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of June 15, 2017.
Premature TCRαβ Expression and Signaling
in Early Thymocytes Impair Thymocyte
Expansion and Partially Block Their
Development
H. Daniel Lacorazza, Carolyn Tucek-Szabo, Ljiljana V.
Vasovic, Kristin Remus and Janko Nikolich-Zugich
J Immunol 2001; 166:3184-3193; ;
doi: 10.4049/jimmunol.166.5.3184
http://www.jimmunol.org/content/166/5/3184
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References
Premature TCR␣␤ Expression and Signaling in Early
Thymocytes Impair Thymocyte Expansion and Partially Block
Their Development
H. Daniel Lacorazza,* Carolyn Tuček-Szabo,* Ljiljana V. Vasović,2* Kristin Remus,* and
Janko Nikolich-Žugich3*†
he main TCR␣␤ thymocyte developmental pathway
transforms a minor population of CD4⫺8⫺ double-negative (DN)4 TCR␣␤⫺ precursors (2– 4% of all thymocytes)
into the mature CD4⫹8⫺ (6 –12%) or CD4⫺8⫹ (3–5%) singlepositive (SP) TCR␣␤high cells via the CD4⫹8⫹ double-positive
(DP) TCRlow intermediates (75– 85%) (reviewed in Refs. 1–3).
Murine DN precursors that generate DP and SP progeny (generative DN (gDN) thymocytes; 90% of all DN thymocytes in a
young thymus) are of the TCR⫺CD24high phenotype. By contrast,
the majority of the CD24⫺ DN cells, which make up ⬍10% of all
DN cells of a 6-wk-old murine thymus, bear intermediate TCR
levels (4, 5). This heterogeneous subset is terminally differentiated
and cannot give DP and SP progeny (6). gDN cells can be further
divided into four developmentally sequential stages, DN1– 4, according to the expression of CD25 (the IL-2R␣ chain; Refs. 7, 8)
and CD44 (Pgp-1; Ref. 9) CD44⫹CD25⫺ (DN1) 3 CD44⫹CD25⫹
(DN2) 3 CD44low/⫺CD25⫹ (DN3) 3 CD44low/⫺CD25⫺ (DN4) (1, 2,
T
*Laboratory of T Cell Development, Immunology Program, Memorial Sloan-Kettering Cancer Center, and †Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021
Received for publication October 31, 2000. Accepted for publication December
29, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by U.S. Public Health Service Grants AI-32064 (to J.N.Ž.) and CA-02583 (Memorial Sloan-Kettering Cancer Center Core Cancer Center
Award); National Cancer Institute Training Grant CA-0914-19 from the National
Institutes of Health; and grants from the PEW Charitable Trust (to J.N.-Ž.), and the
DeWitt Wallace Fund (to J.N.-Ž.).
2
Current address: Department of Pathology, Residency Program, The Lenox Hill
Hospital, New York, NY 10021.
3
Address correspondence and reprint requests to Dr. Janko Nikolich-Žugich, Immunology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New
York, NY 10021. E-mail address: [email protected]
4
Abbreviations used in this paper: DN, double-negative; DP, double-positive; gDN,
generative DN; FCM, flow cytometry; mDN, mature DN; SP, single-positive; i.t.,
intrathymic(ally); Tg, transgenic; wt, wild type.
Copyright © 2001 by The American Association of Immunologists
10, 11). Commitment to the T cell lineage occurs at DN2–3, with the
rearrangement of Tcr-d, g, and, slightly later, b genes (Ref. 12; reviewed
in Ref. 11). Tcr-a genes rearrange much later, at the DP stage (13–15).
The reason for this late rearrangement is incompletely understood, although at least one advantage of later TCR␣ expression is that it allows
for the selection and propagation of only those thymocytes bearing productively rearranged Tcr-b genes (16–18).
The in-frame Tcr-b rearrangement and TCR␤ protein expression
are essential for production of the large numbers of TCR␣␤ cells
(19). The TCR␤ protein pairs with the surrogate ␣-chain, pT␣
(20), to form the pre-TCR complex that appears instrumental for
expansion and/or survival (20) of cells progressing to DN4 (reviewed in Ref. 21). DN4 thymocytes are actually early DP cells;
they are of the CD4lowCD8lowTCR␤low phenotype and exhibit the
cell-autonomous capability to become DP in a matter of hours
(22–25). Rearrangement of the Tcr-a locus occurs in DP cells.
Once a functional TCR␣ protein is produced, it pairs with TCR␤
at the cell surface. Two mechanisms appear responsible for the
attenuation of pT␣ expression and replacement with TCR␣. As
recently shown by Trop et al. (26), TCR␣ has a much higher affinity for TCR␤, and this difference is likely to exclude pT␣ protein from pairing with TCR␤. Moreover, upon ligation of TCR␣␤
by MHC ligands, pT␣ transcription is down-regulated (27).
TCR␣␤ then guides DP thymocytes through positive and negative
selection (28) and mediates recognition of antigenic peptide-MHC
complexes by the peripheral T cells. Contrary to the events coupled with the pre-TCR signaling and the TCR␣␤ signaling in peripheral T cells, TCR ligation during intrathymic (i.t.) positive selection is not coupled to proliferation (28).
