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Department of Anatomy, Division of Immunity and Infection, Medical School, University of Birmingham, B15 2TT, Birmingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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-deficient mice, it is clear that the IL-7
receptor (IL-7R) plays a critical role during the initial stages of
intrathymic CD4-8- precursor development. In
contrast, the role of IL-7R in later stages of thymocyte development
are unclear. Here, we have used various approaches to investigate
directly the role of the IL-7R in thymocyte positive selection and the
recently described phase of postselection proliferation. First, we show
that positive selection involves selective up-regulation of IL-7R-
and IL-7R
-chains, with the majority of CD4+ and
CD8+ cells being IL-7R+. Second, MHC class
II+ thymic epithelium—which drives postselection
proliferation—expresses IL-7 mRNA. Finally, analysis of positive
selection and postselection proliferation in thymocytes from
IL-7R-/- neonates shows that positive selection occurs
normally, whereas postselection expansion is drastically reduced. Thus,
our data provide the first evidence that, as well as playing a role
during early phases of thymic development, IL-7R mediates intrathymic
expansion of positively selected thymocytes, which may aid in
establishment of the neonatal peripheral T cell
pool. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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ß form of the TCR are generated most efficiently in
the thymus, where immature precursors undergo a program of events
involving lineage commitment, somatic recombination of Ag receptor
genes, and cellular expansion to generate a large cohort of
CD4+8+ thymocytes bearing a
random repertoire of TCR specificities (1). Maturation of
thymocytes beyond the
CD4+8+ stage relies upon
the specificity of the TCRs expressed: cells bearing receptors with no
affinity for self peptide/MHC molecules die by neglect, whereas cells
bearing TCRs capable of peptide/MHC recognition are subject to two
distinct selection events. Thus, negative selection removes cells
bearing TCRs with a high affinity for peptide/MHC, whereas positive
selection rescues cells making low affinity TCR-peptide/MHC
interactions, and triggers their differentiation to the
CD4+8- and
CD4-8+ stages
(2, 3, 4). These cells then emigrate from the thymus to
generate the peripheral T cell pool, which recognizes peptides derived
from foreign Ags in a self-MHC restricted manner (5). It is becoming increasingly clear that positive selection is a multistage process, which includes phases of initiation, phenotypic differentiation, acquisition of functional competence, and postselection proliferation (6, 7, 8, 9, 10, 11). The initiation phase of positive selection, characterized by expression of the early activation marker CD69 on CD4+8+ thymocytes, is known to be strictly dependent upon ligation of the TCR complex by peptide/MHC complexes expressed by thymic epithelial cells (9, 12). In contrast, it is now clear that differentiation of CD4+8+69+ cells to the single-positive CD4+ and CD8+ stages and the subsequent postselection proliferation, although dependent upon the continued presence of thymic epithelium, does not require ongoing TCR ligation (9). In this study, we have focused on the molecular mechanisms mediating post positive selection proliferation, which may be an important intrathymic event allowing expansion of newly positively selected thymocytes, thereby aiding establishment of the neonatal peripheral T cell pool. We provide evidence that IL-7, a factor which has previously been shown to play a critical role in development and expansion of early CD4-8- thymocyte precursors (13, 14, 15), also plays a previously unidentified role in driving the Ag-independent proliferation of single-positive neonatal thymocytes following positive selection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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BALB/c (H-2d) mice, used as a source of
thymocytes and thymic stromal cells, together with C57BL/6
(H-2b) mice were bred and maintained at the
Biomedical Services Unit, Birmingham University. Neonatal
IL-7R-/- (C57BL/6,
H-2b) mice (16) were kindly provided
by Dr. Michael Owen (ICRF, Lincoln’s Inn Fields, London, U.K.).
Abs and immunoconjugates
The following Abs were coated onto anti-rat IgG or
streptavidin-coated Dynabeads (Dynal, Wirral, U.K.) as appropriate:
biotinylated anti-CD69 (clone H1.2f3; PharMingen, San Diego, CA),
anti-CD8 (clone YTS169.4; Sera-Lab, Crawley Down, Sussex, U.K.),
anti-CD3 (clone KT-3; Serotec, Oxford, U.K.). Abs used for flow
cytometric analysis are as follows: PE-conjugated anti-CD4 (GK1.5;
PharMingen), FITC or APC-conjugated anti-CD8 (clone 53-6.7;
PharMingen), anti-IL-7R (clone B12-1; PharMingen),
anti-common -chain (clone 4G3; PharMingen), anti-IL-2R
(clone 7D4), FITC-conjugated anti- 5-bromo-2'-deoxyuridine
(BrdU)3 (clone 3D4; PharMingen).
