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*
Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957; and
Department of Veterinary Sciences, University of Texas M. D. Anderson Cancer Center, Science Park-Veterinary Division, Bastrop, TX 78602
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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transgenic
and hCD3/cyclin D1 double transgenic mice remain blocked at the
K8+5+ maturation stage. However, enforced
cyclin D1 expression does expand the developmental window during which
K8+5+ cells can differentiate in response to
normal hemopoietic precursors. Thus, enhancement of thymic function may
be achieved by manipulating the growth and/or survival of TEC
precursors within the K8+5+
subset. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported that the normal adult murine thymus contains two
cortical TEC populations identified by keratin (K) expression patterns
(10). The major cortical subset expresses K8 and K18 but
fails to express K5 or K14. The minor cortical subset, which is highly
enriched at the corticomedullary junction, expresses K8, K18, and K5
but not K14. These latter cells comprise the predominant epithelial
subset in the poorly organized, primitive thymus that develops in mice
expressing a high copy number of a human CD3 (hCD3) transgene
that blocks thymocyte development at the
CD4-8-44+25-
precursor stage (11). In contrast,
Rag-1-/- mice, which sustain a later block in T
cell development at the
CD4-8-44-25+
stage, have a well-organized thymic cortex that consists predominantly
of
K8+K18+K5-K14-
TECs (11, 12). Reconstitution of newborn hCD3
transgenic thymi with Rag-1-/- thymocyte
precursors induces organization of the thymic cortex and development of
K8+K18+K5-K14-
TECs (10). These data suggest that cortical TECs
differentiate from
K8+K18+K5+K14-
precursors to
K8+K18+K5-K14-
progeny in a process dependent on T cell lineage commitment.
Interestingly, other investigators have shown that hCD3 transgenic
thymi are refractory to reconstitution with hemopoietic precursors
beyond the early neonatal period (13, 14). Attempts to
reconstitute adult hCD3 thymi with nontransgenic bone marrow cells
resulted in aberrant T cell development associated with the occurrence
of inflammatory bowel disease (IBD) (13). Therefore, the
developmental potential of TEC progenitors is apparently limited to a
critical time frame during which interactions with T lineage-committed
thymocytes must occur to promote TEC maturation.
In contrast to the severe thymic hypoplasia observed in hCD3
transgenic mice, expression of a cyclin D1 transgene targeted to
epithelial cells by a K5 promoter induces profound thymic hyperplasia
(15). The present report demonstrates that expression of
the cyclin D1 transgene in the
K8+K18+K5+K14-
progenitor TEC subset expands the entire TEC compartment and, as a
consequence, augments thymocyte development due to enhanced
availability of microenvironmental niches. The generation of
hCD3/cyclin D1 double transgenic mice revealed that enforced
expression of cyclin D1 is unable to overcome the arrest in cortical
epithelial development imposed by the T cell maturational block,
underscoring the importance of TEC-thymocyte interactions in thymic
histogenesis. Nevertheless, transplantation experiments revealed that
expression of the cyclin D1 transgene does extend the developmental
window during which the primitive hCD3 thymic epithelium can respond
to normal hemopoietic precursors and differentiate to form a
microenvironment that supports normal thymocyte differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J, hCD3 transgenic, RAG-1-/-,
and Nu/J mice were purchased from The Jackson Laboratory Animal
Resource Unit, Bar Harbor, ME. The cyclin D1 transgenic line was
produced as previously described (15).
Abs and lectin
Rabbit antisera specific for mouse K5 or K14 were developed as described by Roop et al. (16) and obtained from Covance Research (Richmond, CA). Troma-1, a mAb that recognizes K8, was kindly provided by Dr. Rolf Kemler (Max-Planck-Institut fur Immunbiologie, Freiburg, Germany) (17). Anti-CD4 (RM4-5) conjugated with PE, anti-CD8 (53-6.7) conjugated with FITC and biotinylated anti-CD3 (145-2C11), anti-CD69 (H1.2F3), anti-CD25 (7D4), anti-CD62L (MEL-14), and anti-CD44 (IM7) were obtained from PharMingen (San Diego, CA). Fluorochrome-conjugated anti-Ig second step reagents were purchased from Jackson ImmunoResearch (West Grove, PA). Binding of biotinylated Abs was detected by allophycocyanin-conjugated streptavidin (APC-SA, Molecular Probes, Eugene, OR). Biotinylated UEA-1 and FITC-conjugated streptavidin were obtained from Vector Laboratories (Burlingame, CA).
