| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
T Cell Development
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß or
TCR. During ß T cell development, it is clearly established
that productive rearrangement at the TCR ß locus in immature
precursor cells leads to the expression of a pre-TCR complex. Signaling
through the pre-TCR results in the selective proliferation and
maturation of TCR ß+ cells, a process that is known as
ß-selection. However, the potential role of ß-selection during
T cell development is controversial. Whereas PCR-RFLP and
sequencing techniques have provided evidence for a bias toward in-frame
VDJß rearrangements in cells (consistent with ß-selection),
cells apparently develop normally in mice that are unable to
assemble a pre-TCR complex due to a deficiency in TCR ß or pT
genes. In this report, we have directly addressed the physiologic
significance of ß-selection during cell development in normal
mice by quantitating intracellular TCR ß protein in cells and
correlating its presence with cell cycle status. Our results indicate
that ß-selection plays a significant (although limited) role in
cell development by selectively amplifying a minor subset of
precursor cells with productively rearranged TCR ß
genes. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß or TCR. During
the development of ß lineage cells, immature precursors undergo
VDJ rearrangement of the TCR ß locus at the
CD4-CD8-CD25+ stage (1, 2, 3). Those
precursor cells that successfully rearrange VDJß are able to express
a pre-TCR complex (composed of TCR ß, pT, and CD3) at the cell
surface (reviewed in refs. 4 and 5). Signaling through the pre-TCR
leads to the down-regulation of CD25 and to the subsequent
proliferation and maturation of ß lineage cells (reviewed in refs.
6–9). Because these events are all linked to productive TCR ß
rearrangement, the process is frequently referred to as ß-selection.
In contrast to the ß lineage, the role of ß-selection during
T cell development is controversial. In this regard, a positive
role for ß-selection in cells has been inferred from PCR-RFLP
and sequencing studies showing that the frequency of in-frame VDJß
rearrangements in these cells is greater than what would be predicted
by random recombination (10, 11). However, the potential importance of
ß-selection in T cell development has been challenged by the
fact that normal (or even elevated) numbers of T cells can
develop in mice that are deficient for the TCR ß (12) or pT (13)
genes. Neither of these approaches is definitive, however, because PCR
techniques only sample cells that have attempted VDJß rearrangement,
whereas knockout mice may compensate unphysiologically for their
pre-TCR deficiency.
In this study, we have directly addressed the physiologic relevance of
ß-selection in normal T cell development by quantitating
intracellular (i.c.)2 TCR ß
protein in cells and by correlating i.c. TCR ß expression with
cell cycle status. Our data indicate that ß-selection plays a
significant (although relatively limited) role during cell
development by promoting the proliferation of a small subset of
precursors with productively rearranged TCR ß genes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6 female mice were obtained from Harlan Olac (Bicester,
U.K.) and used at 5–6 wk of age. TCR ß- and TCR -deficient mice
were obtained from the Jackson Laboratory (Bar Harbor, ME) and used at
6–12 wk of age. Lymph node (LN), spleen, and
CD4-CD8-CD25- thymocytes were
prepared as described previously (14). Thymocytes from TCR
ß-/- mice were directly stained without depletion
steps. During subsequent FACS analysis and/or sorting, contaminating
CD4+, CD8+, CD25+, and
ß+ cells were positively eliminated where appropriate
by staining with FITC-conjugated mAbs to these surface markers.
Flow cytometry and cell sorting
Thymocyte subsets were analyzed on a FACScan (Becton Dickinson,
San Jose, CA) and sorted on a FACStar+ (Becton Dickinson).
Analysis and sorting for simultaneous surface and i.c. proteins was
performed essentially as described elsewhere (15). Specifically,
surface staining was performed with two colors using a pool of direct
FITC conjugates as described above or TCR -FITC (PharMingen, San
Diego, CA) together with either direct tricolor or CyChrome conjugates
such as TCR -tricolor (Caltag Laboratories, Burlingame, CA) or
CD44-CyChrome (PharMingen). All other FITC conjugates were purified and
conjugated from hybridomas grown in this laboratory and have been
described elsewhere (14). After fixation in 2% paraformaldehyde and
permeabilization with 0.5% saponin, i.c. TCR proteins were stained
with direct phycoerythrin (PE) conjugates as follows: TCR -PE
(GL3), TCR ß-PE (H57.597), CD3
-PE (17A2), and hamster Ig-PE
isotype control (all purchased from PharMingen).
Cell cycle analysis
DNA content of the thymic subsets was determined by initially sorting the selected populations defined by both surface and i.c. protein expression as described above. Propidium iodide (PI) staining of DNA was performed using standard procedures as described previously (16), except the fixation step was omitted. Briefly, sorted cells were treated with 3N HCl, washed in PBS, neutralized with 0.1 M Na2B4O7, and treated with RNase A before the addition of PI. FACS analysis was performed on a FACScan using a doublet discrimination module.
