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* Memorial Sloan-Kettering Cancer Center, New York, NY 10021;
Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021; and
Monash University Medical School, Department of Pathology, Melbourne, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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14 days (reviewed in Ref.
12), these multilineage progenitors are induced to undergo
a series of differentiative events that lead to T lineage commitment
(13). In addition, a great deal of proliferative expansion
occurs, such that each progenitor entering the thymus gives rise to
approximately one million immature progeny (12), thus
generating the cells that are subsequently subjected to TCR-mediated
screening. In general, lineage commitment and proliferation are not cell autonomous processes, but rather occur in response to combinations of signals (morphogens, growth factors, hormones, etc.) that progenitor cells receive from the external microenvironment. The nature of signals that drive lineage commitment and proliferation in early intrathymic progenitors remains largely unknown. A few such signals have been identified in the form of Notch-1 (10, 14, 15), c-kit (16, 17), and the IL-7R (18), although the exact functions (i.e., differentiation, proliferation, survival) in some cases remain controversial. Regardless, it seems unlikely that this relatively small number of signals is adequate to explain the complex sequence of events that lead to production of a large number of T lineage progeny from a relatively few undifferentiated progenitors. To evaluate what other signals might be involved, we began from the assumption that early precursors receive specific signals from their external microenvironment that lead to T lineage commitment and/or proliferative expansion. This led us to define a stratified pattern of precursor distribution in the thymic cortex (19), wherein the earliest progenitors (CD4/8 double-negative stage 1, DN1)4 are in the deep cortex, the next stage (DN2) in the midcortex, and the last stage (DN3) predominates in the outer third of the cortex. Transition to the early CD4/8 double-positive (pre-DP) stage, together with the most obvious wave of proliferation (20, 21), correlates with localization to the subcapsular zone (22). This is followed by a reversal in the polarity of migration, with movement of expanded DP progeny progressively deeper into the cortex toward the medulla (23).
There are two distinct categories of functions that are implicated by
the behaviors described above. The first is the stratified distribution
of signals that induce sequential stages of lineage commitment and
proliferation in the thymic cortex. The second, and the topic of the
present study, is a mechanism for moving cells between them.
Directional migration of cells within tissues indicates the presence of
a number of biochemical requirements. Among these are adhesion
receptors on the migrating cells, as well as a stable matrix of their
ligands or counterreceptors to provide the traction for cell movement.
In this study, we characterize the presence of adhesion receptors on
lymphoid cells traveling outward through the cortex, as well as their
ability to adhere to matrix ligands predicted by these receptors. Our
data suggest that migrating lymphoid progenitors use a cellular, rather
than extracellular, matrix for adhesion and migration. Combined
functional and in situ analysis suggests that this cellular matrix
consists of a subset of cytokeratin+ cortical
stromal cells that express the 
4 integrin
ligand VCAM-1. Together, our findings demonstrate a close association
of early lymphoid progenitors in the thymus with the stromal elements
on which their differentiation depends. Furthermore, in addition to
revealing unreported heterogeneity among
cytokeratin+ stromal cells of the thymic cortex,
these studies suggest that early lymphoid progenitors and other, more
mature lymphoid cells that occupy the same cortical space may
nonetheless interact with very distinct stromal cell types.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Precursor thymocytes were prepared by sorting of
lineage-negative thymocytes isolated from 4- to 8-wk-old C57BL/6 mice,
as previously described (21). Thymic stromal cells were
isolated as previously described (24). The cloned thymic
stromal cell line 100-4 (25) was grown in DMEM
supplemented with 10% FBS, 2 mM glutamine, and 5 x
10-5 M 2-ME. Purified anti-integrin Abs were
clone 9C10 (anti-4), clone 5H10-27
(anti-5), clone GoH3
(anti-6), clone 9EG7
(anti-
1), clone 346-11A
(anti-4), and clone M293
(anti-7), all from BD PharMingen (San
Diego, CA). Secondary Ab used for integrin detection was biotinylated
goat F(ab')2 anti-rat IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA), followed by
PE-streptavidin (Molecular Probes, Eugene, OR). Anti-cytokeratin Ab
was clone C-11 conjugated to FITC (Sigma-Aldrich, St. Louis, MO),
recognizing cytokeratins 4, 5, 6, 8, 10, 13, and 18. Abs used to define
cortical stromal cells by flow cytometry were M 5/114.15.2 and 6C3, as
previously described (26). Anti-VCAM-1 Ab used for in situ
analysis was clone 429 conjugated to biotin (BD PharMingen).