Immunohistology
Serial frozen sections (5 µm) from 6- to 9-wk-old mice were air dried for 30 min before acetone fixation. Thin sections were blocked with normal serum and, if necessary, an avidin-biotin blocking kit (Vector Laboratories) and subsequently incubated with optimal dilutions of primary Abs for at least 1 h at 25°C before washing and incubation with appropriate fluorochrome-conjugated secondary reagents. Controls included slides incubated with nonimmune species matched Ig or isotype-matched mouse Ig. For double staining, the sections were incubated simultaneously with primary Abs from different species. Microscopic analysis was performed with an Olympus ProVis AX70 microscope (Olympus, Melville, NY).
Flow cytometry
For three-color immunofluorescence analysis, cells in HBSS containing 1% BSA and 0.1% sodium azide were incubated with directly conjugated or biotinylated Abs on ice for 30 min followed by three washes. Binding of biotinylated Ab was detected with APC-SA. The cells were fixed in 1% paraformaldehyde before analysis. For determination of DN subsets, thymocytes were stained with a mixture of FITC-conjugated Abs to lineage markers CD4, CD8, CD3, B220, CD11b, and Gr-1 as well as with anti-CD44-PE and anti-CD25-biotin (PharMingen). After washing, the cells were incubated with APC-SA. FITC-negative cells were selected by electronic gating and analyzed for CD44 and CD25 expression. Cells were analyzed with a Coulter Epics Elite flow cytometer (Miami, FL) equipped with an argon laser (488 nm) for FITC and PE excitation and a helium-neon laser (633 nm) for APC-SA excitation. Data were collected on 10–20 x 103 viable cells (or 105 for DN analysis) using a four-decade log amplifier and were stored in list mode for subsequent analysis using Coulter Elite Software.
Transplantation of thymic grafts
Thymi from adult hCD3 transgenic or hCD3/cyclin D1 double
transgenic mice were grafted under the kidney capsule of anesthetized
adult nude recipients. A small incision was made in the peritoneal
cavity, and the left kidney was exposed. With an i.v. cannula, two
thymic lobes from individual donors were positioned under the kidney
capsule. The wound was closed with wound clips.
5-Bromo-2'-deoxyuridine (BrdU) and TUNEL labeling
Mice were injected i.p. with 1 mg BrdU in HBSS, and thymi were obtained 1 h later. To detect BrdU incorporation, deparaffinized thymic sections were incubated in 1 N HCl for 20 min at room temperature. After washing in 0.1% albumin, Tris-buffered saline, the sections were incubated with mouse anti-BrdU (B-D Sciences, San Jose, CA) for 1 h at room temperature followed by incubation with HRP-conjugated anti-mouse-IgG (Jackson ImmunoResearch), and the sections were developed with 3,3'-diaminobenzidine. To detect TUNEL-positive cells, 5-µm frozen sections were processed using an In Situ cell death detection kit (Boehringer Mannheim) per manufacturer’s instructions. Positive control slides were incubated in 10 µg/ml DNase (Sigma) in 4.2 mM MgCl2, 150 mM NaCl in H2O, pH 5.0, for 20 min at 37°C. Negative control slides were incubated in reaction mixture without TdT.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of a cyclin D1 transgene targeted to epithelial cells
by a K5 promoter induces a mild hyperplastic phenotype in the skin
(15). However, the prominent phenotype in transgenic mice
is severe thymic hyperplasia that is readily apparent by 4 weeks of age