Calculation of predicted frequency of in-frame VDJß
rearrangements in T cells
The model is illustrated schematically in Fig. 3
. If nine
precursor cells attempt VDJß rearrangement randomly on both
chromosomes, four will fail because both alleles will be out of frame
(ß-). The remaining five cells will succeed
(ß+) and, assuming allelic exclusion halts further
rearrangement at the TCR ß locus, will contain five in-frame
(ß+) alleles, two out-of-frame (ß-)
alleles, and three germline (ß°) alleles (see ref. 11 for details).
Alternatively, if allelic exclusion at the TCR ß locus does not
function in lineage cells, the five productively rearranged
cells will contain six ß+, four ß-, and no
ß° alleles. Because the model in Fig. 3 predicts that these 9
precursor cells will ultimately give rise to 15 productively rearranged
and 4 nonproductively rearranged mature cells, the resulting
frequency of in-frame VDJß rearrangements among cells can be
calculated from the total number of ß+ and
ß- alleles in these 19 cells (i.e., 52%
(15ß+14ß-) assuming allelic exclusion and
47% (18ß+20ß-) assuming no allelic
exclusion).
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T
cells with in-frame TCR ß rearrangements, we decided to stain these
cells for i.c. TCR ß protein. Accordingly,
CD4-CD8-CD25- thymocytes were
surface-stained in one color with mAbs to TCR and in the second
color with a mixture of mAbs including CD4, CD8, CD44, and TCR ß
to remove all contaminating ß+ T cells. An excess of
unlabeled anti-CD3 mAb was also added to block surface CD3
molecules. Following fixation and permeabilization, the samples were
stained in the third color with mAbs to TCR ß or, as a positive
control, to TCR or CD3. As shown in Fig. 1, 
15% of CD44low thymic
cells, which correspond to the major thymic T cell
lineage, stained brightly for i.c. TCR ß. As expected, 100% of
thymic cells were positive for i.c. TCR and i.c. CD3.
The specificity of the i.c. TCR ß staining in cells was
further confirmed by the complete absence of i.c. TCR ß staining in
CD44low thymic cells from TCR ß-deficient mice
(Fig. 1). A similar analysis of mature ß thymocytes in normal mice
revealed that they did not express detectable i.c. TCR protein
although, as expected, all stained brightly for i.c. TCR ß and i.c.
CD3 (Fig. 1).
|
cells express i.c. TCR ß
protein does not support the hypothesis that ß-selection plays an
important role in T cell development as suggested previously
(10, 11). However, it remained possible that thymic T cells
expressing i.c. TCR ß would have a selective advantage (either in
survival, proliferation, or export) over their TCR ß-
counterparts. To address this issue, we analyzed peripheral T
cells in the LN and spleen for the expression of i.c. TCR ß. As shown
in Table I., the proportion of peripheral
T cells expressing i.c. TCR ß was identical with that observed
in the thymus. A similar frequency (15%) of i.c. TCR
ß+ thymic and peripheral T cells was observed in
TCR -deficient mice (Table I); these mice were the source of the
cells reported previously to have an extremely high frequency of
in-frame VDJß rearrangements (10). Thus, it is clear that i.c. TCR
ß expression does not confer any selective advantage to mature
cells.
|
cells constituted a
relatively minor population, it was nevertheless of interest to
investigate their proliferative status. In the ß T cell lineage,
precursor cells with productive TCR ß rearrangements proliferate
selectively due to signals mediated by the formation of a pre-TCR
complex consisting of TCR ß, pT, and CD3. To determine whether
productive TCR ß rearrangement might also influence the proliferation
of lineage cells, we sorted surface + thymic T
cells according to their expression of i.c. TCR ß (see Fig. 1
) and
reanalyzed the sorted subpopulations for DNA content. As shown in Fig. 2, i.c. TCR ß+ T
cells contained a significantly higher proportion of cycling cells as
compared with their i.c. TCR ß- counterparts. In 10
independent experiments, the mean percentage of cycling (S plus
G2/M) cells was 2.5-fold higher in i.c. TCR
ß+ as compared with i.c. TCR ß- thymic
cells. An influence of productive TCR ß rearrangement on
T cell proliferation was independently confirmed by assessing
the cell cycle status of thymic cells in TCR
ß-/- mice. As shown in Fig. 2, a comparable percentage
of cells were cycling in these mice as compared with i.c. TCR
ß- cells in wild-type (wt) controls.