TCR␣␤ transgenic (Tg) mice were instrumental in advancing
our understanding of T cell development (29). Although many
aspects of T cell development in TCR␣␤ Tg mice appear normal,
for unknown reasons the thymi of most such animals are smaller
and less cellular than their normal counterparts. Such mice also
exhibit a substantial accumulation of mysterious TCR⫹DN cells
that share many characteristics with the CD24⫺DN cells of normal
0022-1767/01/$02.00
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In thymocyte ontogeny, Tcr-a genes rearrange after Tcr-b genes. TCR␣␤ transgenic (Tg) mice have no such delay, consequently
expressing rearranged TCR␣␤ proteins early in the ontogeny. Such mice exhibit reduced thymic cellularity and accumulate
mature, nonprecursor TCRⴙCD8ⴚ4ⴚ thymocytes, believed to be caused by premature Tg TCR␣␤ expression via unknown
mechanism(s). Here, we show that premature expression of TCR␣␤ on early thymocytes curtails thymocyte expansion and impairs
the CD8ⴚ4ⴚ 3 CD8ⴙ4ⴙ transition. This effect is accomplished by two distinct mechanisms. First, the early formation of TCR␣␤
appears to impair the formation and function of pre-TCR, consistent with recently published results. Second, the premature
TCR␣␤ contact with intrathymic MHC molecules further pronounces the block in proliferation and differentiation. These results
suggest that the benefit of asynchronous Tcr-a and Tcr-b rearrangement is not only to minimize waste during thymopoiesis, but
also to simultaneously allow proper expression/function of the pre-TCR and to shield CD8ⴚ4ⴚ thymocytes from TCR␣␤ signals
that impair thymocyte proliferation and CD8ⴚ4ⴚ 3 CD8ⴙ4ⴙ transition. The Journal of Immunology, 2001, 166: 3184 –3193.
The Journal of Immunology
Materials and Methods
Mice
C57BL/6 (B6, H-2b, Thy-1.2, Ly-5.1) and B6.Ly5.2 (H-2b, Thy-1.2, Ly5.2) mice were purchased from the National Cancer Institute breeding program (Frederick, MD). B6.PL-thy1a-Cy (B6.PL, H-2b, Thy-1.1, Ly-5.1)
mice were bred in the Memorial Sloan-Kettering Cancer Center Core Animal Facility from the breeding pairs obtained from The Jackson Laboratory (Bar Harbor, ME). The breeding pairs of ␣␤TCR Tg lines 2C (39),
H-Y (40), and OT-1 (41) were obtained from H. von Boehmer (Hopital
Necker, Paris, France), D. Loh (Roche, Nutley, NJ), F. Carbone (Monash
University, Melbourne, Australia), and W. Heath (The W. and E. Hall
Institute, Melbourne, Australia), respectively. TCR Tg mice were bred and
maintained in the Memorial Sloan-Kettering Cancer Center Core Animal
Facility, and were backcrossed to B6 for a minimum of 14 generations. The
screening for the presence of the transgenes was performed by PCR using
the oligonucleotides that span the V␣J␣ junction (see RT-PCR) and/or by
flow cytometry (FCM) analysis of the PBLs stained with V␣- and V␤specific fluorochrome-conjugated Abs. Thy-1.1⫹ 2C mice were generated
by producing the (B6.PL⫻2C) F1 mice that were screened for the presence
of the TCR transgene. All mice were used at 6 –12 wk of age and were ageand sex-matched within experiments.
Abs, cell preparation, and FCM analysis
Single-cell suspensions of thymocytes were stained with the indicated Abs
and 5–50 ⫻ 104 cells/sample were analyzed using a FACScan (Becton
Dickinson, Mountain View, CA) instrument and CellQuest 3.1 or LYSYS
II software. CD8⫺4⫺ (DN) cells were prepared from total thymocytes by
two cycles of mAb ⫹ C⬘-mediated depletion as described (42). FITCconjugated anti-CD8, CyChrome-conjugated anti-CD4 and FITC- or PEconjugated anti-V␣2 mAb were purchased from PharMingen (San Diego,
CA). mAbs 1B2 (anti-2C ␣␤ Id, Ref. 43); T3.70 (H-Y TCR␣-chain Id, Ref.
44); F23.1 (anti-V␤8, Ref. 45); IM7.8 (anti-CD44, Ref. 9), and PC61 (antiCD25, Ref. 7) were purified from ascites and conjugated to biotin or FITC
in our laboratory. PE-labeled streptavidin was purchased from Caltag
(South San Francisco, CA).
Bone marrow chimera
Single-cell bone marrow suspension from TCR Tg and control mice were
prepared from the leg bones using a mortar and pestle. Cells were depleted
of mature T cells by C⬘-mediated cytotoxicity in the presence of J1J (antiThy1.2 mAb; American Type Culture Collection, Manassas, VA, as described; Ref. 42). Bone marrow cells were then mixed at a 1:1 ratio and
injected i.v. (5 ⫻ 106 cells/recipient) into supralethally irradiated (11.5 Gy)
B6 or B6 congenic mice. After 5–10 wk, the mice were sacrificed and the
thymi were analyzed. The chimerism always exceeded 90%, and was routinely ⬎95%.
Sorting of T cell precursors and i.t. injection
The DN cells, obtained from ␣␤TCR Tg and normal mice as described
above, were stained for the expression of CD44 and CD25 and sorted into
CD44⫺CD25⫹ cells, or with the TCR␣-clonotype and CD25 to sort
TCR␣⫹CD25⫹ and TCR␣⫺CD25⫹ cells. Sorted cells were kept on ice in
5% FCS/HBSS medium until used for i.t. injection or for mRNA analysis.
For i.t. injection, sorted cells were washed with PBS and injected i.t. into
each thymic lobe of lethally irradiated and bone marrow-reconstituted
B6.PL female mice, as described (46). After 9 days, mice were sacrificed
and the thymi were analyzed by FCM. Alternatively, before the analysis,
the donor cells were enriched by mAb ⫹ C⬘-killing using anti-Thy1.1
(19E12) mAb and analyzed by FCM.
RT-PCR
Total DN thymocytes or sorted DN subsets were used for RNA extraction
using the RNAzol method. cDNA was synthesized using the Stratagene Kit
(Stratagene, La Jolla, CA) following the manufacturer’s protocol. PCR was
then performed using primer oligonucleotides complementary to the sequences in the 5⬘ and 3⬘ of: pT␣ (5⬘-CTGCAACTGGGTCATGCTTC-3⬘
and 5⬘-TCAGACGGGTGGGTAAGATC-3⬘; Ref. 20) and the ␤-actin
(Stratagene). Amplification was performed for different cycles (20) at an
annealing temperature of 55°C using a thermal cycling machine (PerkinElmer/Cetus, Norwalk, CT). After amplification, 10 ␮l of the reaction mixture was resolved on a 1.3% agarose gel, blotted to nylon membrane, and
hybridized with a purified fragment specific for each cDNA.