and ultimately results in premature death by 5–6 mo due to
cardiorespiratory failure (15). Fig. 1
A shows that thymic weight in
cyclin D1 transgenic mice exceeds that of nontransgenic littermates by
4 wk of age and continues to increase with age. Histological analysis
revealed that gross thymic architecture is preserved in hyperplastic
cyclin D1 transgenic thymi (Fig. 1B). The transgenic thymi
contain well-organized cortical and medullary regions, although the
medullary areas appear somewhat dispersed compared with
nontransgenic thymi. Furthermore, both previously described
medullary subsets (i.e., stellate
K8-18-K5+K14+UEA-1-
and globular
K8+18+K5-K14-UEA-1+)
are present in transgenic as well as nontransgenic littermates (Fig. 1B and data not shown) (10). The cortex in
cyclin D1 transgenic and nontransgenic mice consists of a predominant
K8+18+K5-K14-UEA-1-
subset (hereafter referred to as
K8+5-). In normal mice, a
minor
K8+18+K5+K14-UEA-1-
subset (hereafter referred to as
K8+5+) is scattered
throughout the cortex and concentrated at the corticomedullary
junction. However, in cyclin D1 transgenic mice, there is a notable
expansion of the K8+5+ TEC
subset. Because TEC progenitors reside within the
K8+5+ subset
(10), the disproportionate expansion of
K8+5+ cells is likely to be
responsible for the profound thymic hyperplasia observed in cyclin D1
transgenic mice.
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Expansion of the thymic epithelial compartment in cyclin D1
transgenic mice supports an extensive increase in thymocyte cellularity
compared with nontransgenic littermates (Fig. 2A). However, the thymic
epithelial hyperplasia does not adversely affect intrathymic T cell
differentiation. As shown in Fig. 2B, there was a normal
distribution of the major thymocyte subsets defined by CD4 and CD8
coreceptor expression in cyclin D1 transgenic mice. Similarly,
transgene expression did not alter the percentage of
CD4-8- precursors defined
by CD25 and CD44 expression (Fig. 2C). Although the relative
frequency of thymocyte subsets in cyclin D1 transgenic mice was
apparently normal, the absolute number of thymocytes in each subset was
greatly increased compared with age-matched littermates (Tables
I and II).
Interestingly, the number of splenic T cells increased 
1.5 fold and
the T:B cell ratio was reversed in 8- to 12-wk-old cyclin D1 transgenic
mice (Table III). The minimal elevation
in splenic T cell number contrasts with the extensive (11-fold)
increase in thymocyte cellularity but is consistent with earlier
studies demonstrating the existence of a strict homeostatic mechanism
controlling the size of the peripheral T cell population (18, 19).
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, the cyclin D1 transgene did not
interfere with the enhanced positive selection of
CD4-8+ thymocytes
typically observed in H-Y TCR transgenic H-2Db
females (20). Furthermore, cyclin D1 transgenic thymi
support efficient negative selection. Thus, despite an increase in
thymocyte cellularity due to expression of the cyclin D1 transgene, the
majority of thymocytes in male H-Y TCR/cyclin D1 double transgenic
thymi express a CD4-8-
phenotype indicative of an active negative selection process (Fig. 3).
Taken together, these results demonstrate that the expanded TEC
compartment in cyclin D1 transgenic mice is capable of supporting
normal thymocyte development and selection.
