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Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsDiscussionReferences |
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genes. In chimeras in which the majority of
thymocytes are blocked at the
CD4-CD8-CD25+ stage (RAG1
deficient), and only a small proportion of T cell precursors are of
wild-type origin, we observed no difference in the maturation of
wild-type CD4-CD8-CD25+ cells to
the CD4+CD8+ stage as compared with control
chimeras. Therefore, the number of cell divisions occurring during this
transition is fixed and not subject to homeostatic regulation. In
contrast, in mixed chimeras in which the majority of thymocytes are
blocked at the CD4+CD8+ stage (TCR-
deficient), an increased efficiency of development of wild-type mature
CD8+ cells was observed. Surprisingly, the rate of
generation of mature CD4+ thymocytes was not affected in
these chimeras. Thus, the number of selectable CD8 lineage thymocytes
apparently saturates the selection mechanism in normal mice while the
development of CD4 lineage cells seems to be limited only by the
expression of a suitable TCR. These data may open the way to the
identification of homeostatic mechanisms regulating thymic output and
CD4/CD8 lineage commitment, and the development of means to modulate
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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and TCR-ß, is
expressed at the cell surface (1). This process is known as ß
selection and increases the proportion of useful precursors that will
subsequently rearrange the TCR- gene. Signaling through
the pre-TCR complex is known to induce these maturation events although
a ligand (if it exists) for the pre-TCR remains to be identified (2).
In recombinase-activating gene 1-deficient
(RAG1°)4 mice
CD4-CD8-CD25+ thymocytes do not
differentiate to the CD4+CD8+ stage as a
consequence of inability to express a pre-TCR (3).
Once CD4+CD8+ thymocytes express the clonotypic
TCR-ß complex they undergo clonal selection processes that result
in a T cell repertoire capable of recognition of self-MHC molecules
("positive selection") while depleted of autoreactive cells
("negative selection") (4, 5, 6). Thymocytes recognizing suitable
MHC-peptide ligands will undergo differentiation events characterized
by the down-modulation of one of the two coreceptors, up-regulation of
the TCR complex, and modulation of the expression of activation and
maturation markers (7, 8, 9, 10). Fully mature T lymphocytes (characterized
by the expression of either CD4 or CD8, high levels of the TCR and
Qa-2, and low levels of HSA) (11, 12) leave the thymus to populate the
peripheral lymphoid organs. Immature CD4+CD8+
thymocytes do not progress to the mature CD4+ or
CD8+ stage in the absence of TCR-, e.g., in
TCR--deficient (TCR°) mice (13).
Several reports have established that only around 3 to 5% of thymocytes fully mature (14, 15). Part of this relatively low efficiency results from the limited number of immature thymocytes with appropriate specificities. Thus, estimates of the proportion of MHC-reactive, preselection CD4+CD8+ thymocytes are in the range of 5% (16). Moreover, in mice lacking thymic negative selection, the output of mature T cells is increased approximately twofold (17, 18). Even in TCR-transgenic mice in which practically all thymocytes express selectable TCRs, not all of the thymic precursors will completely mature. The availability of a limited number of "selecting niches" in the thymus has been suggested to be responsible for this observation (14). Finally, limiting the availability of MHC class II- or class I-expressing thymic stromal cells in vitro has been shown to cause a reduction in the development of mature CD4+ and CD8+ cells, respectively (19). Thus, both TCR specificity-related and unrelated mechanisms may limit the thymic output.
To investigate in vivo whether the rate of thymic precursor cell
generation and of positive selection is subject to homeostatic
regulation in mice with a normal immature TCR repertoire, we studied
the development of immature and mature thymic populations in mixed
hemopoietic chimeras containing limited numbers (approximately 10%) of
selectable wild-type precursors diluted in mutant precursors unable to
mature to the immature CD4+CD8+ (RAG1°) or
mature CD4+ or CD8+ (TCR-°) stage. In all
cases selectable precursors were derived from B6.PL mice, so that TCR
specificity-determined clonotypic competition (20) would not influence
the results. If homeostatic mechanisms exist that limit progression
through a particular developmental stage, the 10-fold reduction in
normal precursors capable of maturation should allow a larger fraction
of those cells to proceed. Our results indicate that the development of
mature CD8+ (but, interestingly, not mature
CD4+ and immature CD4+CD8+)
thymocytes is subject to homeostatic regulation.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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C57BL/6 mice were obtained from Harlan (Zeist, the Netherlands).
