By
From the * Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland; the Lymphocyte Biology Section, Laboratory of Immunology, National
Institutes of Health, Bethesda, Maryland 20892-1892; and § Laboratory of Cellular and Molecular
Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892-1892
Interactions between major histocompatibility complex (MHC) molecules expressed on stromal cells and antigen-specific receptors on T cells shape the repertoire of mature T lymphocytes emerging from the thymus. Some thymocytes with appropriate receptors are stimulated to undergo differentiation to the fully mature state (positive selection), whereas others with strongly autoreactive receptors are triggered to undergo programmed cell death before completing this differentiation process (negative selection). The quantitative impact of negative selection on the potentially available repertoire is currently unknown. To address this issue, we have constructed radiation bone marrow chimeras in which MHC molecules are present on radioresistant thymic epithelial cells (to allow positive selection) but absent from radiosensitive hematopoietic elements responsible for negative selection. In such chimeras, the number of mature thymocytes was increased by twofold as compared with appropriate control chimeras. This increase in steady-state numbers of mature thymocytes was not related to proliferation, increased retention, or recirculation and was accompanied by a similar two- to threefold increase in the de novo rate of generation of mature cells. Taken together, our data indicate that half to two-thirds of the thymocytes able to undergo positive selection die before full maturation due to negative selection.
Tlymphocytes express a heterodimeric To avoid potentially pathogenic responses to self-peptide-MHC complexes, precursor T lymphocytes are monitored for strong autoreactivity during intrathymic development and after emergence in the periphery (8, 9). Many
self-reactive T cells are physically deleted by induction of
programmed cell death. While some clonal deletion can
occur in response to MHC-peptide ligands expressed on
thymic epithelium (10), the main cell type involved in
this process is known to be of hematopoietic origin (2). In
normal mice expressing endogenous mouse mammary tumor virus (MMTV)1-encoded superantigens that interact
with TCRs containing particular V Mice.
Wild-type mice were C57BL/6 (H-2b haplotype) (see
Fig. 1; Jackson Laboratories, Bar Harbor, ME; other experiments,
Harlan Netherlands, Zeist, The Netherlands). Mice deficient in
MHC class I expression (MHC I°) because of a targeted disruption of the
Chimeras.
Chimeras were prepared essentially as described
previously (29). In brief, hosts were lethally irradiated (1,000 rads)
with a Cs137 source and injected the next day i.v. with 10-20 × 106 bone marrow cells depleted of T cells by complement killing
using anti-Thy1 antibody JIJ (30) and anti-Ly1 antibody C3P0
(31) in the experiment shown in Fig. 1 and anti-Thy1 antibody
AT83 (32) in other experiments.
Three-color Flow Cytometry.
Single-cell suspensions of thymocytes
were incubated on ice with saturating concentrations of the following antibodies: Fig. 1, anti-CD8-FITC, anti-TCR-PE (PharMingen, San Diego, CA), and anti-CD4-Red613 (GIBCO BRL,
Gaithersburg, MD); Figs. 2 and 4, anti-TCR-FITC (H57-597),
anti-CD4-PE (Boehringer-Mannheim, Mannheim, Federal Republic of Germany), and anti-CD8-Red613 (GIBCO BRL, Gaithersburg, MD). The samples were analyzed on a FACScan® using LYSYS II software (Becton Dickinson, Mountain View, CA).
Four-Color Flow Cytometry.
Single-cell suspensions of thymocytes were incubated on ice with saturating concentrations of the
following antibodies: FITC-labeled anti-CD44 (Pgp-1) mAb
IM7.8.1 (33), FITC-labeled anti-heat soluble antigen (HSA) mAb
M1/69 (34) or FITC-labeled anti-CD69 mAb H1-2F3 (35), combined with anti-TCR-PE (PharMingen, San Diego, CA), antiCD8-Red613 (GIBCO BRL, Gaithersburg, MD), and anti-CD4-
bio mAb GK1.5 (36) followed by streptavidin-APC. Four-color
flow cytometry was performed on a FACStar Plus® using LYSYS
II software (Becton Dickinson, Mountain View, CA).
Kinetics of Generation of Mature Thymocytes.