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As a consequence of the hCD3 transgene-induced block in
thymocyte development, hCD3 transgenic mice contain severely
hypoplastic thymi with prominent cysts and a poorly organized
epithelium consisting predominantly of
K8+5+ TECs (10, 11). To determine whether enforced cyclin D1 expression would
reverse thymic hypoplasia and promote TEC differentiation, the cyclin
D1 transgene was crossed onto the hCD3 transgenic background. Gross
examination revealed that cyclin D1 expression induced only slight
enlargement of the thymus, although the hCD3/cyclin D1 double
transgenic thymi tended to be less cystic than age-matched hCD3
single transgenic thymi (data not shown). Fig. 4A shows that enforced cyclin
D1 expression induced an increase in the frequency of DNA-synthesizing
cells detected by BrdU incorporation. The increase in cycling cells was
accompanied by an increased frequency of apoptotic cells in double
transgenic thymi, suggesting that enforced expression of cyclin D1
couples cell cycle progression and cell death pathways (21, 22). Moreover, the cyclin D1 transgene failed to induce
substantial differentiation of the TEC compartment (Fig. 4B). Thus, in contrast to the
Rag-1-/- cortex which consists primarily of
K8+5- TECs, the
K8+5+ subset predominates
in both hCD3 and hCD3/cyclin D1 thymi. Importantly, thymocytes
from hCD3/cyclin D1 double transgenic mice remain blocked at the
CD4-8-44+25-
stage (data not shown) indicating that expression of the cyclin D1
transgene in TECs does not abrogate the T cell developmental block
imposed by the hCD3 transgene. Taken together, these data underscore
the fact that TEC differentiation from a
K8+5+ stage to a
K8+5- stage is dependent
on T cell lineage commitment and development beyond the
CD4-8-44+25-
stage (10).
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TECs expands the
developmental window for induction of normal thymic histogenesis
Previous studies have shown that reconstitution of hCD3
transgenic mice with nontransgenic bone marrow cells can restore thymic
architecture, intrathymic T cell maturation and peripheral T cell
function, but only if normal hemopoietic progenitors are transplanted
within a narrow developmental period (up to 8 days after birth)
(13, 14). Attempts to reconstitute adult hCD3
transgenic mice with normal bone marrow progenitors failed to restore
normal thymic architecture and resulted in aberrant intrathymic T cell
development, the accumulation of activated peripheral T cells and the
induction of IBD (13, 23). To determine whether expression
of the cyclin D1 transgene affects the developmental window within
which the hCD3 thymic epithelium is responsive to induction of
differentiation by normal hemopoietic progenitors, thymi from
adult hCD3 or hCD3/cyclin D1 transgenic mice were
transplanted under the kidney capsule of athymic nude recipients (Fig. 5A).
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thymic transplants
developed a wasting syndrome associated with severe diarrhea and
colonic enlargement typical of IBD (Fig. 5
B). None of these
mice survived beyond 12 wk after receiving a hCD3 thymic graft.
Histological analysis revealed marked alterations in the large
intestine including severe mucosal thickening, distorted crypt
architecture, infiltration of the mucosa and submucosa, with a mixed
inflammatory exudate and formation of abscesses and granulomatous
lesions (Fig. 5C). These changes were generally restricted
to the large intestine although inflammatory changes were also noted in
the small intestine of mice that developed IBD within 8 wk after
transplantation. In contrast, recipients of adult hCD3/cyclin D1
transgenic thymic grafts remained healthy with no overt symptoms of
IBD. Histological evaluation as late as 5 mo after transplantation
revealed minimal alteration in the gastrointestinal tract. There was no
apparent thickening of the mucosa, although moderate mononuclear
infiltration of the submucosa was noted in some animals. These minor
alterations were not restricted to the large intestine but were
observed throughout the entire gastrointestinal tract and seemed to
regress in older animals.
Fig. 6 shows that adult hCD3
transgenic thymic grafts recovered from nude recipients retained an
abnormal thymic architecture reflecting an aberrant epithelial
compartment composed predominantly of
K8+5+ TECs. Consistent with
previous reports, the hCD3 transgenic thymic microenvironment
generated peripheral T cells that displayed an activation phenotype.