C57BL/6 mice deficient in the expression of the genes encoding
TCR- (13) and RAG1 (3), as well as C57BL/6
congenic mice expressing the Thy1.1 allele (B6.PL), were purchased from
The Jackson Laboratory (Bar Harbor, ME).
Bone marrow chimeras
Lethally irradiated (1000 rad 
-irradiation, 137Cs
source) C57BL/6 hosts were reconstituted by i.v. injection of 1 to
2 x 107 bone marrow cells depleted of T cells using
anti-Thy1.2 (AT83) (21) and anti-Thy1.1 (HO-22.1.1) (22) Ab and
complement (Saxon Europe Ltd, Suffolk, U.K.). The chimeras were kept on
antibiotic-containing water (0.2% Bactrim, Roche, Basel, Switzerland)
for the duration of the experiment (usually 6 wk).
Abs
Flow cytometric analyses were performed using the following abs: anti-Thy1.2-FITC (AT15) (21); anti-TCR-ß-FITC (H57-597, PharMingen, San Diego, CA); anti-H-2Kb-FITC (AF6-88.5, PharMingen); anti-TCR-ß-PE (H57-597, PharMingen); anti-CD25-PE (PC61, Caltag, San Francisco, CA); anti-CD4-PE (H129.19, Boehringer Mannheim, Mannheim, Germany); anti-Thy-1.2-biotin (AT15, revealed with streptavidin-PE, Caltag); anti-CD4-Red613 (H129.19, Life Technologies, Gaithersburg, MD); anti-CD8-Red613 (53-6.7, Life Technologies); anti-CD25-Red613 (3C7, Life Technologies); anti-CD4-APC (RM4-5, PharMingen); and anti-CD8-APC (53-6.7, PharMingen). Analyses were performed using FACScan and FACStarPlus flow cytometers (Becton Dickinson, San Jose, CA).
Postirradiation thymic reconstitution
Hemopoietic chimeras were sublethally irradiated (700 rad gamma irradiation, 137Cs source) 6 wk after engraftment. Animals were analyzed 9 to 13 days later. Mature thymocytes were CD4+CD8-TCRßhigh or CD4-CD8+TCRßhigh. The fraction of these cells that were of B6.PL (Thy-1.1) origin was determined by Thy-1.2 staining. Four-color experiments in which the mature, B6.PL-derived cells were identified as CD4+CD8-H-2Kb-highThy-1.2- and CD4-CD8+H-2Kb-highThy-1.2- yielded similar results.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Lethally irradiated C57BL/6 hosts were reconstituted with a
mixture of B6.PL and RAG1°- or C57BL/6-derived bone marrow ((RAG1°
+ B6.PL) 
B6 and (B6 + B6.PL) B6 chimeras). The number and
proportion of injected B6.PL-derived bone marrow cells was kept
constant (10%) and the expected degree of chimerism (10–20%) was
observed among CD4-CD8-CD25+
thymocytes (Table I
). We investigated
whether the reduced number of total selectable precursors in (RAG1° +
B6.PL) B6 chimeras (Fig. 1, A and B, Table I) allows those cells to compensate by
increasing the degree of cellular expansion taking place during the
CD4-CD8-CD25+ to
CD4+CD8+ transition. The ratio of the absolute
number of B6.PL-derived (Thy-1.1)
CD4+CD8+TCRlow to
CD4-CD8-CD25+ thymocytes was used
as an indication of the degree of expansion occurring between these two
stages. Sublethal irradiation causes most thymocytes to die, followed
by the expansion and synchronized differentiation of a surviving
precursor population (23). To avoid measuring long-term accumulations,
we sublethally irradiated chimeras and analyzed them 10 to 13 days
later. As shown in Figure 2A,
no difference in the ratio of the number of B6.PL-derived
(Thy1.2-)
CD4+CD8+TCRlow to
CD4-CD8-CD25+ thymocytes was
observed in the two types of chimeras. This lack of a compensatory
increase in cellular expansion is accompanied by the significantly
lower number of total thymocytes observed in (RAG1° + B6.PL)
B6 than in (B6 + B6.PL) B6 chimeras (Fig. 2B). Thus, the rate of proliferation occurring during the
CD4-CD8-CD25+ to
CD4+CD8+ transition is apparently not limited
by homeostatic mechanisms.
