (8.1/8.2/8.3), 9, and
17a were prepared in this laboratory by
conjugation of the purified proteins with the Phycolink PE conjugation
kit (Prozyme, San Leandro, CA); mAbs to TCR Vs 4, 8.3,
10b, 11, and 12 were purchased from Caltag
(Burlingame, CA); and those to TCR Vs 3, 5.1/5.2, 7, 8.1/8.2, and 13
were purchased from PharMingen. The percentage ic TCR
V+ of total ic TCR C+
for DN3 (CD25+CD44-) or
DN4 (CD25-CD44-)
thymocytes was calculated using the CellQuest (Becton Dickinson)
program. For ISP thymocytes, three-color surface staining with
anti-CD8-CyChrome, anti-TCR -PE (PharMingen), and anti-CD24-FITC
(prepared in this laboratory) was followed by fixation and ic staining
with anti-TCR V-PE conjugates. ISP thymocytes were defined as
CD8+CD24+TCR
low. Since ISP thymocytes have been previously
shown to be 100% ic TCR C+ (17),
the percentage of ic V+ ISP was calculated
directly.
Statistical analysis
The percentages of ic V+ DN3 thymocytes
as well as the ratios of ic V+ DN4:DN3 or
ISP:DN3 thymocytes were compared pairwise for all V domains using
the Student t test. Values of p < 0.001 were
considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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staining in immature thymocytes
Although several groups have identified immature fetal or adult
thymocytes expressing TCR protein based on ic staining with
anti-TCR C mAb (17, 20, 21, 22, 23), very little is known
about the relative expression of TCR V domains before positive and
negative selection. To address this issue, we prepared a panel of
PE-conjugated anti-V mAbs and used them in conjunction with
CyChrome-conjugated anti-C mAb in two-color ic staining of
immature CD4-CD8- (DN)
adult thymocytes. Surface staining with a Cy5-conjugated mAb to CD25
and a FITC-conjugated mAb mixture to CD44 and a panel of mature
thymocyte markers (see Materials and Methods) was used in
the other two colors to define
CD44-CD25+ (DN3) and
CD44-CD25- (DN4) subsets
(Fig. 1
). As reported previously
(17), a clearly defined subset of ic TCR
+ cells was distinguishable in both DN3 and
DN4 subsets (25 and 75%, respectively). In addition, a small
proportion of double-staining ic TCR V5+ ic
C+ cells (corresponding to 11 ± 2% of
ic TCR C+ cells) was detectable in both the
DN3 and DN4 subsets (Fig. 1). This staining was shown to be specific,
since immature thymocytes from C57BL/6 (Vb)
mice stained positively for V8.2 but negatively for
V17a (Fig. 2).
In contrast, immature thymocytes from congenic C57BL/6 mice of the
Va haplotype stained positively for
V17a but negatively for V8.2 (Fig. 2).
These results are compatible with the Va
genotype, which harbors a large deletion including V8.2, as well as
a polymorphism that allows expression of V17a
(24, 25).
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repertoire in DN3 thymocytes
The DN3 subset is the earliest stage at which full-length VDJ
transcripts and TCR C protein are expressed (16, 26, 27, 28, 29, 30, 31). It was therefore of interest to determine whether
biases in V usage, which are pronounced after positive and negative
thymic selection (32, 33, 34, 35, 36), could already be detected at
the DN3 stage. Fig. 3 represents a
summary of ic V staining in DN3 thymocytes of C57BL/6 mice for the
16 V domains analyzed in this study. With the exception of
V5.1/5.2 (which cannot be distinguished by the mAb used) and
V17a (which is not expressed in the
Vb haplotype), the other 13 individual V
domains were used at frequencies varying between 2.9% (for V12) and
8.6% (for V8.2) in the DN3 subset. A pairwise statistical
comparison of these 13 V domains indicated that they could be
subdivided into four groups based on their level of utilization in the
DN3 subset: V8.2 > V4, V6, V8.3 > V2, V3,
V8.1, V10, V11 > V7, V9, V12, V13. Thus
there is a considerable (up to 3-fold) variation in the probability of
utilization of a particular V domain at this early developmental
stage.