|
T cells, when peripheral T cells
were isolated according to i.c. TCR ß expression and analyzed for
their proliferative status, both i.c. TCR ß+ and i.c. TCR
ß- subsets contained <5% cells in cycle (data not
shown). These data are in agreement with a recent study showing that
although the majority of thymic T cells are virtually all
labeled by 5-bromodeoxyuridine within 2 days, most peripheral T
cells are not cycling (17). Moreover, these findings explain why the
increased cycling of i.c. TCR ß+ thymic T cells
does not result in a higher frequency of i.c. TCR ß+
T cells in the periphery. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T
cell development. Previous attempts to determine the frequency of
productive VDJß rearrangements in cells using PCR-RFLP and
sequencing techniques have been inconclusive (10, 11, 18, 19, 20, 21), with
estimates of in-frame rearrangements varying widely (from 30 to
70%). Irrespective of the source of these numerical discrepancies,
it is important to note that molecular analyses of VDJß rearrangement
in cells at the population level give only limited information,
because the frequency of rearranged alleles (upon which the calculation
is based) is not known. Instead, we have chosen to directly measure the
frequency of cells with productively rearranged TCR ß genes by
quantitating the i.c. TCR ß protein in these cells. Our results
indicate that only 15% of cells in normal mice are i.c. TCR
ß+, thus setting an upper limit for the impact of
ß-selection on T cell development.
Although it is formally possible that the expression of some
productively rearranged TCR ß-chains is repressed in i.c. TCR
ß- cells, we believe this is highly unlikely
because no evidence for TCR ß transcriptional silencing in
cells has been reported. Therefore, we favor the hypothesis that all
i.c. TCR ß- cells harbor either nonproductively
rearranged or germline VDJß alleles, which is consistent with earlier
quantitative Southern blot analyses (18) (see below).
Despite the relatively low proportion of i.c. TCR ß+
thymic cells, approximately threefold more of these cells were
in cycle compared with their i.c. TCR ß- counterparts.
This result in turn implies that productive TCR ß rearrangement
selectively favors the proliferation of cells of the lineage. By
analogy with ß T cells, it is tempting to speculate that an
increased proliferation of i.c. TCR ß+ cells
occurs as a result of signaling via the pre-TCR. However, this scenario
would require that thymic cells (or their precursors) express
pT, which is an essential component of the pre-TCR complex. Although
mature cells reportedly do not express pT as assessed by PCR
(22), no such studies have been performed on precursors due to a
lack of appropriate phenotypic markers to identify such cells. An
alternative possibility would be that expression of TCR ß protein in
the absence of pT is still able to provide a signal that
significantly enhances cell proliferation.
Aside from the issue of ß-selection in T cells, our data have
more general implications for models of ß and lineage
commitment. Because TCR , , and ß genes appear to rearrange at
about the same time during development (23), it has been proposed that
successful TCR or TCR ß rearrangement (and the subsequent
production of a TCR or pre-TCR protein) may be instrumental in
the commitment of a common precursor cell to the or ß
lineage, respectively (reviewed in ref. 5). In this context, our
finding that a significant fraction of cells express i.c. TCR
ß protein refutes the hypothesis that productive TCR ß
rearrangement alone is sufficient to commit a common precursor to the
ß lineage. However, the existence of i.c. TCR ß+
cells remains compatible with a model in which productive TCR
rearrangement commits a common precursor to the lineage
irrespective of its TCR ß rearrangement status.
Finally, the i.c. TCR ß staining and cell cycle analysis of
cells presented here allows us to propose a simplified model for the
role of TCR ß rearrangement and ß-selection in T cell
development that is consistent with our own results as well as almost
all available published data. According to this model (Fig. 3), only 10% of lineage precursor
cells would attempt VDJß rearrangement. For a cohort of nine such
precursor cells (assuming random VDJß reading frames after
recombination on both alleles), five would undergo productive
rearrangement (TCR ß+), whereas the other four would be
TCR ß-. Consistent with the cell cycle analysis, each
TCR ß+ precursor would give rise, on average, to
three TCR ß+ progeny via ß-selection, whereas
TCR ß- or unrearranged (TCR ß°) precursors would
produce only a single mature cell. As a result of this scenario,
the mature thymic cell population would contain our observed
frequency of 15% i.c. TCR ß+ cells as well as 4% TCR
ß- and 81% TCR ß° cells (Fig. 3). Hence, the
predicted proportion of cells that have attempted VDJß
rearrangement would be 19%, which is in close agreement with a
quantitative Southern blot analysis estimating a 20% loss of germline
Vß genes in DNA isolated from purified thymic cells (18).
Moreover, the predicted frequency of in-frame VDJß rearrangements
among cells according to our model (see Materials and
Methods for calculations) would be either 52%, assuming allelic
exclusion at the TCR ß locus, or 47%, assuming no allelic exclusion.