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B6 chimeras, in which practically normal numbers
of B6.PL-derived mature thymocytes are present (Fig. 1
C,
Table I), no significant numbers of CD4+CD8+
RAG1° thymocytes were observed (Fig. 1E, Table I). As
expected, C57BL/6- and B6.PL-derived precursors develop into
CD4+CD8+ thymocytes in both types of chimeras
(Fig. 1, C, D, and F). Development of mature CD8+ but not CD4+ thymocytes is limited by a homeostatic mechanism
The next stage in the differentiation of thymocytes, the
development of CD4+ or CD8+ mature thymocytes
from CD4+CD8+ precursors, requires the
expression of the TCR-ß heterodimer. The involvement of
homeostatic mechanisms in thymic-positive selection was similarly
assessed using hemopoietic chimeras in which a minority (10–15%,
Table I) of the CD4+CD8+ precursors expressed
the TCR-ß heterodimer and were therefore potentially selectable
(B6.PL, Thy-1.1). The remaining (Thy-1.2)
CD4+CD8+ thymocytes did not express TCR-
protein because of a targeted mutation at the TCR C locus
(13). As expected, the number and proportion of B6.PL-derived
CD4+CD8+ thymocytes were comparable in the
(TCR-° + B6.PL) B6 and (B6 + B6.PL) B6 chimeras (Fig. 3, A and B, Table I). Moreover, while a normal proportion
of C57BL/6-derived thymocytes in (B6 + B6.PL) B6 chimeras
fully matured, no mature TCR--deficient thymocytes were seen (data
not shown).
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° + B6.PL) B6 chimeras than
in (B6 + B6.PL) B6 mice (Fig. 3, A and
B, Table I). In contrast, the steady state number of mature
CD4+ thymocytes was similar in the two types of chimeras.
Since steady state numbers of mature thymocytes do not necessarily
reflect the rate of generation of those cells, we also analyzed their
de novo generation using the postirradiation reconstitution assay (17, 23). While the rate of generation of mature CD8+ thymocytes
was increased approximately twofold, the rate of generation of
CD4+ cells in (TCR-° + B6.PL) B6 chimeras was
similar to that of control (B6 + B6.PL) B6 mice (Fig. 4, Table I). These data confirm those of the
steady state and indicate that the increase in the number of
CD8+ thymocytes results from an increased generation,
rather than thymic retention, of these cells.
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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ß is required. Using mixed bone marrow chimeras in which only
a minority of precursors is selectable, we show here evidence that the
differentiation of mature CD8+ but not CD4+ or
immature CD4+CD8+ thymocytes is limited by
homeostatic mechanisms.
ß Selection is accompanied by proliferation that is responsible for
the generation of most thymocytes, and by an up-regulation of
expression of CD4 and CD8. The pre-TCR that mediates ß selection
contains a monomorphic pT -chain and a clonotypic TCR ß-chain (1),
although other "pre-TCR" compositions have been proposed, all of
them containing products of genes requiring rearrangement for their
expression (26). Rearrangement-defective mice contain few or no
CD4+CD8+ thymocytes, presumably because of the
failure to produce a pre-TCR (3, 27). However, SCID and RAG°
thymocytes have been shown to express CD4 and CD8 after irradiation
(28, 29, 30) and SCID thymocytes can complete this differentiation step in
the presence of mature T cells (24, 25). Despite the presence of
practically normal numbers of B6.PL-derived mature thymocytes in
(RAG1° + B6.PL) B6 chimeras, we did not observe significant
numbers of RAG1° thymocytes expressing CD4 and CD8. This result
indicates that the expression of CD4 and CD8 induced by mature T cells
on SCID-derived thymocytes is specific for this mutation (which allows
a low level of productive rearrangements) (31) and does not occur in
strictly rearrangement-deficient RAG1° thymocytes (3). Therefore, a
pre-TCR containing the product of a rearranging gene is absolutely
required for progression to the CD4+CD8+ stage
during normal thymocyte development.