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repertoire in DN3 thymocytes is independent of
proximity to DJ segments
At the IgH locus, there is a large body of evidence indicating
that proximal VH segments rearrange
preferentially to DJH segments during ontogeny
(37, 38, 39, 40, 41, 42, 43, 44). Thus it is possible that the variations observed
in V usage among DN3 thymocytes reflect proximity to the DJ
segments. However, as shown in Fig. 3
(in which the V segments are
ordered according to their positions on the chromosome), there is no
correlation between frequency of V usage and chromosomal
localization. Indeed, the most DJ-proximal V segments (V7 and
V3) are used at a relatively low frequency, whereas the most
frequently used segment (V8.2) is relatively distal to DJ.
Moreover, within each group of V domains defined by statistically
similar usage in DN3 thymocytes (see above), the individual members
appear to be located randomly within the V locus.
V repertoire in DN3 thymocytes is independent of -selection
As mentioned previously, VDJ rearrangement and TCR protein
expression first occur at the DN3 stage of adult thymus development.
Since DN3 thymocytes also express pT and CD3 components (45, 46), de novo synthesized TCR protein can presumably be
rapidly incorporated into a pre-TCR complex. Formation of a pre-TCR
will in turn signal the cell to enter cell cycle. As a result, DN3
thymocytes are heterogeneous in size (as measured by forward light
scatter), with 70% small (i.e., resting) and 30% large (i.e.,
cycling) cells (Fig. 4A). As
expected, the majority of large DN3 cells (65 ± 7%) but very few
small DN3 cells (12 ± 2%) express ic TCR protein (Fig. 4A). Most large DN3 cells are thus " -selected,"
while the minor subset of small ic TCR + DN3
cells presumably represents cells that either have failed to assemble a
pre-TCR complex (due to the inability of their TCR -chains to pair
with pT) or have not yet entered the cell cycle despite the presence
of a functional pre-TCR (47). In either case, these small
DN3 cells would be considered to be not selected.
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selection significantly alters the V
repertoire of DN3 thymocytes, we compared ic V staining in ic
C+ DN3 cells gated according to size. As shown
in Fig. 4B, the hierarchy of ic V usage observed
previously in total ic C+ DN3 thymocytes was
preserved in the small and large DN3 subsets. Moreover, analysis of the
TCR V repertoire of DN3 thymocytes from pT-deficient mice
(15), which are genetically incapable of forming a
pre-TCR, indicated no significant differences in ic V usage as
compared with wild-type mice (Table I).
Taken together, these data demonstrate that the biased V repertoire
of DN3 thymocytes does not result from the preferential ability of
certain V domains to form a functional pre-TCR.