Results
Uneven T cell development in mixed (TCR TgA ⫹ TCR TgB) 3
P chimera occurs at the level of DN f SDP transition
To address the importance of the delayed Tcr-a rearrangement in
T cell development, we took advantage of TCR Tg mice that express rearranged TCR␣ and TCR␤ proteins early and simultaneously in development. Several groups concluded that TCR Tg
thymocytes exhibit signs of reduced differentiation into the DP
cells (35–37, 47). However, only two studies actually assessed
expansion and precursor function of early TCR Tg thymocytes (33,
38) but to a limited extent (33) or using indirect methods (38). To
directly compare the in vivo kinetics of early T cell development
under normal circumstances, where Tcr-a rearrangement occurs
well after TCR-b rearrangement, or in TCR Tg mice, where both
genes are rearranged and the TCR␣␤ is expressed early, we analyzed thymocyte expansion in mixed bone marrow irradiation chimera. Chimera were generated by injecting a 1:1 mixture of OT-1
(41), H-Y (40), or 2C (43) bone marrows, or of one of them and
the control non-Tg bone marrow, into supralethally irradiated congenic (B6.PL, H-2b, Thy-1.1, Ly-5.1) mice. Each of the marrows,
injected alone, yielded good thymic reconstitution, with donorderived cells making up ⬎90% of the total thymocytes 5–10 wk
following reconstitution (data not shown). When two wild-type
(wt) bone marrows, each marked with a separate congenic marker,
were used for reconstitution {[B6 (H-2b, Thy-1.2, Ly-5.1) ⫹
B6.Ly-5.2 (H-2b, Thy1.2, Ly-5.2)] 3 B6.PL(H-2b, Thy-1.1, Ly5.1)}, they generated two populations of thymocytes that developed evenly and with identical kinetics, repopulating the thymus at
a 1:1 ratio (as detected by FCM using allele-specific mAbs, Fig. 1,
top). By contrast, whenever a TCR Tg marrow was used, uneven
thymic reconstitution was observed. This uneven reconstitution
was not random; thymocytes developing from normal bone marrow always numerically dominated over any of the three TCR Tg
counterparts (as shown for the B6 ⫹ OT-1 combination, Fig. 1);
OT-1 outcompeted both H-Y and 2C, and H-Y was better than 2C
(Fig. 1). Analysis of the four main thymocyte subsets in mixed
chimera provided further insight into the dominance phenomenon.
In a mixed chimera produced from two non-Tg marrows, all four
main thymocyte subsets exhibited equal chimerism, including the
most immature DN cells (Fig. 2A, DN cells; Fig. 1, top, total cells;
all other subsets were represented at ratios between 0.89 and 1.16
relative to each other). A different situation was observed in the wt
⫹ TCR TgA or TCR TgA ⫹ TCR TgB mixed chimera (OT-1 ⫹
2C in Fig. 2). As assessed by the expression of the clonotypic
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mice (30 –33). These cells never go through the DP stage of development (32–34). Although many features of these cells (referred here to as mature DN (mDN) cells) are reminiscent of the
TCR␥␦ thymocytes (35–37), their other characteristics are similar
to the normal CD44⫺25⫺ TCR⫺ counterparts (38).
We studied thymocyte expansion and development in TCR Tg
mice. We discovered that DN CD25⫹ thymocytes interpret preTCR and TCR␣␤ signals in a fundamentally different manner. Although the pre-TCR signals allowed thymocyte expansion and development of DP cells, the mere expression of TCR␣␤ interfered
with these signals. Moreover, signals via the TCR␣␤ were poorly
conducive for thymocyte expansion and DP thymocyte production,
and diverted many cells into the nonprecursor mDN subset. The
intensity of TCR␣␤ signaling appeared to determine the extent of
the expansion block and of the mDN diversion, as it was dependent
on the i.t. TCR-MHC interactions. These results indicate that
TCR␣␤ expression and signaling are detrimental to thymocyte expansion and DP thymocyte development and suggest that one of
the key functions of late Tcr-a rearrangement may be to shield
expanding DN cells from TCR␣␤ signals.
3185
3186
CONTROL OF EARLY THYMOCYTE DEVELOPMENT BY PRE-TCR AND TCR
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FIGURE 1. Thymocyte dominance in mixed bone marrow chimera.
Mixed non-Tg or TCR Tg chimera were produced as described in Materials and Methods, and the thymocyte repopulation was analyzed at 8 –10
wk by FCM. Total thymocytes were stained with the allelic Ly-5 mAbs (to
detect thymocytes of non-Tg origin) and/or with clonotype-specific mAbs
(H-Y or 2C) or the Tg TCR␣-chain-specific mAbs (OT-1). The average of
seven mice per group ⫾ SD for each chimera are shown, representative of
over 40 mice per type analyzed. Control single bone marrow chimera revealed ⬎95% reconstitution with donor marrow (data not shown).