Unconjugated Abs were detected using sequential incubations in
biotinylated anti-rat IgG (Caltag, South San Francisco, CA) and
streptavidin APC (PharMingen).
Cell purification
Thymocytes. Methods to isolate thymocyte subsets at pre- and intermediate-stages of positive selection have been described in detail elsewhere (6, 7). Briefly, CD4+8+69+ cells were prepared by immunomagnetic selection from neonatal BALB/c mice by isolation of CD69+ cells and then further positive selection using anti-CD8-coated beads, a technique which yields a highly purified population of CD4+8+69+ cells (6). Preselection thymocytes were obtained by depleting newborn suspensions of CD3+ cells, followed by the isolation of CD4+8+ cells, again using anti-CD8-coated beads.
Thymic stromal cells. Enriched preparations of thymic epithelial cells were prepared as described (17), by culturing 15-day gestation BALB/c or C57BL/6 thymus lobes in the presence of 1.35 mM 2-deoxyguanosine for 5–7 days, followed by trypsinization to form a single-cell suspension (7).
Reaggregate thymus organ cultures (RTOCs)
RTOCs were prepared from 1:1 mixtures of freshly prepared thymic stromal cells and thymocytes by depositing the combined cell slurry onto the surface of a 0.8-µm Nuclepore (Costar, High Wycombe, U.K.) filter under organ culture conditions (18).
Analysis of post-positive selection expansion
To analyze thymocyte proliferation in RTOCs simultaneously with expression of CD4 and CD8, cultures were pulsed with 5 µg/ml BrdU (Sigma, St. Louis, MO) for the final 18 h of a 6-day culture period. RTOCs were subsequently teased apart and viable thymocytes counted. Suspensions were labeled for surface expression of CD4 and CD8, with BrdU incorporation being detected as described by Tough and Sprent (19).
PCR
Total RNA was extracted from 
5 x
105 cells using TRIzol (Life Technologies,
Paisley, Scotland) according to the manufacturer’s instructions. RNA
samples were treated with Rnase-free DNase I (Pharmacia Biotech,
Uppsala, Sweden) to remove any contaminating genomic DNA. Reverse
transcription was conducted according to Montgomery and Dallman
(20). RT-PCR was then performed as described
(21) with ß-actin as a housekeeping gene to obtain
equivalent amounts of cDNA in each sample. The sequences for ß-actin,
IL-7, and IL-15 oligonucleotides are as follows: ß-actin: sense,
5'-GTTACCAACTGGGACGACA-3'; anti-sense,
5'-TGGCCATCTCCTGCTCGAA-3'; IL-7: sense,
5'-ACTACACCCACCTCCCGCA-3'; anti-sense,
5'-TCTCAGTAGTCTCTTTAGG-3'; IL-15: sense,
5'-GAGGAATACATCCATCTCGTGC-3'; anti-sense, 3'-GCTCGCATGCAGTCAG
GAC-3'.
At the indicated cycles, samples were cooled to 4°C before removing 10-µl aliquots, to allow for semiquantitative analysis. PCR products were analyzed by ethidium bromide agarose gel electrophoresis and identified by fragment size.
Flow cytometry
Two- and three-color flow cytometric analyses were performed using a dual laser Coulter Epics Elite machine (Coulter, Hialeah, FL), with forward and side scatter gates set so as to exclude nonviable cells (6, 7).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Although the generation of single positive
CD4+ and CD8+ cells from
CD4+8+ precursors does not
initially involve cell division, a wave of proliferation that involves
multiple cell divisions has been observed in newly formed single
positive cells, particularly in the neonatal thymus (22, 23), which may serve in the expansion of the newly generated
neonatal repertoire before thymic export. Interestingly, this phase of
cellular proliferation is independent of signaling through the
ßTCR complex (7) but dependent upon thymic epithelial
cells (23), suggesting that this event is regulated by
additional but as yet undefined signals. Because cytokines are known to
be key molecules in both early thymocyte and mature T cell
proliferation (24, 25), we hypothesized that particular
cytokines may be involved in the regulation of this late phase of
intrathymic proliferation. Thus, we conducted an initial series of
experiments to analyze expression of various cytokine receptors and
associated molecules implicated in mediating signaling by these
receptors.