These theoretical values are very close to those measured
experimentally by Mertsching et al. (19, 20) (42%) and Burtrum et al.
(18) (50–55%) but are very different from those reported by Dudley et
al. (10, 11) (70%) and Vicari et al. (21) (30%). Lastly, because our
model implies that ß-selection affects only a small subset of
cell precursors in normal mice, it is not surprising that cell
development appears to be unaffected in TCR ß-/- and
pT-/- mice, in which ß-selection cannot occur.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: i.c., intracellular; PI, propidium iodide; wt, wild type; LN, lymph node(s); PE, phycoerythrin.
Received for publication May 13, 1998. Accepted for publication July 21, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß T cell development in the thymus of normal and genetically altered mice. Curr. Biol. 9:263.
ß TCR+ and TCR+ T cells from a common precursor. Semin. Immunol. 9:171.[Medline]
ß and T cells can share a late common precursor. Curr. Biol. 5:659.[Medline]
and ß block thymocyte development at different stages. Nature 360:225.[Medline]
gene in the development of ß but not T cells. Nature 375:795.[Medline]
ß transgenic mice. Dev. Immunol. 2:85.[Medline]
determines clonal deletion or induction of developmental program. J. Exp. Med. 177:707./ T cells. J. Exp. Med. 187:357. cell development. J. Immunol. 157:4293.[Abstract]
-expressing fetal mouse thymocytes are generated without TCR Vß-selection. Eur. J. Immunol. 26:804.[Medline]
gene rearrangement and transcription in adult thymic cells. Eur. J. Immunol. 27:389.[Medline]
TCR+NK1.1+ thymocytes specifically produce interleukin-4, are major histocompatibility complex class I-independent, and are developmentally related to ß TCR+ NK1.1+ thymocytes. Eur. J. Immunol. 26:1424.[Medline]
gene. Eur. J. Immunol. 25:1877.[Medline]
gene rearrangement and T early (TEA) expression in immature ß lineage thymocytes: implications for ß/ lineage commitment. Immunity 4:37.[Medline]
This article has been cited by other articles:
|
|
 |
|
  J. Inoue, T. Kanefuji, K. Okazuka, H. Watanabe, Y. Mishima, and R. Kominami Expression of TCR{alpha}beta Partly Rescues Developmental Arrest and Apoptosis of {alpha}beta T cells in Bcl11b-/- Mice J. Immunol., May 15, 2006; 176(10): 5871 - 5879. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Gerber, L. Boucontet, and P. Pereira Early Expression of a Functional TCR{beta} Chain Inhibits TCR{gamma} Gene Rearrangements without Altering the Frequency of TCR{gamma}{delta} Lineage Cells J. Immunol., August 15, 2004; 173(4): 2516 - 2523. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Laurent, N. Bosco, P. N. Marche, and R. Ceredig New insights into the proliferation and differentiation of early mouse thymocytes Int. Immunol., August 1, 2004; 16(8): 1069 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Renno, A. Wilson, C. Dunkel, I. Coste, K. Maisnier-Patin, A. Benoit de Coignac, J.-P. Aubry, R. K. Lees, J.-Y. Bonnefoy, H. R. MacDonald, et al. A Role for CD147 in Thymic Development J. Immunol., May 15, 2002; 168(10): 4946 - 4950. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kang, A. Volkmann, and D. H. Raulet Evidence That {{gamma}}{{delta}} versus {{alpha}}{beta} T Cell Fate Determination Is Initiated Independently of T Cell Receptor Signaling J. Exp. Med., March 12, 2001; 193(6): 689 - 698. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. D. Lacorazza, C. Tucek-Szabo, L. V. Vasovic, K. Remus, and J. Nikolich-Zugich Premature TCR{{alpha}}{{beta}} Expression and Signaling in Early Thymocytes Impair Thymocyte Expansion and Partially Block Their Development J. Immunol., March 1, 2001; 166(5): 3184 - 3193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Wilson, C. Marechal, and H. R. MacDonald Biased V{beta} Usage in Immature Thymocytes Is Independent of DJ{beta} Proximity and pT{{alpha}} Pairing J. Immunol., January 1, 2001; 166(1): 51 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Radtke, I. Ferrero, A. Wilson, R. Lees, M. Aguet, and H. R. MacDonald Notch1 Deficiency Dissociates the Intrathymic Development of Dendritic Cells and T Cells J. Exp. Med., March 27, 2000; 191(7): 1085 - 1094. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Wilson, M. Capone, and H. R. MacDonald Unexpectedly late expression of intracellular CD3{epsilon} and TCR {gamma}{delta} proteins during adult thymus development Int. Immunol., October 1, 1999; 11(10): 1641 - 1650. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