FIGURE 2. The dominance in the mixed chimera occurs at the DN 3
DP stage. FCM analysis of the purified DN cells from [(B6 ⫹ B6. Ly-5.2)
f B6. PL ] chimera (A) and of various thymocyte subsets from the [(OT-1
⫹ 2C. Thy-1.1) f B6. Ly-5.2] chimera (B and C) was performed 8 wk
after chimera construction following staining for the congenic Thy-1
and/or Ly-5 markers. A, DN cells were purified by mAb ⫹ C⬘ depletion
(⬎95% purity) and analyzed using Ly-5 and Thy-1 markers. Results represent x៮ ⫾ SD for five mice. B, Experiment was performed exactly as in A,
but for the indicated TCR Tg chimera and using TCR Tg␣-chain expression to trace chimerism. In C, the chimera was of the [(OT-1 ⫹ 2C. Thy1.1) f B6. Ly-5.2] type. DN cells were isolated and the congenic markers
(Thy1.1 or 1.2) analyzed in donor-derived (Ly5.1⫹) CD25⫹ DN, DP, and
CD4 and CD8 SP thymocytes. Similar results were obtained in two other
experiments.
TCR␣-chain and/or the expression of Thy-1 or Ly-5 markers, the
dominance was present at the level of DP, CD8⫹ SP, and CD4 SP
thymocytes (for the wt ⫹ OT-1 chimera, the wt/OT-1 distribution
was 94:4% among DP, 88:3% among CD8 SP, and 95:1% among
CD4 SP cells, respectively; for the OT-1 ⫹ 2C chimera, results are
shown in Fig. 2C). By contrast, the percentage of the two donor
populations among the DN cells was less disparate in the TCR Tg
mixed chimera, with little or no dominance by either population
(for the wt ⫹ OT-1 chimera, the wt/OT-1 distribution was 48.4:
46.1% among the DN cells; for the OT-1 ⫹ 2C chimera, see Fig.
2B). A caveat to this experiment is that in TCR Tg mice the majority of the DN cells express the Tg TCR␣␤ receptor and are not
The Journal of Immunology
3187
Makeup of the DN compartment in TCR Tg mice
Because wt thymocytes always outcompeted the TCR Tg ones, the
above results suggested that there is a block in DN3 DP transition
in TCR Tg mice. The surprising hierarchy of dominance between
different TCR Tg thymocytes (non-Tg ⬎ OT-1 ⬎ H-Y ⬎ 2C)
further indicated that the extent of this block was different in each
TCR Tg strain. In searching for clues to the reasons behind this
block, we investigated the distribution of cell subsets and their
detailed phenotype among total thymocytes and the DN cells of
TCR Tg mice. By percentage, 2C mice exhibited the most dramatic overaccumulation of mDN cells, followed by H-Y and OT-1
(Table I). Thus, a correlation existed between the extent of mDN
cell accumulation and the poor ability to compete in a mixed chimera. Consistent with the available literature (33, 36, 37), this
prompted us to hypothesize that the block was caused by the diversion of many of the potential precursors to the alternative developmental pathway that yields mDN cells. Furthermore, we also
hypothesized that an unknown factor, perhaps related to the TCR
specificity, would determine the extent of the block in each of the
Tg lines. (To simplify our analysis, we focused further studies
FIGURE 3. In vitro differentiation potential of the TCR Tg DN cells.
DN cells were purified from total thymocytes of indicated strains by mAb
⫹ C⬘-mediated killing using anti-CD8 and anti-CD4 Abs, and their phenotype was analyzed in A. Following incubation of purified DN cells from
A at 37°C in the presence of 10% FCS for 18 –20 h, the expression of CD4
and CD8 was evaluated by FCM, and the results are shown in B. Similar
results were obtained in five other experiments.
Table I. Percentages and absolute numbers of cell subsets among the DN thymocytes of normal and TCR
Tg mice
Total
DN
CD44⫹25⫺
CD44⫹25⫹
CD44⫺25⫹
CD44⫺25⫺
B6
2C
HY
OT-1
139 ⫾ 43
2 ⫾ 0.5b (2.7)a
11 ⫾ 3 (0.3)
6 ⫾ 2 (0.2)
54 ⫾ 7 (1.5)
28 ⫾ 6 (0.7)
29 ⫾ 9
28 ⫾ 4 (8.1)
4 ⫾ 1 (0.3)
3 ⫾ 1 (0.2)
12 ⫾ 3 (1.0)
80 ⫾ 5 (6.5)
58 ⫾ 6
16 ⫾ 3 (7.5)
3 ⫾ 2 (0.2)
3 ⫾ 2 (0.2)
5 ⫾ 1 (0.4)
88 ⫾ 4 (6.6)
64 ⫾ 25
10 ⫾ 1 (6.3)
2 ⫾ 0.5 (0.1)
2 ⫾ 1 (0.1)
8 ⫾ 5 (0.6)
87 ⫾ 4 (5.5)
a
a
Seven to 10 mice of each type were analyzed by a combination of vital dye cell counting and multicolor FCM. Results are
expressed as average ⫾ SD.
b
Absolute thymocytes numbers ⫻ 106.
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capable of developing into the DP thymocytes, as previously
shown for the H-Y mice (33). To directly assess the representation
of true precursor gDN cells in mixed chimera and to avoid possible
bias introduced by using the TCR Tg␣-chain as the population
marker, we injected a mixture of congenic marker-labeled OT-1
(Thy-1.2, Ly-5.1) and 2C (Thy-1.1, Ly-5.1) marrow into irradiated
B6.Ly-5.2 (Thy-1.2, Ly-5.2) recipients. We then compared the
representation of DN CD25⫹ early precursor cells derived from
each donor using Thy-1 allele-specific mAbs. In such a mixed
chimera the OT-1 cells heavily dominated over the 2C at the level
of DP and both types of SP thymocytes, but there was no difference in the percentage of the two types of CD25⫹ gDN precursors
(Fig. 2C). The wt ⫹ OT-1 chimera exhibited a 45:51% ratio
among the DN CD25⫹ cells, and similar data were obtained in the
wt ⫹ H-Y chimera. Thus we conclude that the dominance of one
TCR Tg population over the other in mixed chimeras must occur
at the gDN3 DP transition.