Anti-VCAM-1 Ab used for in vitro blocking was clone M/K2.7. Abs used to
identify precursor thymocytes in situ were clones PC-61 (anti-CD25)
or ACK-2 (CD117) coupled to biotin. Detection of VCAM-1 or CD117 by
immunohistochemistry or immunofluorescent microscopy was performed
using tyramide signal amplification (NEN, Boston, MA).
Assay for adhesion to purified extracellular matrix (ECM) ligands
Murine fibronectin (FN) and laminin 1 (LN1) were purchased from Life Technologies (Carlsbad, CA). Ninety-six-well trays (Nunc 473768; Nalge Nunc International, Rochester, NY) were coated with ECM proteins by overnight incubation at 4°C with 1 (FN) or 5 µg (LN1) purified protein in 100 µl PBS. These concentrations were determined by measurement of optimal binding efficiency using unsorted DN thymocytes (i.e., lineage-depleted thymocytes). Blocking of excess protein binding was performed by incubating wells in a solution of heat-inactivated BSA (1% in PBS) for 1 h at room temperature. Purified DN precursors were added in PBS/BSA and allowed to settle for 30 min at 4°C. In any given experiment, the wells contained identical numbers of cells for each developmental stage, although the absolute number of cells varied between experiments, ranging from 4–5 x 105 cells/well. Plates were then incubated at 37°C for 30 min, followed by repeated washes in PBS/BSA. After a final wash in PBS, cells were fixed using 4% formaldehyde for 30 min at room temperature, followed by washing in PBS, treatment with 20% methanol in water (10 min at room temperature), and staining with 2% crystal violet. A single field at the center of each well was photographed, and the digital image was analyzed using the colony count function of Quantity One software (Bio-Rad, Hercules, CA). For an estimation of total cells in the well before washing, a phase-contrast image of the center of the well was used.
VCAM-1 adhesion assay
The 100-4 cells were grown in eight-well glass slide chambers (Lab-Tek; Nalge Nunc International), as described above. Purified T cell precursors (4–5 x 105/well) were added in DMEM supplemented as described above, followed by incubation at 37°C for 1 h. The glass slide was removed from the chambers and washed by stirring in a beaker containing PBS. Fixation was then performed as described above, followed by staining in Harris’ Modified Hematoxylin (Fisher Scientific, Pittsburgh, PA) and mounting. For blocking experiments, anti-VCAM-1 Ab (5 µg/ml) was added to the adherent monolayers 30 min before addition of lymphoid cells, and left in the medium during subsequent incubation.
Flow cytometry and microscopy
Thymic sections (4 µM) were prepared by cryosectioning after embedding in OCT. Immunohistochemical detection was performed using the VectaStain ABC kit (Vector Laboratories, Burlingame, CA). Staining for immunofluorescent microscopy was performed as previously described (19). Tissue counterstains were hematoxylin (immunohistochemistry) or 4',6'-diamidino-2-phenylindole (DAPI; immunofluorescent microscopy). Digital microscopy was performed using an Olympus (Melville, NY) BX-50 microscope equipped with a mercury light source. Flow cytometric analysis was performed using a three-laser LSR cytometer (BD Biosciences, San Jose, CA).
Anti-VCAM-1 administration in vivo
Animals were injected daily for 5 days with 100 µg/day mAb recognizing VCAM-1 (clone MK-2.7) or control (nonspecific) Ab. Following this, animals were euthanized and the thymus was removed carefully. One lobe was frozen immediately for immunohistochemical and/or immunofluorescent analysis, while a single cell suspension was prepared from the other lobe. The latter was used for determination of total cellularity (by hemacytometer counting) as well as for phenotypic analysis of developmental stage by flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous work has shown that early intrathymic precursors migrate outward through the cortex before differentiating into CD4+8+ cells (19). In an effort to understand the mechanisms of transcortical migration, we sought to analyze the expression of integrins by defined stages of intrathymic differentiation. Expression of integrins is implicit in cell migration through tissues, and although the mere presence of an integrin does not necessarily imply functional activity, it does indicate potential for adhesion to the corresponding ligand. It should be noted that while a number of studies have evaluated integrin expression on thymocytes, especially fetal progenitors or total DN cells, integrin expression relative to defined stages of differentiation in the postnatal progenitors has not been characterized, nor has binding to the corresponding ligands. Given the recent description of stratified regions through which progenitors migrate during differentiation (19), these were the goals of the experiments described in this work.
