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-Chain Cytoplasmic Tail1
Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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cytoplasmic domains by transgenic expression of wild-type or
cytoplasmic deletion mutants of IL-7R in the thymi of
IL-7R-/- mice. We show an essential requirement for
the tyrosine-containing carboxyl-terminal T domain in restoring thymic
cellularity, pro-/pre-T cell progression, and survival. In contrast,
the functional differentiation of TCR 
cells and the development
of TCR 
cells are partially independent of the T domain. Thus,
separate cytoplasmic domains of the IL-7R chain differentially
control distinct functions during T cell development, whereas normal
IL-7R-dependent thymic development requires the integrated activity of
all these domains. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and the common -chain
(c),3
is required for normal T cell and B cell development (1, 2) and is essential for the production of TCR lineage
cells (3, 4). The IL-7/IL-7R interaction functions to
stimulate the proliferation of developing thymocytes (5),
confer resistance of pro- and pre-T cells to apoptosis
(6, 7, 8), promote pro-T cell progression
(9, 10), and induce rearrangement and transcription of TCR
-chain genes (11, 12). The IL-7R initiates multiple
signaling pathways through several nonreceptor kinases that associate
with the cytoplasmic tail of the IL-7R chain. Three IL-7R
domains, A (acidic), S (serine-containing), and T
(tyrosine-containing), serve as potential docking regions for these
kinases (Refs. 13 and 14 , and see Fig. 1
A). IL-7 activates c-associated
JAK3 and IL-7R-associated JAK1, presumably via the S region
(14, 15), leading to the activation of primarily STAT5
and, to a lesser extent, STAT1 and STAT3. STAT5 directly associates
with phosphotyrosines in the T domain of IL-7R (14, 16, 17). IL-7 also induces the activation of phosphatidylinositol
3-kinase (PI3-kinase), which is dependent on the
phosphorylation of tyrosine 449 in the T region of IL-7R (18, 19). In addition, triggering the IL-7R in pre-B cell and some
T cell lines leads to the activation of the src family
tyrosine kinases p56lck and
p59fyn, which associate with the A region of
IL-7R (20, 21, 22).
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cell differentiation
appears to be partially dependent on IL-7R-induced STAT5, because
expression of constitutively activated STAT5 in
IL-7R-/- precursors led to the appearance of
TCR cells in thymic organ culture (23). IL-7R T
region-associated PI3-kinase and STAT5 activities
have also been implicated in promoting thymic cellularity and survival
signals of developing T cells (24). However, in all these
cases, examination of IL-7R signaling domains and pathways has relied
on assessment of their activity in vitro. In turn, the contribution of
IL-7R signaling to T cell development may have been over- or
underestimated through the use of constitutively active signaling
proteins and/or fetal thymic organ culture to characterize the
development of human precursor cells. In contrast, the present study
directly characterizes the contribution of individual IL-7R
cytoplasmic subdomains to T cell development in vivo. This issue was
addressed by transgenic expression of wild-type (WT) and cytoplasmic
deletion mutants of IL-7R in the thymi of
IL-7R-/- mice. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Full-length WT IL-7R cDNA was prepared by RT-PCR using total
RNA from the 70Z pre-B cell line and cloned into the BamHI
cloning site of the p1017 vector, which places the cDNA under the
control of the proximal lck promoter
(25), using BglII restriction sites
incorporated into both the forward
(5'-TCCAGATCTAGAATGATGGCTCTGGGTAGAGCTTTC) and reverse
(5'-TCCAGATCTTCATTTGTTTTGGTAAAAACTAGA) IL-7R PCR
primers. Mutants lacking the T and S regions (
ST) or
only the T region (T) of the IL-7R cytoplasmic tail (see Fig. 1
A) were prepared by introducing stop codons using the
Quickchange site-directed mutagenesis kit (Stratagene, San Diego, CA)
according to the manufacturer’s instructions, with WT IL-7R in
p1017 as template DNA. The forward mutagenic oligonucleotides were
5'-TCAGTCAGCCCACCATAAACAGTTAGAAGAGAGTCACC for ST and
5'-TACAGAGATGGTGACTGAAATAGGCCTCCTGTG for T. All constructs were
verified by DNA sequence analysis.
Mice
To produce transgenic mice, expression cassettes containing WT
or mutated IL-7R constructs in p1017 were microinjected into
1-day-old embryos of (B6 x SJL)F2 mice (The
Jackson Laboratory, Bar Harbor, ME), which were then transplanted into
pseudopregnant (B6 x DBA)F2 mice.
Transgenic founders were identified by Southern blot analysis of
BamHI-digested genomic DNA using a full-length coding
32P-labeled IL-7R cDNA probe and were
backcrossed for three to five generations to
IL-7R-/- mice on a predominantly C57BL/6
genetic background (back-crossed for five generations to C57BL/6 mice).
In some experiments, C57BL/6 mice were used as normal control animals.