Chimeras were sublethally irradiated (720 rads, Cs137 source) 6 wk after engraftment
and thymocytes analyzed 9-13 d later by flow cytometry. CD4
single-positive (CD4SP) cells are CD4+CD8 To determine the quantitative impact of thymic clonal
deletion on the T cell repertoire, we have examined thymocyte development in radiation bone marrow chimeras
of H-2b haplotype in which MMTV superantigen-mediated deletion is not a significant factor (18, 19). Chimeras
in which the donor bone marrow was from wild-type (wt)
mice and the lethally irradiated host animals were deficient
in both MHC class I and II expression (wt Experiments were then conducted to examine the development of mature thymocytes in the absence of the major
characterized source of negative selection signals (2). To
this end, chimeric mice were produced in which MHC
class II was expressed on the radioresistant thymic epithelium to support positive selection of CD4SP cells, but not
on the negatively selecting radiosensitive hematopoietic elements (MHC II° Several explanations other than the lack of negative selection could account for the increased number of CD4SP
cells in MHC II°
Analysis of cell number in the steady-state thymus measures the accumulation rather than the rate of de novo generation of thymocytes. Because the latter parameter more
directly addresses the issue of whether additional cells are surviving the selection/differentiation process in the absence
of hematopoietic MHC-mediated negative selection, we
analyzed the kinetics of development of mature thymocytes in MHC-deficient chimeric mice. Most thymocytes are
highly radiosensitive and die upon sublethal irradiation,
whereas a small radioresistant CD4 Collectively, our results indicate that a remarkably large
fraction (half to two-thirds) of thymocytes recognizing ligands able to mediate their positive selection do not reach
maturity due to overriding negative selection in the presence of physiological ligands expressed on hematopoietic
cells. These data, combined with those on the kinetics of
thymocyte development (53, 54), imply that ~90% of developing thymocytes die of neglect and 5% die due to deletion, whereas the remaining 5% survive to populate the periphery. In considering this calculation, one caveat is that
the proportion of negatively selected thymocytes could
theoretically be larger if some cells are normally deleted despite being unable to be positively selected. However, we
observe no increase in the absolute number of CD4+CD8+
thymocytes in deletion-deficient chimeras (data not shown),
which is inconsistent with the existence of any appreciable
number of such cells. Therefore, when considering the entire
thymocyte precursor pool, effective signaling for positive
selection places a much greater constraint on the repertoire
than negative selection. This conclusion is also consistent
with a study by Surh and Sprent (55), which showed that
the extent of intrathymic death caused by negative selection is less than that due to neglect, although no quantitative
estimate of the extent of deletion could be inferred from
these data. However, if one considers only those thymocytes whose interaction with self-peptide-MHC ligands
can initiate the positive selection process necessary for contribution to the functional repertoire, the data presented
here clearly show that many of these cells never complete
maturation, but are eliminated by recognition of the same
class of MHC as that responsible for initiating their differentiation.
Two very recent studies bear on the issue of the frequency of positively selected thymocytes that subsequently
undergo negative selection. Using transgenic mice expressing a single peptide-MHC class II complex, Ignatowicz et al.
(22) found that 65% of T cell hybridomas established from
positively selected CD4+ cells reacted with wild-type MHC
class II molecules presented by APCs. These data were interpreted to mean that 65% of positively selected thymocytes in these transgenic mice would normally have undergone negative selection. However, in another transgenic
model in which MHC class II was expressed only on thymic cortical epithelium, Laufer et al. (25) estimated the frequency of positively selected CD4+ cells reactive with MHC
class II on APCs to be only 5% by limiting dilution analysis.
The apparent discrepancy in the frequency estimates of
thymocytes that are potentially subject to negative selection in these two studies could result from differences in the
transgenic models and/or assay systems used. Whatever the
explanation, it should be emphasized that neither study directly addresses the extent of thymic clonal deletion, because the fate of the MHC-reactive cells in these transgenic
systems in vivo could have been either deletion or induction of anergy.