A number of integrins were not found at any appreciable level on DN
cells, including 1,
2, 3, and
v. The latter is particularly informative,
because it eliminates potential involvement of
3-, 5-,
6-, and 8-containing
heterodimers in the precursor migration process. Likewise, the
involvement of 2 integrins has been largely
ruled out by gene-targeting experiments (27), thus leaving
1-, 4-, and
7-containing heterodimers as the major areas
of interest. Analysis of these integrins and their partners reveals
substantial heterogeneity among the various stages of differentiation,
as illustrated in Fig. 1
. For instance,
all DN stages express 4 integrin, although DN1
express it in a bimodal pattern, and at levels that are lower than
either DN2 or DN3. Likewise, 5 integrin is
expressed by all DN stages, although a bimodal distribution is again
noted, this time in DN2, but not DN1 or DN3 cells. Thus, although these
two integrins are consistently expressed on DN cells, they are not
uniformly expressed; the relevance of this finding is addressed by
other experiments in this manuscript, and in Discussion.
Expression of integrin 6 is fairly uniform on
all DN cells, as is that of 1. However,
heterogeneity is again revealed by analysis of both
4 integrin, which is up-regulated upon
transition from DN1 to DN2, and 7 integrin,
which appears to be largely specific for DN2 cells. Thus, the
functional heterodimers that can be expressed by different stages of
intrathymic precursor differentiation are identifiable, and can be used
to predict the potential ligands for adhesion at each stage of
transcortical migration. The major predicted ligands, as summarized in
Table I, were the ECM proteins FN, LN1,
and LN5, as well as VCAM-1, which is generally implicated in cell-cell
rather than cell-matrix interactions. The ability of integrin
heterodimers on DN precursors to bind these ligands was subsequently
tested, as described in the next section.
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To confirm the integrin expression data shown in Fig. 1
, and to
further characterize the nature of the matrix for transcortical
migration of lymphoid precursors, static adhesion to purified ECM
proteins was performed (Fig. 2). Although
static adhesion does not allow measurement of absolute affinity for a
given ligand, it can be used to determine relative affinity of cells
expressing a given set of receptors. Adhesion to LN5 was not tested,
because it is found only in the basal layers of the thymic capsule and
thymic blood vessels (28, 29), neither of which are
primary sites for DN localization in the cortex (19). For
the remaining ECM ligands, namely FN and LN1, 96-well tissue culture
trays were coated with optimal levels of purified ECM proteins (see
Materials and Methods), and equal numbers of cells at each
stage were added. Following incubation and washing, relative levels of
binding were determined. Absolute quantitation of such assays is
complicated by an accumulation of cells at the edge of the well, in
which the hydrodynamic forces of washing are greatly reduced.
Consequently, counting was restricted to a single microscopic field at
the center of the well. The number of cells in this field was
quantitated using the colony-counting function of Quantity One software
(Bio-Rad) and compared with the total cells present before washing (see
Materials and Methods). Several observations were made using
this assay. First, the most efficient binding was of DN3 cells to FN,
with 70–80% of cells bound on average (numerous experiments were
performed, but not all DN populations were examined in all
experiments). DN1 also bound efficiently to FN, with a similar
proportion of cells bound (60–70%). However, despite expressing
multiple FN receptors (Table I), DN2 cells did not bind with
appreciable frequency to FN (10–15% of cells bound). For all
populations, the frequency of binding to LN1 was less than that of FN,
although appreciable numbers of cells still bound at both the DN1 and
DN3 stages (30–35%). However, binding of DN2 cells to LN1, although
significant by the Student’s t test for paired samples, was
only trivially higher than binding to BSA alone. Together, these
findings suggest that DN1 and DN3 cells express active FN receptors
and, to a lesser extent, LN1 receptors, while DN2 cells do not bind
with high affinity or frequency to either of these, despite expressing
the appropriate receptors.