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repertoire is not significantly affected by pre-TCR-mediated
expansion of immature thymocytes
Following productive VDJ rearrangements at the DN3 stage, the
TCR -chain pairs with the invariant pT-chain and CD3 components
to form the pre-TCR complex (8). As a consequence of
pre-TCR signaling, DN3 thymocytes shut off further VDJ
recombination, down-regulate the expression of CD25, and enter a phase
of rapid proliferation (DN4). Proliferating DN4 thymocytes subsequently
express CD8 at the ISP stage before becoming
CD4+CD8+ cells and
initiating VJ rearrangement. To determine whether the V
repertoire is selected during this pre-TCR-mediated expansion phase of
thymocyte development, we measured ic V expression in DN4 and ISP
thymocyte subsets and compared these values with those obtained in the
preexpansion (DN3) stage. As shown in Fig. 5, only very slight changes in the V
repertoire were observed in DN4 and ISP subsets, since the ratio of
DN4:DN3 or ISP:DN3 ic TCR V+ cells was not
significantly different from 1 in virtually all cases. These data
indicate that the dramatic expansion of immature thymocytes that occurs
between the DN3 and ISP stages of development does not depend upon the
V domain utilized by the pre-TCR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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repertoire early in development before
positive and negative selection events. Although it is known that the
expressed TCR V repertoire is modified during both positive
(32, 35) and negative (33, 34, 36) thymic
selection, the contribution of other factors (such as rearrangement
frequency and probability of pairing with pT) to TCR V repertoire
formation has not been previously evaluated. Our data indicate that the
TCR V repertoire is already significantly skewed at the earliest
stage of adult thymus development in which VDJ rearrangements can be
detected. Moreover, and in marked contrast to the
VH repertoire in immature B cells, the bias in
V repertoire observed in immature T cells does not reflect any
obvious preference for rearrangement of proximal V segments or for
pairing with the pT-chain. Thus, although the general parallels in B
and T cell development are striking, the mechanisms responsible for
controlling V and VH usage at early
developmental stages are quite distinct. Technical considerations
Previous studies of VH repertoire formation in immature B cells have mainly relied upon PCR amplification and sequencing at the population level. Such techniques have the advantage of detecting both nonproductive and productive IgH rearrangements. However, they are subject to biases due to the variable efficiency of amplification by different PCR primers and do not evaluate rearrangements at the single-cell level. The latter problem can be overcome using single-cell PCR (42, 48), but this method introduces sampling errors due to the relatively small number of cells that can be analyzed.
To assess the TCR V repertoire in immature thymocytes, we have
developed a two-color ic staining procedure using a panel of
PE-conjugated anti-V mAbs in conjunction with
CyChrome-conjugated anti-C mAb. Anti-V mAbs represent a
powerful tool to analyze the TCR V repertoire, since (at least in
the mouse) mAbs are available for the majority of V-chains. Indeed,
the panel of anti-V mAbs used here detected 70% of the total
V repertoire in both DN3 and DN4 subsets as assessed by ic staining.
ic staining is, however, necessary to reliably detect TCR protein
expression at early stages of thymus development, since the pre-TCR is
expressed at very low levels on the cell surface and can only be
detected using elaborate multistage staining protocols
(49). Moreover, simultaneous assessment of ic V and ic
C protein expression in individual cells allows the proportion of ic
V+ cells to be measured with greater
precision, particularly in immature subsets (such as DN3) where only a
minority of cells express TCR protein.
Intracellular V staining detects only productive VDJ
rearrangements
Imprecise joining of V, D, and J gene segments during VDJ
recombination leads to premature stop codons and/or an incorrect
translational reading frame for the C domain in two of three cases.
Nevertheless, the V domain could still in theory be translated
correctly in cases of nonproductive rearrangement, leading to a
truncated TCR protein. Two-color ic staining for V and C
domains argues against the presence of stable truncated TCR
proteins, since (for all 16 V domains analyzed) ic V staining was
only detected in cells that stained positively for ic C. Moreover,
the clear quantitative correlation between ic V and ic C staining
in individual cells (manifested as a diagonal staining pattern in
two-color plots) demonstrates that the two mAbs are binding to the same
molecule and hence (by implication) that there are no detectable
truncated TCR proteins expressing V but not C.
There are several possible explanations for the absence of detectable
truncated ic TCR proteins arising from nonproductive VDJ
rearrangements. First, although out-of-frame TCR and BCR rearrangements
are clearly transcribed, several studies indicate that the resulting
mRNAs are considerably less stable than mRNAs arising from in-frame
rearrangements (50, 51, 52). Second, even if they are
translated, truncated ic TCR proteins may be degraded much more
rapidly than full-length proteins. Finally, truncated ic TCR
proteins (even if relatively stable) may not fold correctly and thus
may not be recognized by anti-V mAbs. Whatever the explanation,
the fact that none of the 15 independent anti-V mAbs used in our
study recognizes truncated ic TCR proteins rules out the
possibility that the ic V staining technique itself introduces a
repertoire bias by selectively detecting certain nonproductive VDJ
rearrangements.