It might be argued that the observed differences in thymocyte
numbers were due to negative selection and death of TCR Tg
thymocytes at the DP stage, rather than to an unequal expansion of
DN precursors. However, measurements of apoptosis (annexin V
expression) among the newly formed DP cells (in a DN f DP
conversion assay, as in Fig. 3) provided no support for this explanation (wt ex vivo isolated DN cells were 9 ⫾ 2% annexin⫹ propidium iodide (PI)⫺, wt overnight in vitro generated DP, 16 ⫾ 4%;
OT-1, 12 ⫾ 2 and 18 ⫾ 1; 2C, 10 ⫾ 3 and 17 ⫾ 3, respectively).
Therefore, we conclude that the presence of TCR␣␤ at the surface
of pre-T cells does not induce negative selection, but rather inhibits
their expansion and conversion into DP cells.
3188
CONTROL OF EARLY THYMOCYTE DEVELOPMENT BY PRE-TCR AND TCR
upon OT-1 and 2C thymocytes, with the rationale that the two
represent the extreme ends of the observed effect. H-Y thymocytes
were included in the analysis when appropriate, to ensure the generality of the observed findings.)
Increased production of mDN cells and decreased production of
DP thymocytes from TCR Tg DN precursors
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To test this hypothesis, we sought to quantify the number of precursors of mDN thymocytes and of DP thymocytes among the DN
cells of the three TCR Tg strains. We first analyzed the phenotype
of the DN cells using CD25 and CD44. In all three strains, ⬎75%
of all DN cells were of the CD44low/⫺CD25⫺ (Table I) phenotype,
which, in TCR Tg mice, mostly demarcates terminally differentiated mDN cells that are not DP thymocyte precursors (33). Owing
to the uniformly high expression of CD24 on TCR Tg mDN cells,
it was impossible to phenotypically distinguish gDN from mDN
cells. However, a distinction could be made functionally by taking
advantage of the in vitro conversion assay. In normal mice, the
vast majority of the CD44⫺25⫺ cells progress to the DP stage
following an overnight in vitro culture in the absence of stimulation (22). In a typical non-Tg mouse ⬃15–25% of the cells become
DP (15% in Fig. 3). This percentage was lower in TCR Tg mice
(Fig. 3); moreover, it directly correlated to the ability of TCR Tg
T cell precursors to compete in mixed chimera; OT-1 DN precursors generated 10% DP cells, H-Y ⬃3%, and 2C 0.5–2%. Therefore, production of DP cells from their immediate precursors was
impaired in each of the three TCR Tg mouse strains, albeit to
different extents.
To directly demonstrate that precursor DN thymocytes from
TCR Tg mice produce not only DP but also mDN progeny in vivo,
we isolated CD25⫹ DN cells from these strains and injected them
i.t. into lethally irradiated and syngeneic bone marrow-reconstituted congenic recipients. Nine days later, the progeny of these
cells were analyzed by three-color FCM for the expression of CD4,
CD8, and of the donor-type marker (Thy-1.2). Consistent with
previous results (42, 48), B6 CD25⫹DN cells yielded exclusively
DP progeny over this time period (Fig. 4). The progeny of OT-1
CD25⫹DN cells was also mostly DP, albeit a discrete population
of DN cells was also evident (Fig. 4). Remarkably, although 2C
CD25⫹DN precursors generated DP progeny, they also yielded an
abundant (41%) DN population, indicating that in these mice many
early precursors generated mDN cells (Fig. 4). These results formally demonstrate for the first time that homogenous precursor
DN cells from TCR Tg mice frequently generate mDN cells. They
also raise the possibility that the same mechanism that impairs the
ability of TCR Tg DN precursors to become DP also dictates their
shunting into the mDN lineage.
TCR expression on, and in vivo expansion of, the TCR Tg
CD25⫹DN thymocyte subsets
As the expression of both TCR␤ and pT␣ is instrumental for an
efficacious thymocyte expansion during the DN f DP transition,
pT␣ mRNA may be indicative of the precursor status and expansion potential of TCR Tg DN cells. In TCR Tg mice, where most
DN cells express TCR␤ mRNA but many are not on their way to
become DP, the expression of pT␣ mRNA might correlate with the
precursor status of the cell because the terminally differentiated
mDN cells would be expected to down-regulate pT␣ mRNA (27).
We examined the expression of pT␣ mRNA among the DN cells
of the three TCR Tg strains. Fig. 5A shows a correlation between
the expression of pT␣ mRNA and the expansion potential of TCR
Tg precursors in mixed chimera. Thus, 2C DN thymocytes expressed barely detectable levels of pT␣ mRNA, followed by H-Y,
OT-1, and the non-Tg littermates.
FIGURE 4. In vivo, Tg DN precursors generate fewer DP and more DN
progeny. CD25⫹ cells were purified from total DN cells (obtained as described in Fig. 2) of B6 and TCR Tg mice by cell sorting, and were i.t.
injected into lethally irradiated and syngeneic bone marrow-reconstituted
B6.PL mice. Recipient mice were sacrificed 9 days later, and their thymocytes were pooled (five mice per group) and depleted of host-type
(Thy1.1⫹) thymocytes with the 19E12 Ab. FCM analysis was performed
on these depleted thymocytes by staining with anti-Thy1.2⫹ (donor
marker) anti-CD8 and anti-CD4 Abs. The CD4/CD8 profile of Thy1.2gated (donor) cells are shown for B6, OT-1, and 2C (top, middle, and
bottom, respectively). The quadrant delineates the DN compartment, and
the numbers correspond to the percentage of cells within it. Results are
representative of two experiments.