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Our previous studies suggest that DN2 cells must be highly
migratory, because they span the midcortical regions between DN1 and
DN3 cells (19). Directional cell migration requires a
matrix for cell adhesion, yet the results shown in Fig. 2 suggest that
DN2 do not bind efficiently to ECM components for which they bear
receptors. DN2 cells also bear multiple receptors for VCAM-1 (Fig. 1
and Table I), a counterreceptor that is generally found in cell surface
form and mediates cell-cell interactions. VCAM-1 has been shown to be
expressed in human thymus by both nonhemopoietic stroma
(30) and macrophages (31). Interestingly, the
stromal cells expressing VCAM-1 in the former case created a reticular
lattice in the cortex. To evaluate the possibility that a cellular
matrix composed of VCAM-1+ cells might form a
substrate for cell migration across the thymic cortex, we first
analyzed expression of VCAM-1 in the mouse thymus (Fig. 3). Similar to previous findings on the
human thymus, we find that VCAM-1 is expressed on reticular cells that
form a radially aligned matrix in the thymic cortex, while expression
in the medulla is restricted to scattered cells with macrophage-like
morphology, or to vascular elements. The nature of various
VCAM-1+ cells is further revealed by dual
staining using an Ab recognizing cytokeratins (Fig. 3). Numerous
phenotypes can be observed, including VCAM-1+
keratin- cells with macrophage/dendritic
morphology (located mainly in the medulla), and a significant number of
VCAM-1- keratin+ stromal
cells in the cortex. However, throughout the cortex there is also a
subset of keratin+ cells that is
VCAM-1+. This finding is confirmed by flow
cytometric analysis, which shows that approximately one-third of
cortical stromal cells (MHC-II+,
6C3+; see Materials and Methods) are
also VCAM-1+ (Fig. 3). Together, the data
presented in Fig. 3 reveal that VCAM-1 is expressed in the thymus on a
subset of stromal cells that form a reticular matrix in the cortex.
Especially given the lack of DN2 binding to ECM components, this raised
the possibility that stromal cells, and in particular
VCAM-1+ stromal cells, might represent the
substrate for precursor migration outward through the cortex, as
evaluated below.
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show that this is, in fact, the case.
An Ab recognizing CD117 (c-kit) was used to identify early
intrathymic precursors (DN1 and DN2) found in the inner and midcortex
(19), together with an Ab recognizing cytokeratins, to
identify the most abundant stromal cells in the cortex. The vast
majority of CD117+ cells were found to be in
direct contact with cytokeratin+ stromal cells,
in support of the above hypothesis. It should be noted that in any
single plane, there were always a few (one or two) precursors that did
not appear to be in contact with a stromal cell, suggesting the
possibility that not all lymphoid precursors remain in contact with the
stromal matrix at all times. However, it is quite possible that these
few cells may have been associated with a stromal cell that was above
or below the plane of the tissue section being examined. In any case,
the majority of precursor cells in any given plane are found to be
directly in contact with a stromal cell. The intimate nature of this
interaction is further revealed by high magnification views at various
depths in a single tissue section (Fig. 4, b and
c). Lymphoid precursors are not only in contact with stromal
cells, but are virtually surrounded by reticular processes from the
stromal cells that they contact. These data not only support the
hypothesis that stromal cells may provide a matrix for migration of
early precursors outward through the cortex, but also show that
intimate lymphostromal contacts are formed during this process,
providing a basis for reciprocal signals that may influence
differentiation, proliferation, and/or survival of either cell type, as
predicted by the findings of others (for examples, see Refs.
32, 33, 34).
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). In this assay, DN1 cells bound in a
relatively nonspecific manner to both the stromal cells and the plastic
substrate; consequently, the ability of DN1 cells to bind specifically
to VCAM-1 cannot be reliably determined. However, both DN2 and DN3
cells bound specifically to these VCAM-1+ cells
in a manner that could be blocked almost completely by preincubation
with anti-VCAM-1. This finding is most revealing in relation to the
DN2 subset, which had poor affinity for ECM ligands under similar assay
conditions (Fig. 2). Because the anatomic location of DN2 cells is
intermediate between that of DN1 (inner cortex) and DN3 (outer cortex),
the reliance of this stage on cell surface-expressed VCAM-1 for
adhesion strongly implicates requirement for a cellular, rather than
extracellular, matrix for transmigration of early precursors between
different cortical microenvironments.
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The data presented to this point show that early lymphoid
precursors directly interact with stromal cells during migration
outward through the cortex, and strongly implicate adhesion to this
stromal matrix via a VCAM-1-dependent mechanism. To confirm the
relevance of this data, an in vivo assay was highly desirable. Several
factors complicate such analysis. First, disruption of lymphoid
receptors for VCAM-1 results in an inability of bone marrow precursors
to intravasate and travel to the thymus (35), making
interference with 4 integrins problematic.