Biases in VDJ rearrangement at the TCR locus
VDJ rearrangements and subsequent expression of a TCR
protein first occur during the DN3 stage of adult thymus development.
Thus DN3 thymocytes would be expected to express a TCR V repertoire
representative of the frequency at which individual V gene segments
recombine during development. As shown here by ic V staining, the
frequency of expressed V segments varies by a factor of 3 among DN3
thymocytes (8.6% for V8.2 vs 2.9% for V12). This variation in
V usage is not due to preferential association of certain
V-chains into a pre-TCR complex, since similar V repertoires were
observed in small and large DN3 thymocyte subsets, which represent
stages before and after pre-TCR selection events (47).
Moreover, V repertoires were not significantly different among DN3
thymocytes from pT-deficient and wild-type mice, formally ruling out
any major influence of pre-TCR-mediated selection on the observed V
bias. Subject to the caveat that ic V staining measures only the
frequency of productively rearranged VDJ alleles (see above), we
conclude that there is a significant developmental bias inherent in
VDJ recombination in adult thymocytes before pre-TCR- or
TCR-mediated selection. Whether a similar bias also applies to fetal
VDJ rearrangement remains to be investigated.
Possible origin of biased VDJ recombination in immature T
cells
There are a number of factors that could influence the frequency
of rearrangement of individual V gene segments in DN3 thymocytes. By
analogy with studies of VDJH recombination in
immature B cells, one of the most obvious possibilities would be the
relative proximity of a given V segment to the DJ cluster.
Indeed, in both fetal and adult mouse pre-B cells, it has been shown
that DJH-proximal VH
segments (such as VH 81X) are preferentially
rearranged (37, 38, 39, 40, 42, 43, 44). In marked contrast to pre-B
cells, the most DJ-proximal V segments in DN3 thymocytes (V7
and V3) are apparently rearranged at relatively low frequency,
whereas the relatively distal V8.2 segment is rearranged most
frequently. These data indicate that physical proximity on the
chromosome does not play an equivalent role in directing VDJ
rearrangement in immature mouse T and B cells.
Several other molecular mechanisms could influence the frequency of
rearrangement of individual V gene segments. For example, subtle
differences in the conserved heptamer/nonamer recombination signal
sequences (RSS) or slightly differing lengths of the conserved 23-aa
spacers could favor recognition or cleavage of certain V segments by
the recombinase machinery (53, 54, 55, 56). In this respect, the
recent availability of the complete nucleotide sequence of the TCR
locus has allowed us to compare RSS and spacer lengths for all V
genes examined in this study (see GenBank data sequences under
AE000663, AE000664, and AE000665). This analysis indicates that there
is no obvious difference in either parameter that correlates with the
observed frequency of V recombination in DN3 thymocytes (A. Wilson
and C. V. Jongeneel, data not shown). Clearly, quantitative
functional analysis of the efficiency of cleavage of these V
recombination substrates in vitro will be required to formally address
this issue.
Finally, it is possible that differences in the frequency of
rearrangement of particular V genes simply reflect differences in
their accessibility to recombinase (53, 56, 57). In this
regard, recent studies have demonstrated that the efficiency of VDJ
recombination is influenced by nucleosomal structure, histone
acetylation, methylation status, and transcriptional activity
(58, 59, 60, 61). Obviously any (or all) of these factors may
contribute to the biased V repertoire of immature T cells.
No evidence for pre-TCR-mediated V-selection
Another factor influencing the VH repertoire in immature B cells is the differential ability of individual IgH chains to form a functional pre-BCR (62, 63). It has been estimated that only 50% of IgH chains in pre-B cells have the capacity to pair with the surrogate L chain and hence to form a pre-BCR (7, 42). This failure to form a functional pre-BCR is at least in part due to structural constraints imposed by the VH domain, since certain VH families (such as VH 7183 and VH Q52) are significantly less represented in the expressed VH repertoire following pre-BCR-mediated selection (42).