In (RAG1° + B6.PL) B6 chimeras, in which the absolute number of
precursors capable of differentiation to the
CD4+CD8+ stage is strongly reduced, the total
number of thymocytes was significantly lower than in (B6 + B6.PL)
B6 mice. The degree of expansion occurring during the transition of
the CD4-CD8-CD25+ to the
CD4+CD8+ stage was identical in mice with high
and low numbers of selectable precursors. Therefore, the number of cell
divisions occurring at this stage seems to be preprogrammed and is not
determined by thymocyte-extrinsic factors. This surprising result
raises the possibility that thymus size is controlled at a very early
stage of development, i.e., by the number of
CD4-CD8-CD25+ thymocytes capable
of expressing a pre-TCR. This number, in turn, may be determined by
intrathymic proliferation at this or at earlier stages of
differentiation, and/or by immigration of precursors into the thymus.
Because of the small number of these precursor cells and the lack of
precise phenotypic markers for their identification, the mixed bone
marrow chimeric system used here cannot reliably be used to investigate
this issue.
The development of mature thymocytes has been suggested to be under the control of TCR-independent rate-limiting mechanisms. In one report, limiting the proportion of MHC-expressing thymic stromal cells in an in vitro system was shown to result in a proportional reduction in the steady state levels of mature thymocytes (19). The results were suggested to indicate that each thymocyte can interact with a single "selecting niche," a concept for which additional supporting data have been reported (32). Although very intriguing, these data do not address the question of whether in a normal thymus the number of selecting niches is limiting for the development of mature CD4+ and/or CD8+ thymocytes.
The notion that the number of thymic niches may be a limiting factor in the development of CD8+ thymocytes was originally proposed in a study addressing the kinetics of TCR transgenic (H-Y/H-2Db) thymocyte development (14). In mixed hemopoietic chimeras containing varying ratios of TCR transgenic to wild-type bone marrow cells, the fraction of TCR transgenic cells that matured inversely correlated with their representation in the immature population. It was proposed that a limited number of selecting niches is available for the positive selection of CD8+ thymocytes. It needs to be kept in mind that these results were obtained using TCR transgenic mice with an immature TCR repertoire in which a very high fraction of cells is positively selectable, in contrast to the normal immature TCR repertoire in which only approximately 5% are estimated to be MHC reactive (16). While this high frequency of selectable cells may saturate any putative rate-limiting selection mechanism in TCR transgenic mice, such limitations may play no detectable role in mice with a normal repertoire.
Our data indicate that in mice with a normal immature TCR repertoire
the development of CD8+, but surprisingly not
CD4+, thymocytes is limited because of the abundance of
selectable CD4+CD8+ precursors. In bone marrow
chimeric mice in which only approximately 5 to 10% of the
CD4+CD8+ thymic precursors can express the
TCR-ß heterodimer and therefore may be positively selected, the
proportion of mature CD8+ but not CD4+ cells is
1.5-fold increased as compared with control chimeras. This steady state
increase is confirmed by a 2-fold increased rate of de novo generation
of mature CD8+ (but not CD4+) cells in these
chimeras. Thus, the number of selectable CD8 lineage thymocytes
apparently saturates the selection mechanism in normal mice while the
development of CD4 lineage cells seems to be limited only by the
expression of a suitable TCR. Our results may at least partially
explain the excess of mature CD4+ over CD8+
thymocytes, although other factors (e.g., immature TCR repertoire)
probably also contribute. Moreover, homeostatic limitations in the
generation of mature CD8+ (but not CD4+)
thymocytes may contribute to the genetically determined differences in
CD4/CD8 ratios observed in certain mouse strains and in man (33, 34, 35).
In any event our results are consistent with data suggesting that
immature thymocytes may differentiate into the CD4 lineage by default
while additional signals seem to be required for development of CD8
lineage thymocytes (36, 37).
