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Expression and Signaling in Early Thymocytes Impair Thymocyte Expansion and Partially Block Their Development
ek-Szabo*
2,*
ugich3,*,
*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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
TCR+CD8-4- 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
CD8-4- 
CD8+4+
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
CD8-4- thymocytes from TCR signals that
impair thymocyte proliferation and CD8-4-
CD8+4+ transition. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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%) single-positive (SP) TCRhigh cells
via the CD4+8+
double-positive (DP) TCRlow intermediates
(75–85%) (reviewed in Refs. 1, 2, 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)
CD44+CD25+ (DN2)
CD44low/-CD25+ (DN3)
CD44low/-CD25- (DN4)
(1, 2, 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, 14, 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, 17, 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
CD4lowCD8lowTCRlow
phenotype and exhibit the cell-autonomous capability to become DP in a
matter of hours (22, 23, 24, 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 mice
(30, 31, 32, 33). These cells never go through the DP stage of
development (32, 33, 34). Although many features of these
cells (referred here to as mature DN (mDN) cells) are reminiscent of
the TCR
thymocytes (35, 36, 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
pre-TCR 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6, H-2b, Thy-1.2, Ly-5.1) and
B6.Ly5.2 (H-2b, Thy-1.2, Ly-5.2) 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 VJ
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.PLx2C) F1 mice that were screened for the
presence of the TCR transgene. All mice were used at 6–12 wk of age
and were age- and sex-matched within experiments.
Abs, cell preparation, and FCM analysis
Single-cell suspensions of thymocytes were stained with the
indicated Abs and 5–50 x 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). FITC-conjugated anti-CD8,
CyChrome-conjugated anti-CD4 and FITC- or PE-conjugated
anti-V2 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-V8, Ref.
45); IM7.8 (anti-CD44, Ref. 9), and PC61
(anti-CD25, 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 (anti-Thy1.2 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 x 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
(Perkin-Elmer/Cetus, Norwalk, CT). After amplification, 10 µl 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P
chimera occurs at the level of DN 
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, 36, 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 donor-derived 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)]
B6.PL(H-2b, Thy-1.1, Ly-5.1)}, 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 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
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 gDNDP transition.
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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.
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Because wt thymocytes always outcompeted the TCR Tg ones, the
above results suggested that there is a block in DNDP 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 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.)
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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.
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As the expression of both TCR and pT is instrumental for an
efficacious thymocyte expansion during the DN 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.
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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
TCRlow 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 120-fold 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 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-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.
TCR inhibits early thymocyte expansion and progression to DP
cells by two mechanisms
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
2-microglobulin x
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 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.
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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 x 106 cells; likewise, OT-1 class
I+ and class I- thymi had
78 ± 9 and 94 ± 12 x 106 cells,
respectively). Surprisingly, the (2C + H-Y) 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 x 106, compared with
65 ± 15 x 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- x
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 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) 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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development emerge from the above results. Our data directly and
formally demonstrate that: 1) the low thymus cellularity in TCR Tg mice
is caused by the block in DN DP transition; 2) this block occurs
because many CD25+DN precursors express TCR
and lose their ability to expand and become DP cells; 3) instead,
TCR+CD25+DN cells
switch off the expression of pT and CD25 and become mature
nonprecursor DN cells; and 4) that expression of TCR interferes
with thymocyte expansion and differentiation at two levels—early
expression of TCR blocks the pre-TCR function (Fig. 5) likely by
competing pT out of the pre-TCR (26) and then TCR
prematurely interacts with the i.t. MHC ligands to pronounce the block
depending on the intensity of the TCR-MHC contact (Figs. 6 and 7). Therefore, the same contacts that
mediate positive i.t. selection at the DP stage also prevent proper
expansion and maturation if administered at an earlier precursor
stage.
|
selection was ushered (17), its
teleological justification was obvious—quality control for the cells
synthesizing a biologically valid TCR-chain and a subsequent
increase in the efficacy of TCR+ cell
production. It appeared that this hypothesis explains the need for
dissociation of Tcr-a and Tcr-b rearrangement.
Subsequently, it was shown that the pre-TCR was also important for
thymocyte expansion (52), allelic exclusion
(53), and lineage commitment (21, 54). Transgene reconstitution experiments (35, 55)
showed that TCR can substitute for the many, but not all,
functions of the pre-TCR. Indeed, when no other TCR is available (as in
pT-/- mice), TCR can promote some
expansion and a relatively normal differentiation, as judged by
steady-state status of thymocyte subsets. But thymi in such mice are
hypocellular, and mDN cells accumulate (52). Our results
clearly show why TCR cannot be used in place of the pT in
early development. Apparently, thymocyte expansion and/or survival at
the DN DP transition require fundamentally distinct signals from
those that govern thymocyte selection at the DP stage. Whatever those
early signals are, if the TCR is present on the developing DN
cells at the same time, pre-TCR formation is disrupted. As demonstrated
recently (26), TCR has a much higher affinity for
TCR than pT, effectively outcompeting pT and, as shown by our
data, blocking the function of pre-TCR. Given the extremely low
expression levels of pre-TCR even at the surface of normal cells, it is
very difficult to ascertain whether pre-TCR and TCR actually coexist at
the surface of TCR Tg DN cells. Perhaps the remaining pre-TCR signals
still allow some proliferation and expansion to occur. However, such
signals can be further compromised, if not overridden, by the signals
resulting from the MHC-TCR interaction. Thus, premature
MHC-TCR interaction and the consequent TCR-propagated
signals are the second mechanism that interferes with normal T
cell development. Such signals further curtail expansion of DN cells
and block their conversion into DP thymocytes, leading to the
production of mDN cells. Analogous results were observed in mice
overexpressing CD8; however, the role of MHC in this phenomenon was not
directly established (56). It is worth noting that a block
in the progression of immature fetal
CD8+4- thymocytes into DP
cells was found under the negatively selecting circumstances
(57) in male H-Y mice but that the outcome of such
signaling in the adult thymus appeared to be clonal deletion
(58). In either case, the critical difference between
these studies and the results presented here is in the presence of a
bona fide negatively selecting ligand in the H-Y male model and the
lack of any evidence for negative selection in our study (see
Results).
It was previously speculated that mDN cells are actually of the
TCR lineage (35, 37, 59). A recent study by
Fowlkes’s group provided very persuasive arguments supporting this
view (60), albeit a decisive proof is difficult to obtain
in the absence of diagnostic markers for the TCR lineage
(59). If indeed the mDN 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 expansion-blocking
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 |
|---|
and H. T. Petrie for
critical reading of the manuscript and D. Nikolich-ugich for help
with FCM. |
Footnotes |
|---|
.) 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.-.), and the DeWitt Wallace Fund (to J.N.-.). 
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-ugich, Immunology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.
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.
Received for publication October 31, 2000. Accepted for publication December 29, 2000.
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± SD for five mice. B, Experiment was performed
exactly as in A, but for the indicated TCR Tg chimera
and using TCR Tg
) and TCR
Tg
) subsets, using the
staining and methods shown in Fig. 5