Based on the requirement for epithelial MHC recognition in positive selection, the negative selection events mediated by hematopoietic MHC molecules here appear to
occur after initiation of this process. This raises the question
of what is different about recognition by partially mature
T cells of MHC ligands on bone marrow-derived cells as
compared with the recognition of possibly the identical peptide-MHC complex on epithelial cells by immature precursors, such that the former leads to cell death and the latter to promotion of differentiation. Two of the most obvious possibilities are the increased surface expression of TCR
by partially mature cells (56, 57, 41), which could change
the quantity or even quality of signals received by the cell,
and the expression of costimulatory ligands such as CD80/
CD86 on the hematopoietic cells. In some models, the latter has been shown to contribute to thymocyte death when
combined with TCR signals (58, 59). Whatever the explanation, these data focus attention on the issue of whether
peptides unique to epithelial cells make a predominant contribution to repertoire development. Whereas no epitheliumspecific peptides could be eluted from class II MHC molecules (60), the existence of such tissue-specific ligands has
been demonstrated by transplant experiments (10).
Finally, central (thymic) and peripheral tolerization by
means other than deletion have been described (61, 62). In
this respect, it will be of considerable interest to determine
whether mature T cells from our chimeras that have escaped negative selection will respond when confronted
with professional APCs expressing the MHC ligands encountered during positive selection.
TCR that
recognizes peptides bound to cell surface molecules
encoded by the MHC. Whereas TCRs expressed on mature CD4+ T helper cells respond to peptides associated
with MHC class II, CD8+ cytotoxic cells express MHC
class I-restricted TCRs (1). For effective maturation in the
thymus, precursor T cells need to undergo a suitable interaction between their clonotypic TCR and MHC-peptide
ligands expressed on thymic cortical epithelium (2, 3),
though minor exceptions to this rule have been reported (4).
elements, or in transgenic mice with predominant expression of both a single
TCR and its cognate ligand, deletion of developing thymocytes can markedly decrease the total number of T cells undergoing full maturation (18). Moreover, in transgenic
mice expressing MHC molecules associated with either a
single or highly predominant peptide, a strong reactivity of
mature T cells to normal syngeneic MHC is observed, indicating that many of these T cells under normal circumstances would have been tolerized during development
(21). It has recently been shown that thymocytes and
peripheral T cells from mice expressing MHC class II molecules only on thymic cortical epithelium react to the same
MHC presented by professional APCs (25), confirming
that thymic medullary epithelial cells and hematopoietic elements normally induce T cell tolerance. However, it remains unclear whether active deletion of thymocytes potentially capable of effective positive selection in the presence
of the usual diverse array of peptide-MHC complexes displayed on thymic stromal cells involves a small or large
proportion of this population. This is a critical issue for a
full understanding of the events involved in intrathymic T cell
development, construction of a useful repertoire, and characterization of the ligands involved in these processes (3).
In this study, we analyze the quantitative impact of thymic
clonal deletion mediated by cells of hematopoietic origin
using bone marrow chimeras with different combinations of MHC deficiencies.
2-microglobulin gene (26) were obtained from Dr.
B. Koller and Genpharm (Mountain View, CA). Mice of H-2b
haplotype (I-E
) with an introduced null mutation in the I-A
b
gene (27), and therefore deficient in MHC class II expression (MHC II°), were obtained from Dr. L. Glimcher and Genpharm
and used in the experiment shown in Fig. 1. In all other experiments, mice deficient in MHC class II expression due to an induced disruption of the I-A
b gene (28) in C57BL/6 stem cells
(I-E
) were used (provided by Dr. H. Bluethmann, Basel, Switzerland). MHC I° and MHC II° animals were interbred in our facilities to obtain MHC I°II° mice. The
2-microglobulin-deficient mice, as well as the I-A
b mutants, were crossed at least six
generations to C57BL/6 mice, after which intercrossing yielded
mice homozygous for the disrupted alleles.
Fig. 1.