Since homeostatic limitation of mature CD8+ thymocyte development is TCR specificity independent, the responsible mechanism seems to regulate either commitment to the CD8 lineage (4) or survival of already committed thymocytes. It will therefore be of considerable interest to identify the homeostatic mechanisms limiting the development of mature CD8+ thymocytes. In this regard, a putative role of the product of the Notch1 gene in the development of mature CD8+ cells has been invoked (38). A transgenic constitutively activated Notch1 protein was shown to favor differentiation of mature CD8+ thymocytes at the expense of mature CD4+ cells. One explanation for our results could therefore be limited availability of the Notch1 ligand.
The thymic output of mature T lymphocytes is determined by 1) the immigration of bone marrow-derived stem cells into the thymus, 2) the intrathymic generation of T cell precursors, and 3) the proportion of those precursors that will complete maturation. During ontogeny and in case of T cell depletion caused by pathologic conditions (e.g., AIDS), the output of the thymus and thus the concentration of peripheral T lymphocytes could potentially be modulated. As shown in this report, given the right conditions, the rate of generation of mature CD8+ T lymphocytes may be significantly increased. It will be of interest to identify the factors limiting the development of mature CD8+ thymocytes, and to assess whether their natural variations or artificial manipulation will lead to increased thymic output.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Genetics and
Microbiology, University of Geneva Medical School, Geneva,
Switzerland.
3 Address correspondence and reprint requests to Dr. H. Robson MacDonald, Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland.
4 Abbreviations used in this paper: RAG°, recombinase-activating gene deficient; TCR-°, TCR- deficient; PE, phycoerythrin.
Received for publication August 21, 1997. Accepted for publication November 24, 1997.
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References |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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and ß block thymocyte development at different stages. Nature 360:225.[Medline]
T cells promote CD4 and CD8 expression by SCID thymocytes. Int. Immunol. 5:991.-radiation induces differentiation of CD4-/CD8- into CD4+/CD8+ thymocytes without T cell receptor ß rearrangement in recombinase activation gene 2-/- mice. J. Exp. Med. 180:1517.This article has been cited by other articles:
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  J. Ribot, P. Romagnoli, and J. P. M. van Meerwijk Agonist Ligands Expressed by Thymic Epithelium Enhance Positive Selection of Regulatory T Lymphocytes from Precursors with a Normally Diverse TCR Repertoire J. Immunol., July 15, 2006; 177(2): 1101 - 1107. [Abstract] [Full Text] [PDF]
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P. Romagnoli, D. Hudrisier, and J. P. M. van Meerwijk Molecular Signature of Recent Thymic Selection Events on Effector and Regulatory CD4+ T Lymphocytes J. Immunol., November 1, 2005; 175(9): 5751 - 5758. [Abstract] [Full Text] [PDF]
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C. E. Busse, A. Krotkova, and K. Eichmann The TCR{beta} Enhancer Is Dispensable for the Expression of Rearranged TCR{beta} Genes in Thymic DN2/DN3 Populations but Not at Later Stages J. Immunol., September 1, 2005; 175(5): 3067 - 3074. [Abstract] [Full Text] [PDF]
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M. M. Freeman and H. K. Ziegler Simultaneous Th1-Type Cytokine Expression Is a Signature of Peritoneal CD4+ Lymphocytes Responding to Infection with Listeria monocytogenes J. Immunol., July 1, 2005; 175(1): 394 - 403. [Abstract] [Full Text] [PDF]
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E. S. Sobel, L. Morel, R. Baert, C. Mohan, J. Schiffenbauer, and E. K. Wakeland Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Evidence for Functional Expression of Sle3/5 by Non-T Cells J. Immunol., October 1, 2002; 169(7): 4025 - 4032. [Abstract] [Full Text] [PDF]
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 A. R.M. Almeida, J. A.M. Borghans, and A. A. Freitas T Cell Homeostasis: Thymus Regeneration and Peripheral T Cell Restoration in Mice with a Reduced Fraction of Competent Precursors J. Exp. Med., August 27, 2001; 194(5): 591 - 600. [Abstract] [Full Text] [PDF]
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 J. P.M. van Meerwijk and H. R. MacDonald In Vivo T-Lymphocyte Tolerance in the Absence of Thymic Clonal Deletion Mediated by Hematopoietic Cells Blood, June 1, 1999; 93(11): 3856 - 3862. [Abstract] [Full Text] [PDF]
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