Second, it is possible that VCAM-1 may be involved not just in the
intrathymic migration process, but in the interaction of blood-borne
progenitors with vascular endothelium in thymic blood vessels, and thus
in entry of early precursors into the thymus. Inhibition of entry into
the thymus and inhibition of early precursor differentiation inside the
thymus could have similar phenotypes, i.e., an overall reduction in
thymocyte number, thus making it difficult to unconditionally
distinguish between these two potential roles for VCAM-1.
With this caveat in mind, we performed such experiments, using in vivo
administration of a mAb against VCAM-1 for 5 consecutive days in mice.
Overall, the size of the thymus was reduced by approximately one-third,
both in terms of cross sectional area (Fig. 6) and cell number (Table II), in animals
receiving anti-VCAM-1 Ab vs a nonspecific
control Ab. The density of cells in the
anti-VCAM-1-injected thymus was different from that of controls,
with the cortex assuming a less densely packed appearance (i.e., more
space between cells). The frequency and anatomic distribution of early
(CD117+) precursors were also influenced, with
fewer cells being present overall, and markedly fewer cells in the mid-
to outer cortex of anti-VCAM-1-injected mice. Overall, the
proportion of total
CD4-8- cells did not
differ substantially in thymuses from anti-VCAM-1 injected vs
controls (Table II). Likewise, the proportion of individual DN subsets,
including the CD117+ stages (DN1 and DN2), was
not dramatically affected (data not shown). This result is to be
expected given the relatively short period of VCAM-1 administration (5
days) compared with the total life span of DN cells (2 wk; see Ref.
12), especially because the transit time through the
CD117+ stages is at least 10 days. Thus, 5
days of anti-VCAM-1 administration can effectively block migration
to the outer cortex without substantially changing the number of
precursor cells that are present. Administration of Ab for longer
periods incurs the risk of anti-Ig immune responses in either
VCAM-1- or nonspecific Ab-treated mice. Nonetheless, 5 days of
anti-VCAM-1 Ab had clear effects on both thymic size and precursor
distribution. The fact that early progenitors were biased toward the
inner cortex in anti-VCAM-1-treated animals further suggests that
VCAM-1 must play a role in intrathymic migration in addition to any
potential role in precursor entry; effects on precursor entry alone
should result in an accumulation of precursors in the outer, rather
than the inner, cortex. Thus, our findings are consistent with a
requirement for VCAM-1 in the intrathymic migration of early
progenitors, although an additional role in precursor entry cannot be
excluded by these studies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
Table I) to predict the mechanical requirements for migration of
immature precursors from the deep cortex to the capsule. Although deep
cortical (DN1) and outer cortical (DN3) stages were capable of
efficient binding to ECM ligands predicted by integrin expression
profiles, DN2 cells that span the midcortex were not. This led us to
evaluate binding to alternative matrix ligands, specifically the cell
surface-expressed integrin counterreceptor VCAM-1. Functional and in
situ studies revealed that lymphoid progenitors migrating outward
through the cortex were found in intimate contact with a radially
aligned stromal cell matrix (Fig. 4), a subset of which was
VCAM-1+ (Fig. 3). Although technical limitations
prevented us from performing direct colocalization studies of VCAM-1
and early precursors in situ, the capacity for these cells to interact
in a VCAM-1-specific manner was demonstrated by in vitro binding (Fig. 5). Furthermore, administration of an anti-VCAM-1 Ab in vivo
resulted in reduced thymic size and cortical density, as well as a
reduction in the presence of early progenitors in the outer cortex
(Fig. 6). This phenotype is consistent with a role for
VCAM-1+ stromal cells in providing the adhesive
matrix for intrathymic precursor migration, although, as mentioned
earlier (see Results), VCAM-1 could also play a role in the
entry of precursors into the thymus. Our data conclusively show that
early lymphoid progenitors form intimate contacts with a matrix
composed of cortical stromal cells (Fig. 4); that they express
receptors for VCAM-1 (Fig. 1 and Table I), which is found on components
of this cellular matrix (Fig. 3); and that they bind specifically to
VCAM-1 on cloned cortical stromal cells in vitro (Fig. 5). Together
with supportive results from in vivo studies (Fig. 6 and Table II),
these findings provide direct evidence for intimate interactions
between stromal cells and early lymphoid progenitors during their
migration across the cortex, and further suggest that VCAM-1 and
4 integrins (respectively) are probably
responsible for this interaction. Although inducible deletion of VCAM-1
has been reported to have no obvious effect on the distribution of
thymic subsets (36), deletion was performed in the
immediate postnatal period, when the embryonic wave of thymic precursor
differentiation predominates (reviewed in Ref. 1). Thus, a
requirement for VCAM-1 in either the extravasation or transcortical
migration of progenitors in the steady state thymus may not be obvious
from such experiments. Furthermore, the overall size of the thymus in
gene-targeted animals was not reported, and consequently the results of
those studies may be completely consistent with those obtained by us
using in vivo Ab administration (Fig. 6 and Table II).