To focus on events following the initiation of positive selection, and
so to exclude possible confusion from cytokine receptor expression by
proliferating
ßTCR-CD4+8+
blast cells (26), we analyzed cytokine receptor expression
on freshly purified
CD4+8+69+
cells, which have been shown to represent developmental intermediates
in the positive selection process (23, 27). Analysis of
cytokine receptor expression on freshly isolated
CD4+8+69+
thymocytes revealed that the majority of cells did not express either
the common -chain (Fig. 1
a)
or the IL-7R-chain (Fig. 1c). Similarly, only a small
proportion of
CD4+8+69+
thymocytes were found to express the IL-2R-chain (Fig. 1b). Subsequent analysis of cytokine receptor expression
during maturation of
CD4+8+69+
cells was achieved by culturing these cells with thymic stromal cells
in RTOCs for 3 days, during which time cohorts of single positive
CD4+ and CD8+ thymocytes
are generated (Fig. 1d), which subsequently undergo a phase
of postselection proliferation (23). Although the cytokine
receptors tested were found to be absent from most freshly purified
CD4+8+69+
cells (Fig. 1, a–c), notable changes were observed in the
pattern of cytokine receptor expression on CD4+
and CD8+ cells generated from
CD4+8+69+
precursors in RTOCs. Thus, the vast majority of cells were found to
express the common -chain (CD132), a component of IL-2, IL-4, IL-7,
IL-9, and IL-15 cytokine receptors (Fig. 1, e and
h). Interestingly, whereas some CD132 partner chains such as
CD25 (IL-2R) were found to be absent from most
CD4+ and CD8+ thymocytes
(Fig. 1, f and i), the majority were found to
express the IL-7R-chain (CD127) (Fig. 1, g and
j). Notably, these cytokine receptors were absent from
virtually all thymocytes that remained at the
CD4+8+ stage after 3 days
in RTOC. Thus, further maturation of
CD4+8+69+
thymocytes to the CD4+ or
CD8+ stage appears to involve selective
up-regulation of particular cytokine receptors, including the CD132 and
CD127 components of the IL-7R complex.
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), we next analyzed cytokine gene
expression within this stromal cell compartment using semiquantitative
PCR analysis. Of the cytokines analyzed, MHC class
II+ thymic epithelial cells were found to express
readily detectable levels of mRNA encoding IL-7 (Fig. 2b) and IL-15 (Fig. 2c), but not IL-2, IL-4, or IL-6 (data not shown).
Interestingly, as well as pairing with CD132 to form the IL-7R complex,
the IL-7R-chain has also been shown to pair with another cytokine
receptor chain, the thymic stromal lymphopoietin (TSLP) receptor
(TSLP-R) (28). TSLP was originally identified as a product
of a thymic stromal cell line (29), and appears to have
both shared and unique functions, when compared with IL-7 (28, 30). However, whereas TSLP has been shown to play a role in
early stages of B cell development (29), unlike the clear
demonstration of the importance of IL-7 in
IL-7-/- mice, the role of TSLP in thymocyte
development is unclear.
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c-containing receptors,
our data do not allow direct analysis of the possible involvement of
individual cytokines in regulation of postselection expansion.
IL-7R-/- CD4+8+ thymocytes
show evidence of normal positive selection but severely impaired
post-selection proliferation
Thus, to directly investigate the requirement for specific
cytokines in positive selection and related postselection
proliferation, we next purified
CD4+8+ thymocytes from
neonatal mice deficient in expression of the IL-7R-chain, thereby
allowing direct discrimination between IL-7R and IL-15R involvement.
Although the role of IL-7/IL-7R interactions during early stages of T
cell development are well described (16, 31), analysis of
the functional importance of these molecules during positive selection
and subsequent postselection events is made difficult by the impaired
proliferation and differentiation observed in mice lacking either IL-7
or IL-7R. However, although adult
IL-7R-/- mice show a marked block in
thymocyte differentiation, predominantly at the
CD4-8- stage, some cells
do reach the CD4+8+ stage,
particularly in fetal and neonatal mice (31).