The Journal of Immunology
Low expression of pT␣ mRNA among the total DN cells of
TCR Tg mice (Fig. 5A) was consistent with three scenarios: 1) it
could have been caused by simple dilution of gDN cells by mDN
cells that overaccumulate in this compartment; 2) it could have
been reflective of fewer cells expressing this molecule, owing to
repression of pT␣ transcription by TCR␣␤; or 3) the number of
cells expressing pT␣ may be unchanged, but mRNA and protein
levels per cell could be lower (again consistent with some form of
repression). Our previous results in the H-Y model (33) indicated
that the absolute number of CD25⫹ precursors is reduced by 70%
compared with the non-Tg littermate mice (the first scenario), but
the expression of pT␣ on these cells has not been studied and,
although it was noted that some of these cells expressed TCR␣␤,
the effects of this expression were not tested. To address these
issues, we studied the TCR␣, ␤, and CD25 expression pattern on
TCR Tg DN thymocytes. Unlike the non-Tg CD25⫹ DN cells,
which do not express TCR␣ and express barely detectable levels of
TCR␤ by FCM, and the vast majority of which are gDN, nearly
half of the OT-1 CD25⫹ DN cells expressed TCR␤ (data not
shown, but fully mirroring the expression of TCR␣; see also Ref.
33) and TCR␣ (Fig. 5B). Even more strikingly, TCR␣⫹ CD25⫹
cells made up over three-fourths of the CD25⫹DN cells in H-Y
and ⬎80% in 2C mice (Fig. 5B). It is important to mention that the
majority of the 2C cells within the TCR␣⫺ gate were actually
TCR␣low rather than negative. Thus, the percentage of TCR␣␤⫹
CD25⫹ DN cells in TCR Tg strains correlated inversely with the
ability of different TCR Tg thymocytes to expand in vivo, suggesting that the early expression of TCR␣ may negatively influence both pT␣ expression and function (expansion and efficient
transition to DP).
The above results allowed us to subdivide the TCR Tg CD25⫹
DN cells into the TCR␣⫹ (actually, ␣␤⫹; Ref. 33) and TCR␣⫺
subsets and directly address two critical questions. First, is the
difference in the precursor activity of TCR Tg DN cells observed
in vitro (Fig. 3) due to a reduced number of precursor cells or to
a lower inherent ability of each precursor to expand and become
DP? And second, what is the influence of TCR␣␤ on the developmental potential of CD25⫹ DN cells? We sorted CD25⫹ DN
cells from each TCR Tg strain into TCR␣⫹ and TCR␣⫺ fractions,
and injected the same number of each cell subset i.t. into irradiated
recipients. Results clearly showed that both the presence of the
TCR␣ and the specificity of the TCR␣␤ receptor expressed on
thymocytes played a major role in their expansion (Fig. 5C). OT-1
TCR␣⫺ precursors expanded vigorously, giving at least an 120fold expansion, comparable to that of wt CD25⫹ TCR␣␤⫺ DN
cells (131 ⫾ 19, n ⫽ 4, data not shown and Refs. 10, 42, 48). By
contrast, OT-1 CD25⫹ DN cells expressing TCR␣ failed to expand
significantly, if at all (the maximum expansion was 2- to 3-fold,
assuming a 70% cell loss at the time of an i.t. injection). Likewise,
neither H-Y nor 2C CD25⫹TCR␣⫹ precursors expanded significantly. In both strains the TCR␣⫹ fractions expanded less (if at all)
than the TCR␣⫺ ones (Fig. 5C). Although the H-Y DN
CD25⫹TCR␣⫺ thymocytes expanded about 10-fold, those from
2C mice failed to expand substantially, most likely because many
of these cells actually expressed low levels of TCR␣ (Fig. 5B). The
above experiments directly established a negative role of the
TCR␣␤ receptor in early thymocyte expansion. Although it could
be argued that the CD25⫹DN TCR␣⫹ and CD25⫹DN TCR␣⫺
thymocytes represent cells at different stages of differentiation, two
facts argue that this is not the case. First, i.t. expression of CD25
is regulated very tightly (7, 8) so that this molecule is transiently
expressed only on specific DN cells auditioning to become DP (7,
48, 49). Second, we have assessed the content of pT␣ mRNA
among the subsets used for i.t. injection and have found that both
CD25⫹TCR␣⫺ and CD25⫺TCR␣⫹ DN cells express it at comparable levels (Fig. 5D). As no other thymocyte subset expressed
pT␣ mRNA in our hands (data not shown), we conclude that the
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FIGURE 5. The relationship between pre-T␣ mRNA and TCR␣(␤) protein expression and the in vivo expansion potential of TCR Tg DN thymocytes. A, Expression of pT␣ mRNA among DN thymocytes of TCR Tg
mice. Total RNA was extracted from DN thymocytes, reverse transcribed,
and analyzed by PCR with oligonucleotide primers to amplify pT␣ and
␤-actin. Serial 10-fold dilutions of the template are shown. Results are
representative of three experiments. B. FCM analysis of TCR Tg␣ expression on Tg CD25⫹DN cells. The percentage of CD25⫹DN cells positive or
negative for the TCR Tg␣-chain is shown, with the total number of
CD25⫹DN cells taken as 100%. The indicated quadrants were also used as
the gates to sort each population for the experiment in C. As mentioned in
the text, virtually all TCR␣⫹ cells were also TCR␤⫹. C. Sorted cells from
A were i.t. injected into B6.PL mice. After 9 days, mice were sacrificed and
the thymocytes were isolated. The enriched donor cells were stained for the
donor marker (Thy1.2), and this percentage was used with the trypan-blue
counts to determine the absolute number of donor-derived cells in the thymus of each recipient. Results are shown as x៮ absolute cell numbers ⫾ SD,
and are representative of two experiments. D. Experiment was performed
as in A, except that we used nonfractionated B6 DN cells, or sorted TCR␣⫺
or TCR␣⫹ subsets of CD25⫹ DN cells from TCR Tg mice, as indicated.
3189
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CONTROL OF EARLY THYMOCYTE DEVELOPMENT BY PRE-TCR AND TCR
two subsets must be at the same or successive stage(s) of differentiation. Therefore, we conclude that the inappropriate expression
of TCR␣␤ at the surface of CD25⫹ DN precursors severely curtails thymocyte expansion and commits many DN thymocytes to
the mDN lineage.
pT␣ mRNA expression and cell cycle status of CD25⫹ gDN
thymocytes in TCR Tg mice
A question arising from the above results is how TCR␣ mediates
its inhibitory effect upon gDN cell expansion and differentiation.