Positive selection depends on expression of MHC molecules
on radioresistant thymic epithelial cells. (A) Thymi from wild-type wild-type, MHC I°II°
I°II°, and wild-type
I°II° chimeras were analyzed by flow cytometry 6 wk after grafting. Thymocytes were stained
with anti-CD8, anti-TCR, and anti-CD4 antibodies and analyzed on a
FACScan® using LYSYS II software. Contour plots are 75% logarithmic
and TCR histograms are from total thymus and from electronically gated cells using the gates indicated in the figure. (B) Percentages of CD4SP
(CD4+CD8
TCRhigh) and CD8SP cells (CD4
CD8+TCRhigh) in thymi
from the indicated chimeras were determined and depicted as percentage
of an age- and sex-matched nonchimeric control mouse ± SD.
[View Larger Versions of these Images (38 + 15K GIF file)]
Fig. 2.
Increased CD4SP (CD4+CD8TCRhigh) thymocytes in chimeras lacking MHC class II expression on hematopoietic elements.
Groups of sex- and age-matched chimeras were analyzed on the same day
6-8 wk after engraftment. Flow cytometry was performed using antiTCR, anti-CD4, and anti-CD8 antibodies. In each experiment, the ratio of CD4SP cells in the indicated groups was calculated. Error bars indicate
SD. The increased ratio of CD4SP cells in MHC II°
MHC I° versus
MHC II+
MHC I° chimeras is statistically significant as assessed by the
Student's t test (P <0.0001), whereas the ratio of CD4SP thymocytes in
MHC I°
MHC I° versus MHC I+
MHC I° chimeras is not significantly increased (P = 0.02).
[View Larger Version of this Image (23K GIF file)]
Fig. 4.
Accelerated kinetics of generation of CD4SP and CD8SP
thymocytes in wild-type hosts lacking MHC class II or I, respectively, on
hematopoietic elements. 6-wk-old wt wt (n = 3, day 9; n = 6, days 10-
13), MHC I°II°
wt (n = 3 each day), and MHC I°
wt (n = 3) chimeras were sublethally irradiated (720 rads) and analyzed on day 9-13 as in
Fig. 2. Data represent average percentage of CD4+CD8
TCRhigh and
CD4
CD8+TCRhigh thymocytes ± SD.
[View Larger Version of this Image (25K GIF file)]
TCRhigh and CD8SP
cells are CD4
CD8+TCRhigh.
MHC I°II°
chimeras) (37, 38) lacked significant numbers of mature
CD4+8
or CD4
8+TCRhigh thymocytes (CD4SP and
CD8SP, respectively; Fig. 1 A). A quantitative assessment
reveals the development of less than 3% of wt levels of
CD4SP cells and less than 8% of CD8SPs in such chimeras
(Fig. 1 B). These results confirm previous data showing that
efficient positive selection depends on expression of MHC
molecules on radioresistant thymic epithelial cells (2, 3) and
indicate that in the system used here the previously reported low level of T cell differentiation induced by hematopoietic elements (4) plays a negligible role.
MHC I° chimeras). A significant (1.7 ± 0.2-fold) increase in the steady-state percentage of mature CD4SP thymocytes was observed in these chimeras as compared with control (MHC II+
MHC I°) chimeras (Fig.
2). As expected, lack of MHC class I on hematopoietic elements (MHC I°
MHC I° chimeras) did not result in significantly increased CD4SP percentages (1.1 ± 0.1-fold) as
compared with appropriate control (MHC I+
MHC I°)
chimeras (Fig. 2). Because thymi from the chimeras had similar total cell numbers (data not shown), these results reflect increases in the absolute number of CD4SP cells.
Thus, in the absence of MHC class II expression on hematopoietic elements, a 1.7-fold increase in mature CD4SP
cells occurs, suggesting that approximately half of the positively selectable thymocytes with receptors specific for selfMHC class II undergo negative selection on the same
MHC molecule.