In contrast to these findings for immature B cells, we find no evidence
for a role of the V domain in pre-TCR-mediated selection. In
particular, large DN3 thymocytes (which are already cycling as a
consequence of pre-TCR signaling) expressed a V repertoire similar
to that of their small, noncycling DN3 counterparts. Moreover, the V
repertoire in DN3 thymocytes of pT-deficient mice, which are
genetically incapable of assembling a pre-TCR, was not significantly
different from that observed in wild-type mice. Even more strikingly,
the V repertoire in DN4 and ISP thymocyte subsets, which have
undergone extensive proliferation as a consequence of pre-TCR
signaling, could not be distinguished from the DN3 V repertoire.
Clearly, these data do not formally exclude the possibility that the
pre-TCR may exert a selective role in forming the TCR -chain
repertoire, perhaps by favoring certain CDR3 lengths or sequence
motifs. Nevertheless, they argue strongly that V domains (unlike
VH domains) do not play an important structural
role in the assembly of the pre-TCR complex. This result might have
been anticipated in view of the fact that a genetically engineered
pre-TCR complex lacking most of the extracellular portion of the TCR
-chain (including the V domain) appears to signal normally in
transgenic mice (64, 65).
Differing requirements for the VH and V
domains in pre-BCR vs pre-TCR selection may have structural
implications. Whereas the pre-BCR contains (in addition to the
surrogate L chain and signaling components) a VpreB component, the
existence of a comparable VpreT element in the pre-TCR has not been
demonstrated. Based on our data, one could speculate that the
hypothetical VpreT chain does not exist and consequently that fewer
molecular constraints are imposed upon V (as opposed to
VH) pairing during pre-TCR and pre-BCR
assembly.
Concluding remarks
In conclusion, we have shown that the overall striking parallels
in T and B cell development extend to biases in V gene segment
recombination at the TCR and IgH loci. However, in contrast to
VH gene biases in immature B cells, V gene
biases in immature T cells do not reflect proximity to D segments and
are not significantly influenced by pairing with the surrogate (pT)
chain. Thus, inherent developmental biases in V gene rearrangement
represent one of the major elements shaping the mature TCR V
repertoire.
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Acknowledgments |
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a-congenic and pT-deficient mice and
Victor Jongeneel (Swiss Institute of Bioinformatics) for help in
alignment of RSS. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. H. Robson MacDonald, Ludwig Institute for Cancer Research, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.
3 Abbreviations used in this paper: BCR, B cell receptor; RSS, recombination signal sequence; ic, intracellular; DN, double negative; ISP, immature single positive.
Received for publication July 19, 2000. Accepted for publication September 27, 2000.
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M. Senoo, L. Wang, D. Suzuki, N. Takeda, Y. Shinkai, and S. Habu Increase of TCR V{beta} Accessibility within E{beta} Regulatory Region Influences its Recombination Frequency But Not Allelic Exclusion J. Immunol., July 15, 2003; 171(2): 829 - 835. [Abstract] [Full Text] [PDF]
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R. Pacholczyk, P. Kraj, and L. Ignatowicz Peptide Specificity of Thymic Selection of CD4+CD25+ T Cells J. Immunol., January 15, 2002; 168(2): 613 - 620. [Abstract] [Full Text] [PDF]
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 D. Gibbons, N. C. Douglas, D. F. Barber, Q. Liu, R. Sullo, L. Geng, H.-J. Fehling, H. von Boehmer, and A. C. Hayday The Biological Activity of Natural and Mutant pT{alpha} Alleles J. Exp. Med., September 4, 2001; 194(5): 695 - 704. [Abstract] [Full Text] [PDF]
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