For example, the mere expression of TCR␣ could interfere with
pre-TCR expression or function: 1) TCR␣ could compete with
pT␣ for pairing with TCR␤, as indicated by recent data from
Zuniga-Pflucker’s group showing that TCR␣ pairs with TCR␤
with much higher affinity than pT␣ (26); 2) TCR␣ could down-
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FIGURE 6. Chimerism in mixed chimera devoid of one or both types of
MHC molecules. Mixed chimera were
constructed as described, except that donors and/or recipients were deficient in
MHC class I or class II molecules, as indicated, as a consequence of the targeted
disruptions of the ␤2-microglobulin and
I-Aa genes, respectively (A–D). In E,
both recipients and donors expressed all
MHC molecules, but the marrows were
obtained from OT-1␣␤ or OT-1␣ Tg
strains. Thymocytes were analyzed, and
results were reported as in Fig. 1, with
five mice per group shown in this figure.
regulate the expression of pT␣ by a feedback mechanism; or 3)
TCR␣␤ could compete with pre-TCR for signaling molecules in
the absence of TCR␣␤ signaling (dominant-negative action). To
gain insight into this issue, we first performed pT␣ mRNA analysis
on CD25⫹ DN subsets separated according to their expression of
TCR␣ (Fig. 5D). This analysis showed essentially no difference in
pT␣ mRNA expression, regardless of whether TCR␣ was expressed at the surface of CD25⫹ DN cells. Therefore, TCR␣ expression does not down-regulate the expression of pT␣ mRNA by
a feedback mechanism. This indicated that TCR␣ expression
blocks thymocyte proliferation by a competitive mechanism at the
protein level. Preliminary studies of cell cycle distribution were
consistent with this explanation because we found that the presence of TCR␣-chain correlated with lower number of cells in the
S phase of the cell cycle in OT-1 mice.
The Journal of Immunology
FIGURE 7. The effect of MHC class I removal on the makeup of gDN
cells in TCR Tg mice. Analysis was performed by compiling percentages
of TCR Tg␣⫹ CD25⫹ DN (䡺) and TCR Tg␣⫺ CD25⫹ DN (f) subsets,
using the staining and methods shown in Fig. 5B. Genotype of each group
is shown under the figure. Results are expressed as x៮ ⫾ SD (n ⫽ 6) and are
compiled from two experiments.
Although the above results directly demonstrated that the existence
of a block in thymocyte expansion and progression to the DP stage
depends on the expression of the TCR␣␤ receptor, it was only
partially clear how the TCR effects this block and it was not clear
why different TCRs induce this block to a different degree. As
previously mentioned, the mere expression of TCR␣ could interfere with pre-TCR expression or function by a competitive or dominant interfering mechanism. A mutually nonexclusive alternative
is that the interaction of TCR with the peptide-MHC ligands in the
thymus (i.e., the intensity of TCR␣␤ signaling) could induce the
developmental block. To address these possibilities, we took away
MHC class I or all MHC molecules (␤2-microglobulin⫺ or ␤2microglobulin ⫻ I-Ab⫺ mice, respectively) from the thymic environment in which thymocytes from mixed chimera developed. In
the first set of experiments, class I⫺ marrow from wt and OT-1
mice was coinjected into class I⫺II⫺ recipients. Under those circumstances, wt thymocytes still developed better than those from
OT-1 mice (Fig. 6A). This result indicated that the mere expression
of TCR␣␤ in the absence of TCR-MHC contact is inhibitory for
DN f DP transition, most likely due to vastly superior affinity of
TCR␣ for TCR␤ compared with pT␣ (26), and the consequent
disruption of pre-TCR formation and function.
We next investigated the role of the early TCR-MHC contact in
DN f DP transition. When we injected mixed TCR Tg bone marrow from the MHC class I⫺ TCR Tg donors into class I⫺ recipients, the results were dependent on the type of the donor mice. In
the absence of class I molecules, 2C and OT-1 thymocyte populations were equally abundant with no signs of domination (Fig.
6B), indicating that these two thymocyte subsets now developed
with equal kinetics. Furthermore, the absolute number of DP cells
and the cellularity of the whole thymus increased substantially in
2C, and somewhat less remarkably in OT-1, class I⫺ animals compared with their class I⫹/⫹ counterparts (2C/class I⫹ thymi had
40 ⫾ 10, whereas 2C/class I⫺ mice had 100 ⫾ 20 ⫻ 106 cells;
likewise, OT-1 class I⫹ and class I⫺ thymi had 78 ⫾ 9 and 94 ⫾
12 ⫻ 106 cells, respectively). Surprisingly, the (2C ⫹ H-Y) f
class I⫺ mixed chimera still exhibited heavy domination, but now
by the 2C cells, which are the inferior partner in normal, class
I-sufficient mice (Fig. 6C). Cellularity of thymi of the H-Y/class I⫺
mice was actually reduced (12 ⫾ 3 ⫻ 106, compared with 65 ⫾
15 ⫻ 106 in H-Y/class I⫹ mice; see also Ref. 50). These results
indicated that the presence of class I molecules heavily inhibits the
expansion of 2C thymocytes (and/or reduces their numbers by negative selection) and suggested that H-Y thymocytes may similarly
be inhibited by the self class II molecules. The latter possibility
was consistent with the recent study (50) showing that the H-Y
receptor has an affinity toward I-Ab and that the presence of this
molecule suppressed T cell development and partially anergized
H-Y T cells in B6 mice. Therefore, we constructed another set of
mixed chimera, using mice that do not express any MHC molecules (␤2-microglobulin⫺ ⫻ I-A␣⫺, referred to as class I⫺ II⫺
mice) as marrow recipients. Although mouse thymocytes express
class I molecules that can readily imprint tolerance and other immunological phenomena (51), they do not express class II molecules, so that the only cells expressing class II in these chimera are
the non-T cells of hemopoietic origin that are much less frequent
in the thymus compared with thymocytes. In such chimera, 2C and
H-Y thymocytes developed rather evenly (Fig. 6D). The average
percentage of the H-Y thymocytes slightly lagged behind 2C in
mixed chimera. This was likely due to the presence of I-Ab⫹ donor
non-T cells of hemopoietic origin (B cells, dendritic cells, macrophages) that affected the expansion of early H-Y TCR⫹ thymocytes contacting them.