MHC I° chimeras. However, proliferation of mature CD4SP does not account for the increase as
cell cycle analysis reveals no augmentation in the number
of CD4SP cells in S+G2/M phase (Fig. 3 A). A change in
their maturation/export rate could result in an increased
retention of CD4SP cells in the thymus. CD69 is a molecule transiently expressed during thymocyte development:
in vivo, it is first expressed on CD4+CD8+ cells that have
undergone MHC-mediated activation and is downmodulated only when the thymocytes have already reached the
SP stage (39, 29). The normal ratio of mature thymocytes from the chimeras expressing high versus low levels of CD69 therefore is an indication of normal thymic
maturation (Fig. 3 B). Expression of HSA on CD4SP thymocytes is also downmodulated during their final maturation (42, 43). Again, the normal ratio of HSAhigh versus
HSAlow CD4SP cells in the chimeras indicates normal maturation. Taken together, the data on the expression of
CD69 and HSA suggest normal thymocyte development
and render the possibility of prolonged retention of CD4SP
cells in the thymus unlikely. Another possibility would be
the recirculation into the thymus of mature peripheral T cells
(44). Peripheral T lymphocytes do not express significant levels of HSA (42, 43) and the lack of an increased
percentage of HSA
CD4SP thymocytes (Fig. 3 B) thus is
inconsistent with a recirculation model. Reentry into the
adult thymus of peripheral T cells is known to be restricted
to activated cells (44). These cells may be expected to
express high levels of the memory/activation marker CD44
(47). No evidence for an increase in CD44high CD4SP thymocytes was found in the chimeras (Fig. 3 B). Collectively, these data strongly suggest that the increased number of
CD4SP cells in chimeras lacking MHC class II on hematopoietic elements is not due to proliferation, to increased retention of thymocytes in the thymus, or to recirculation of
peripheral T lymphocytes.
Fig. 3.
Increase in CD4SP thymocytes in MHC II° MHC I° chimeras is not due to (A) proliferation or (B) recirculation of peripheral T
lymphocytes. (A) Cell cycle analysis (PI incorporation) was performed on
ethanol-fixed total thymocytes and electronically sorted CD4+CD8
TCRhigh cells (purity
95%). Representative results are shown. The statistics represent mean percentage cells in S+G2/M phase ± SD from the indicated number of experiments. (B) Four-color flow cytometry was
performed using anti-CD4, anti-CD8, and anti-TCR antibodies combined with anti-CD44, anti-HSA, or anti-CD69. The CD44, HSA, and
CD69 histograms are of electronically gated CD4+CD8
TCRhigh cells.
Representative results are shown. The statistics represent mean percentage ± SD from the indicated number of experiments.
[View Larger Versions of these Images (14 + 16K GIF file)]
CD8
precursor population will repopulate the thymus upon such treatment. In
this model system, CD4+CD8+ immature thymocytes appear around day 6 after irradiation and 3-4 d later mature
SP cells can be observed (41, 48). Using this post-irradiation repopulation model, we studied the kinetics of generation of mature thymocytes in bone marrow chimeras
(Fig. 4). In particular, 6 wk after grafting, MHC I°II°
wt,
MHC I°
wt, and control wt
wt chimeras were sublethally irradiated (720 rads) and their thymi analyzed 9-13 d
later. A significant, approximately twofold greater, generation of CD4SP thymocytes was observed in the absence of
MHC class II expression on bone marrow-derived cells,
whereas in the absence of MHC class I, CD4SP cells developed at the same rate as in control chimeras (Fig. 4).
CD8SP thymocytes were also generated more efficiently in
the absence of their negatively selecting ligand (MHC class I)
on hematopoietic elements (Fig. 4). Thus, in MHC I°
wt
and MHC I°II°
wt chimeras a two- to threefold increase
in generation of CD8SP thymocytes was observed. During the reconstitution no differences in the number of total
thymocytes were seen between the different types of chimeras (data not shown), again indicating that absolute increases in SP cells were occurring in these circumstances.
These data confirm that the increase in the number of
CD4SP cells seen in the steady-state thymus of mice lacking MHC class II on hematopoietic cells is due to the lack of negative selection rather than to alterations in proliferation, homeostasis, or recirculation of these cells. Moreover,
they reveal a similarly large impact of negative selection on
the development of CD8SP thymocytes.
Address correspondence to Dr. H.R. MacDonald, Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.
Received for publication 23 September 1996
1Abbreviations used in this paper: CD4SP, CD4+CD8We thank E. O'Connell, C. Eigsty, A.-L. Peitrequin, and P. Zaech for expert technical help and Drs. H. Bluethmann, L. Glimcher, and B. Koller for providing MHC mutant mice.
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