Thus, as shown in Fig. 7A,
CD4+ lymph node T cells recovered from nude mice
8–12 wk after transplantation of hCD3 transgenic thymic grafts
expressed diminished levels of CD62L and elevated levels of CD44, CD25,
and CD69. In striking contrast, the hCD3/cyclin D1 thymi recovered
from nude recipients revealed restoration of apparently normal thymic
architecture, including clearly distinguishable cortical and medullary
regions containing numerous thymocytes (Fig. 6). Staining for keratin
expression demonstrated that, similar to the pattern in normal adult
thymus, the reconstituted adult hCD3/cyclin D1 thymic cortex
consisted predominantly of
K8+5- TECs, and the
medullary regions contained the previously described subsets
(10). Furthermore, generation of differentiated cortical
and medullary epithelia in the hCD3/cyclin D1 thymi was accompanied
by the development of normal relative frequencies of DN, DP, and SP
thymocyte subsets (Fig. 7B). Consistent with these findings,
Fig. 7A shows that the majority of peripheral T cells
recovered 5 mo after transplantation of adult hCD3/cyclin D1 thymi
expressed a naive rather than an activated phenotype.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although both cortical and medullary TECs were greatly expanded in cyclin D1 transgenic thymi, there was a disproportionate increase in the K8+5+ subset that is typically concentrated at the corticomedullary junction. The K8+K5+ population is likely to be a heterogeneous population consisting of epithelial cells at various maturation stages each of which may be differentially affected by enforced cyclin D1 expression. In the skin, the basal epithelial population contains stem cells, characterized by a high self-renewal capacity, and their immediate progeny, transit amplifying cells, that produce daughter keratinocytes which undergo terminal differentiation (24, 25, 26). Whereas epidermal stem cells divide infrequently in vivo, the transit amplifying subset contains a relatively high percentage of dividing cells. By analogy, the K8+K5+ TEC subset may consist of stem cell progenitors as well as transit amplifying-like cells that differentiate to produce cortical and medullary TECs. Expression of the cyclin D1 transgene in K8+K5+ TECs could expand the epithelial compartment by promoting stem cell proliferation and/or lowering a signaling threshold required for differentiation of stem cells to transit amplifying-like cells. Alternatively, or in addition, transit amplifying-like cells may undergo additional rounds of replication before differentiating or become refractory to growth-inhibitory signals in the cyclin D1 transgenic mice. Either scenario is consistent with the well-recognized role that cyclin D1 plays in regulating G1 progression in epithelial cells by activating cyclin-dependent kinases that phosphorylate retinoblastoma (Rb) family proteins (reviewed in Ref. 27). Cyclin D1 also can act as a transcriptional regulator independently of its ability to inactivate Rb. For example, cyclin D1 activates the estrogen receptor in a ligand-independent manner via recruitment of transcriptional coactivators (28).
Regardless of the mechanism that governs the disproportionate increase
in K8+K5+ TECs, it is
important to note that cyclin D1 transgene expression alone is not
sufficient to support the differentiation of
K8+K5+ precursors to
K8+K5- progeny. These data
support the notion that TEC differentiation requires inductive signals
from T cell lineage committed thymocytes. Thus, TECs from
hCD3/cyclin D1 double transgenic mice remain blocked at the
K8+K5+ developmental stage
resembling the predominant TEC subset in hCD3 transgenic mice
(10). TECs in the double transgenic mice fail to undergo
normal differentiation and expansion despite the increase in DNA
synthesizing cells that occurs as a consequence of cyclin D1
expression. This apparent dichotomy may be explained by the finding
that the cyclin D1 transgene also increases the fraction of apoptotic
cells in double transgenic thymi. Previous studies have shown that
overexpression of cyclin D1 can induce apoptosis in various cell types
(21, 22). Thus, during normal thymic development,
thymocytes and TEC interactions may regulate the TEC developmental
program by activating epithelial proliferation via a cyclin
D1-dependent pathway and simultaneously inducing intracellular signals
that block a potentially apoptotic pathway.
Although enforced cyclin D1 expression is not permissive for TEC
maturation when thymocyte development is blocked at the
CD4-8-44+25-
stage, the cyclin D1 transgene does expand the developmental window
during which K8+5+
progenitors respond to hemopoietic-derived signals that induce TEC
differentiation and normal thymic histogenesis. Transplantation of
adult hCD3 transgenic thymi under the kidney capsule of nude
recipients not only failed to restore TEC differentiation but also
resulted in the generation of aberrantly activated peripheral T cells
associated with development of fatal IBD. In contrast, adult
hCD3/cyclin D1 transgenic thymic transplants were capable of
supporting TEC development and thymocyte differentiation. Moreover, the
peripheral T cell compartment in recipients of adult hCD3/cyclin D1
thymic transplants did not display activation markers and the mice did
not succumb to IBD. It is not clear why induction of TEC
differentiation by hemopoietic precursors is restricted to a discrete
neonatal period in hCD3 transgenic mice. Perhaps the most primitive
TEC progenitors do not survive or fail to respond to differentiation
signals in the absence of sustained interactions with thymocyte
precursors. Enforced cyclin D1 expression beyond the neonatal period in
the K8+5+ subset may
maintain stem cell potential and/or responsiveness in the absence of
such interactions.