These results strongly suggested that an early TCR-MHC contact plays an inhibitory role in DN thymocyte expansion and commitment to DP lineage. If so, TCR␣␤ Tg DN precursors would be
expected to exhibit a more profound DN f DP block than TCR␣
Tg DN cells because only in the former mouse strain would all
cells express TCR␣␤ capable of contacting i.t. MHC molecules.
This hypothesis was tested in a mixed chimera of the [(OT-1
TCR␣␤ Tg ⫹ OT-1 TCR␣ Tg) f wt] type. The two types of
progeny were identified by double-staining with Tg-specific TCR␣
and TCR␤ mAbs (double-positive cells were TCR␣␤ Tg, whereas
those staining only with TCR␣ were derived from single Tg origin). As predicted, the presence of a complete Tg TCR␣␤ induced
a more profound block in early thymocyte expansion and differentiation than the TCR␣ transgene (Fig. 6E).
To further assess the extent to which TCR␣␤ interaction with
MHC molecules impacts upon the gDN compartment, we investigated the distribution of TCR␣ on CD25⫹ DN cells in TCR Tg
MHC-sufficient or MHC class I-deficient mice. The rationale here
was that if class I deficiency partially alleviates the TCR␣␤-mediated block, one would expect that in these mice fewer CD25⫹
DN precursors would be TCR␣⫹. Indeed, the removal of MHC
molecules changed the ratios between TCR␣⫹ and TCR␣⫺
CD25⫹ DN cells in H-Y and 2C mice, decreasing them from ⬎4:1
to ⬍2:1. The ratio in OT-1 mice did not change. These results
suggest that the block in expansion and differentiation of gDN cells
and the accumulation of mDN cells in these mice is mostly caused
by the impairment of pre-TCR assembly and function by the prematurely expressed TCR␣-chain and that the OT-1 TCR interaction with the i.t. pep-MHC molecules plays only a subtle modulatory role in pronouncing the block. By contrast, a much stronger
block, as seen in H-Y and, in particular, 2C mice, is caused by a
strong interaction of these two TCRs with i.t. pep-MHC molecules.
All of the above results establish a negative role for the early
TCR-MHC interaction in determining the extent of prothymocyte
expansion and thymic cellularity, and definitively show that early
TCR␣␤ expression disrupts thymocyte expansion and differentiation by two distinct mechanisms.
Discussion
Several novel points pertinent to the control of TCR␣␤ development emerge from the above results. Our data directly and formally demonstrate that: 1) the low thymus cellularity in TCR Tg
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TCR␣␤ inhibits early thymocyte expansion and progression to
DP cells by two mechanisms
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CONTROL OF EARLY THYMOCYTE DEVELOPMENT BY PRE-TCR AND TCR
cells are related to ␥␦ cells, our results, along with those of others
(52, 61) showing that pT␣ signals are not required for the development of TCR␥␦ cells, indicate that the pre-TCR is essential in
ensuring the domination of the TCR␣␤ lineage over the TCR␥␦
cells in the thymus. pTCR signals are exclusively available to the
former (62), leading to their expansion, whereas the latter develop
without the benefit of major expansion. Even when TCR␣␤ operates in the early development, as in TCR Tg mice or TCR␣␤reconstituted pT␣⫺/⫺ mice, the mDN (supposedly ␥␦-like) lineage
seems to be gaining ground.
Most importantly, our results, together with those of Trop et al.
(26), suggest an additional mechanistic explanation why TCR␣
protein should be produced later in ontogeny than TCR␤ protein.
It is indeed a good “business decision” to delay the expression of
TCR␣ to test ␤-chains for proper folding and function, and thus
maximize T cell production (17). However, TCR␣ could accomplish the same “testing” task instead of pT␣, albeit with more
waste. But the more important reason for delaying Tcr-a rearrangement and the consequent TCR␣ protein production and TCR␣␤
assembly is to allow pre-TCR to mediate thymocyte expansion and
to simultaneously shield CD25⫹ thymocytes from expansionblocking TCR␣␤ signals initiated by the TCR-MHC contact. Unlike the pre-TCR in thymocytes, or the TCR␣␤ in mature T cells,
the TCR␣␤ in thymocytes is not compatible with transmission of
proliferative signals, consistent with our data that TCR␣␤ transmits signals that block proliferation of developing DN cells and
keep them from becoming DP. Indeed, similarities in the outcome
of this signal and the positively selecting signal transduced by the
TCR␣␤ at the DP stage are striking. In DP thymocytes, there is no
proliferation associated with productive selection, and the initial
consequence of the signal is a down-regulation of CD8 and CD4
(58). Thus it is likely that the mechanism of the CD8 and CD4
down-regulation and loss would be similar to the one identified by
Takahama and Singer (57).
Acknowledgments
We thank Drs. S. Vukmanović and H. T. Petrie for critical reading of the
manuscript and D. Nikolich-Žugich for help with FCM.
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Whatever those early signals are, if the TCR␣␤ is present on the
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expansion to occur. However, such signals can be further compromised, if not overridden, by the signals resulting from the MHCTCR␣␤ interaction. Thus, premature MHC-TCR␣␤ interaction
and the consequent TCR␣␤-propagated signals are the second
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