It should be noted that stromal cells in the thymic cortex are
scattered (Fig. 4), such that not every cortical lymphocyte (which are
mostly DP cells) can be in contact with a stromal cell at any one
time. Consequently, the finding that early progenitors do remain in
contact with stromal cells during their migration across the cortex may
reveal important principles about the differentiation process, as
follows. Stromal cells are generally believed to be responsible for
establishing the thymic microenvironment (reviewed in Refs.
37 and 38), and consequently, for inducing
the steady state production of mature T lymphocytes from uncommitted
progenitors. Our previous work has shown that although stromal cells
from various cortical regions may be morphologically indistinct, they
can be functionally differentiated, and establish a series of
stratified microenvironments in which distinct stages of early
precursor differentiation occur (19). Many of the signals
that induce cellular differentiation, including those known to be
important for intrathymic differentiation, such as Notch and
c-kit ligands (see above), exist as cell surface proteins.
Thus, successful differentiation of early lymphoid progenitors may
require direct, sequential interactions with stromal cell-expressed
ligands in each cortical region (for an example, see Ref.
22). Interstitial migration, i.e., migration along an ECM,
could leave many such interactions to chance, while migration along a
matrix of the very cells that generate these signals would ensure the
efficiency of required interactions. This does not mean that the
cortical stromal matrix exists merely to support DN migration and
differentiation, because DP cells undoubtedly have a requirement for
stromal cells as well. In fact, given that VCAM-1 expression reveals
clear heterogeneity among cytokeratin+ cortical
stroma (Fig. 3), it is conceivable that DN and DP cells may use quite
different stromal cell types, despite being present in the same
cortical regions. Likewise, the ECM plays a required role in the
differentiation, proliferation, and/or survival of lymphoid progenitors
(39), either by direct signaling to the progenitors
themselves, or through the organization of other stromal cells in their
corresponding microenvironments.
A requirement for cell migration in progenitor differentiation is not
unique to the steady state thymus. Of course, migration into and/or
away from different microenvironments is a well-known paradigm of
differentiation in the embryo. In addition, progenitor migration
between different extracellular microenvironments is an integral
component of steady state differentiation in a variety of postnatal
tissues, including the epidermis (40), intestinal crypts
(41), germ cells (42, 43), and even bone
marrow (44, 45). Frequently, the divergence of independent
cell fates, i.e., commitment to one lineage vs another, or self-renewal
vs differentiation, is intricately linked to the position of a given
stem/progenitor cell relative to other cells in its microenvironment.
It is likely that progenitors in the thymus undergo a similar process,
because they not only migrate, but make numerous cell fate choices as
well, including (for instance) divergence of / vs 
/
lineages, or the decision to differentiate or remain (albeit
temporarily) in an undifferentiated state (46). The
signals that regulate this asymmetry in the thymus are largely unknown.
However, it is worth noting that integrin expression analysis reveals
heterogeneity among otherwise homogenous DN1 and DN2 subsets, in which
many such cell fate decisions occur (see Fig. 1). It is interesting to
speculate that such changes in integrin expression may, in fact, be
linked to asymmetry of cell fates, as it is in differentiating
epidermis (see Ref. 40). However, not only are integrins
nonhomogenously expressed on DN cells, but their ligands are
nonhomogenously distributed in the thymus (this study, and Refs.
28, 29 and 47). Further structural and
biochemical mapping of specific regions in which asymmetric cell fate
decisions take place thus represents an important next step in
deciphering the signals for steady state T cell production in the
postnatal thymus.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA 17033.