A dramatic augmentation of thymocyte production accompanied expression of the cyclin D1 transgene in the epithelial compartment. Enhanced thymocyte cellularity may be the result of highly efficient seeding of transgenic thymi by bone marrow-derived precursors due to greater availability of microenvironmental niches and/or elevated levels of TEC-derived chemoattractants (29). Consistent with either premise, there was an increase in the absolute number of CD4-8-44+25- thymocyte progenitors in cyclin D1 transgenic thymi. Alternatively, cyclin D1 expression in TECs may expand the CD4-8-44+25- progenitor subset by enhancing production of IL-7 and/or other cytokines that promote survival and proliferation of early thymocyte subsets (30, 31). In either case, the mutually inductive interactions that occur between the expanded epithelial and thymocyte compartments are likely to account for the profound thymic hyperplasia characteristic of the cyclin D1 transgenic mice.
Phenotypic and functional analyses revealed that T cell differentiation and selection processes are unperturbed in cyclin D1 transgenic thymi. Despite the overall increase in thymocyte cellularity, cyclin D1 transgenic thymi contain normal proportions of the four major subsets defined by CD4 and CD8 coreceptor expression. In addition, the relative distribution of DN subsets defined by CD44 and CD25 expression is equivalent in transgenic and nontransgenic littermates. These data indicate that thymocyte maturation follows a typical developmental sequence in cyclin D1 transgenic mice. Furthermore, the data obtained from H-Y TCR/cyclin D1 double transgenic mice demonstrated that the cyclin D1 transgene did not impair the capacity of the thymic microenvironment to support positive and negative selection. Thus, cyclin D1 transgenic mice are a potentially useful tool for studying T cell population dynamics and provide an intrathymic environment that not only promotes expansion of primitive hemopoietic precursors but also supports the selection processes that shape the TCR repertoire.
The involution of the thymus that occurs during aging is generally considered to be a causative factor in the decline of immune responsiveness in older individuals. Although a recent report demonstrated that the adult human thymus supports thymopoiesis to some degree and generates a relatively diverse TCR repertoire (4), thymocyte production and export are generally diminished as a function of age (5, 6, 7). Reduced thymic function as a consequence of involution in adult patients presents a further obstacle in attempting to reconstitute the peripheral T cell pool of patients after chemotherapy, radiation, or HIV infection. Because the cyclin D1 transgene prevents thymic involution, delineation of the signal transduction pathways induced by cyclin D1 expression in K8+5+ TECs may suggest strategies for enhancing thymic function in immune compromised individuals.
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Acknowledgments |
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Footnotes |
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2 D.B.K. and E.C. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Ellen R. Richie, University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957. E-mail address:
4 Abbreviations used in this paper: TEC, thymic epithelial cell; K, keratin; hCD3, human CD3; APC-SA, allophycocyanin-conjugated streptavidin; IBD, inflammatory bowel disease; BrdU, 5-bromo-2'-deoxyuridine; Rb, retinoblastoma.
Received for publication November 8, 1999. Accepted for publication December 9, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and nontransgenic (
) littermates.
B, Comparison of thymic architecture and TEC subsets in
cyclin D1 transgenic and nontransgenic (NT) mice. Serial 5-µm frozen
thymic sections were stained with hematoxylin-eosin (H&E) or incubated
with Abs specific for K8, K5, or K14 followed by appropriate
second-step reagents as described in Materials and
Methods. Note the increased frequency of cortical
K8+5+ TECs which appear yellow in the costained
section of a cyclin D1 transgenic thymus. Original magnification,
x100.