Thus, RTOCs were set up combining thymic stromal cells with either
wild-type (WT) or IL-7R-/-
CD4+8+ thymocytes, and,
after 5 days in culture, lobes were pulsed with BrdU to allow
investigation of proliferation following positive selection. Fig. 3 shows that cells recovered from RTOCs
initiated with either WT or IL-7R-/-
CD4+8+ thymocytes show
evidence of positive selection, with cohorts of
CD4+8- and
CD4-8+ cells observed in
both cultures (Fig. 3, a and b). Indeed, when
expressed as ratios (Table I), WT and
IL-7R-/-
CD4+8+ thymocytes were
found to generate similar proportions of
CD4+8- as compared with
CD4-8+ cells (2.8:1 WT,
2.2:1 IL-7R-/-). In contrast, cell
recoveries were reduced overall in IL-7R-/-
compared with WT cultures, and, in addition, analysis of BrdU
incorporation also revealed that at least part of this lower cell
recovery was due to differences in the extent of postselection cell
division. Thus, both
CD4+8-IL-7R-/-
and
CD4-8+IL-7R-/-
thymocytes showed considerable reductions in the proportion (Fig. 3, c–f), and absolute cell number (Fig. 4, a and b) of
proliferating (BrdU+) cells, as compared with WT
controls. Interestingly, proliferation of
CD4+8- cells appears to be
affected more than that of
CD4-8+ cells by lack of
IL-7R expression, with ratios of 1.9:1 WT and 13:1
IL-7R-/- for nondividing
CD4+8-:dividing
CD4+8- cells, and 0.8:1 WT
and 3.5:1 IL-7R-/- for nondividing
CD4-8+: dividing
CD4-8+ cells (Table I).
Thus, these data suggest that, although positive selection to the
CD4+8- and
CD4-8+ stage occurs
similarly in both WT and IL-7R-/-
thymocytes, the phase of postpositive selection expansion operating on
these newly selected thymocytes is dramatically impaired in
IL-7R-/- thymocytes, and so highlights a
previously unidentified role for the IL-7R in mediating expansion
during later stages of thymocyte development. However, as mentioned
previously, as both IL-7 and TSLP can utilize surface receptors
containing the IL-7R-chain (28), it may be the case
that both of these cytokines can play a role in this late stage of
thymocyte expansion.
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Recently, we and others have identified a phase of post-positive
selection proliferation operating on both
CD4+8- and
CD4-8+ thymocytes, which
appears to be thymic epithelial cell-dependent but independent of
TCR-mediated signaling, because it occurs in the presence of
MHC-deficient thymic stromal cells (7). Moreover, as
compared with adult thymocytes, postselection proliferation of mature
thymocytes is far more evident in neonatal mice (22, 23),
where it may act as a mechanism to expand the newly selected neonatal T
cell repertoire, thereby aiding in the establishment of the neonatal
peripheral T cell pool. Here, we have investigated the mechanism of
postselection expansion and provide direct evidence that the IL-7R
complex plays a key role in this phase of thymocyte differentiation.
First, we have shown that transition from the
CD4+8+69+
intermediate stage to the
CD4+8- and
CD4-8+ stages involves
selective up-regulation of both the - and -chains of the IL-7R
complex, suggesting that, following the generation of single positive
thymocytes by positive selection, these cells may become receptive to
IL-7/IL-7R-mediated signals. Second, by directly comparing positive
selection and subsequent postselection expansion in WT and
IL-7R-/- thymocytes, we show that lack of
expression of the IL-7R complex, although not affecting the
differentiation of CD4+8+
into CD4+8- and
CD4-8+ cells, results in a
dramatic reduction in post-positive selection proliferation.
Collectively then, whereas the cells which regulate postselection
expansion—MHC class II+ thymic epithelial
cells—are known to express a variety of cytokines (21),
including IL-15 as shown here, our data provide direct evidence for an
essential requirement for IL-7/IL-7R interactions during post-positive
selection expansion in the neonatal thymus.
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Acknowledgments |
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-/- mice, Sonia Parnell for RT-PCR analysis, and
Katharine Partington for expert technical assistance. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Graham Anderson, Department of Anatomy, Division of Immunity and Infection, Medical School, Vincent Drive, University of Birmingham, B15 2TT, Birmingham, U.K.
3 Abbreviations used in this paper: BrdU, 5-bromo 2'-deoxyuridine; RTOC, reaggregate thymic organ culture; TSLP, thymic stromal lymphopoietin; WT, wild type.
Received for publication March 20, 2000. Accepted for publication June 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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