3 Address correspondence and reprint requests to Dr. Howard T. Petrie, Memorial Sloan-Kettering Cancer Center, Box 341, 1275 York Avenue, New York, NY 10021. E-mail address: petrieh{at}mskcc.org
4 Abbreviations used in this paper: DN, double negative; DAPI, 4',6'-diamidino-2-phenylindole; DP, double positive; ECM, extracellular matrix; FN, fibronectin; LN, laminin.
Received for publication July 3, 2002. Accepted for publication August 13, 2002.
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References |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1 in fetal thymocyte commitment and differentiation. Science 268:1906.RII/III contains precursors of T lymphocytes and natural killer cells. Cell 69:139.[Medline]
or - lineages can occur just prior to the onset of CD4 and CD8 expression among immature thymocytes. Eur. J. Immunol. 22:2185.[Medline]
versus T cell lineage decision. Cell 88:833.[Medline]
gene recombination: dissociation from cell cycle regulation and developmental progression during T cell ontogeny. J. Exp. Med. 185:1549.. J. Exp. Med. 179:1495.2 integrin leukocyte function-associated antigen-1. J. Immunol. 157:5375.[Abstract]
41 (VLA4) integrin in T-cell development. Blood 89:2461.4 integrins during fetal and adult hematopoiesis. Cell 85:997.[Medline]
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 M. Drumea-Mirancea, J. T. Wessels, C. A. Muller, M. Essl, J. A. Eble, E. Tolosa, M. Koch, D. P. Reinhardt, M. Sixt, L. Sorokin, et al. Characterization of a conduit system containing laminin-5 in the human thymus: a potential transport system for small molecules J. Cell Sci., April 1, 2006; 119(7): 1396 - 1405. [Abstract] [Full Text] [PDF]
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S. Vielkind, M. Gallagher-Gambarelli, M. Gomez, H. J. Hinton, and D. A. Cantrell Integrin Regulation by RhoA in Thymocytes J. Immunol., July 1, 2005; 175(1): 350 - 357. [Abstract] [Full Text] [PDF]
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M.-L. Arcangeli, C. Lancrin, F. Lambolez, C. Cordier, E. Schneider, B. Rocha, and S. Ezine Extrathymic Hemopoietic Progenitors Committed to T Cell Differentiation in the Adult Mouse J. Immunol., February 15, 2005; 174(4): 1980 - 1988. [Abstract] [Full Text] [PDF]
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M. Zamisch, B. Moore-Scott, D.-m. Su, P. J. Lucas, N. Manley, and E. R. Richie Ontogeny and Regulation of IL-7-Expressing Thymic Epithelial Cells J. Immunol., January 1, 2005; 174(1): 60 - 67. [Abstract] [Full Text] [PDF]
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 A. Misslitz, O. Pabst, G. Hintzen, L. Ohl, E. Kremmer, H. T. Petrie, and R. Forster Thymic T Cell Development and Progenitor Localization Depend on CCR7 J. Exp. Med., August 16, 2004; 200(4): 481 - 491. [Abstract] [Full Text] [PDF]
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T. M. Schmitt, M. Ciofani, H. T. Petrie, and J. C. Zuniga-Pflucker Maintenance of T Cell Specification and Differentiation Requires Recurrent Notch Receptor-Ligand Interactions J. Exp. Med., August 16, 2004; 200(4): 469 - 479. [Abstract] [Full Text] [PDF]
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S. E. Prockop and H. T. Petrie Regulation of Thymus Size by Competition for Stromal Niches among Early T Cell Progenitors J. Immunol., August 1, 2004; 173(3): 1604 - 1611. [Abstract] [Full Text] [PDF]
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J. Plotkin, S. E. Prockop, A. Lepique, and H. T. Petrie Critical Role for CXCR4 Signaling in Progenitor Localization and T Cell Differentiation in the Postnatal Thymus J. Immunol., November 1, 2003; 171(9): 4521 - 4527. [Abstract] [Full Text] [PDF]
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H. E. Porritt, K. Gordon, and H. T. Petrie Kinetics of Steady-state Differentiation and Mapping of Intrathymic-signaling Environments by Stem Cell Transplantation in Nonirradiated Mice J. Exp. Med., September 15, 2003; 198(6): 957 - 962. [Abstract] [Full Text] [PDF]
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 R. F. de Pooter, S. K. Cho, J. R. Carlyle, and J. C. Zuniga-Pflucker In vitro generation of T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors Blood, September 1, 2003; 102(5): 1649 - 1653. [Abstract] [Full Text] [PDF]
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