All animals were housed under virus Ag-free conditions. Animal
experiments were approved by the University of Miami animal care and
use committee.
Reagents and Abs
Complete medium was RPMI 1640 supplemented with 10% FCS, 100
U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME. FITC-CD4
(GK1.5), -CD8 (53-6.7), -CD25 (7D4), -CD44 (IM7), biotinylated CD8,
and -CD44 were prepared in our laboratory. FITC-CD43 (S7), CyChrome
(CY)-CD3
(145-2C11), -CD4, -CD8, biotinylated B220 (RA3-6B2),
-TCR (GL3), PE-streptavidin, CY-streptavidin, PE-annexin V, and
mAb specific for bcl-2 (3F11) and TNP (A19-3) were purchased from
PharMingen (San Diego, CA). Polyclonal rabbit anti-mouse bax (P-19)
was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Purified
mAbs specific for IL-2R (5H4) (26) and IL-7R (A7R34;
kindly provided by S. Nishikawa, Kyoto University, Kyoto, Japan)
(27) have been described previously. For IL-7-binding
studies, binding medium consisted of RPMI 1640 with 2.5% FCS, 15 mM
HEPES, and sodium azide (1 mg/ml).
Flow cytometry
To detect IL-2R and IL-7R expression, three-step staining
was done using mouse-adsorbed biotinylated-anti-rat IgG
(PharMingen) revealed with PE- or CY-streptavidin after blocking with
whole rat Ig (Pierce, Rockford, IL) to prevent cross-reactivity with
other surface stains. In some experiments, following surface staining,
intracellular staining for bcl-2 and bax was performed as described
(8). Cells were fixed for 45 min at 25°C in 1 ml 2%
paraformaldehyde solution and permeabilized in PBS with saponin (5
mg/ml) before staining with anti-bcl-2, anti-bax, anti-TNP,
or whole rabbit Ig for 30 min at 25°C. A mixture of
FITC-anti-hamster IgG clones (G70-204 and G94-56; PharMingen) was
used to visualize anti-bcl-2 and anti-TNP, while
human/mouse/rat-adsorbed polyclonal goat anti-rabbit Ig
(PharMingen) was used for anti-bax and rabbit Ig control-stained
cells. Forward and side scatter axes and propidium iodide exclusion
were used to set live lymphocyte gates from whole-cell preparations.
Between 5 and 10 x 103 gated events were
collected per sample on a Becton Dickinson FACScan (Becton Dickinson,
San Jose, CA), and results were analyzed using CellQuest software
(Becton Dickinson). WT C57BL/6 mice or nontransgenic littermates were
analyzed for comparison.
IL-7-binding
Thymocytes (2.5–10 x 106) in 100 µl of binding medium were incubated in 100 µl of 1 nM 125I-labeled IL-7 for 1 h at 4°C with constant mixing. In some cases, the cells were pretreated with 200 nM unlabeled IL-7 for 15 min at 4°C before adding the 125I-IL-7. Cells were washed two times in binding medium before measuring cell-associated radioactivity with a gamma counter. Bound counts from competitively inhibited samples were subtracted from noncompeted sample counts to determine specific IL-7-binding.
Lymphocyte proliferation
Whole thymocytes (5 x 105/well) were
cultured in triplicate in 200 µl of complete medium with
anti-CD3 mAb (5 µg/ml), IL-2 (50 U/ml), or IL-7 (30 ng/ml) in
96-well plates for 48 h. Wells were then pulsed with 1 µCi of
[3H]thymidine, and cells were harvested 6
h later with a Tomtec 96 automated cell harvester (Wallac,
Gaithersburg, MD). Radioactivity was then quantified using a Betaplate
1205 beta counter (Wallac). Replicate values did not differ by >10%
in individual experiments.
Apoptosis of triple-negative (TN) cells
To determine cell surface levels of the early apoptosis marker
phosphatidylserine in stage 2–3 pro-T cells, thymocytes were stained
ex vivo or after 6 h of culture in media at 37°C in 7%
CO2-humidified air. Following surface staining
with CY-CD3, -CD4, -CD8, and FITC-CD25, cells were stained with
PE-annexin V in staining buffer containing 2.5 mM
CaCl2.
TCR -chain gene rearrangements
Genomic DNA from unfractionated thymocytes was used in PCR with
primers to V (V1.2, 5'-CTTCCATATTTCTCCAACACAGC; V2,
5'-AAGGAGTACAAGAAAATGGAGGCAAGT) or J (J1,
5'-CGGGATCCCAGAGGGAATTACTATGAGC; J2,
5'-ACTATGAGCTTTGTTCCTTCTG) gene segments, respectively, to amplify
DNA across rearranged TCR gene sequences. Reactions consisted of 30
cycles of 30 s at 94°C, 1 min at 55°C, and 90 s at
72°C. PCR products were visualized in 2% agarose gels with ethidium
bromide staining. Nonrearranged TCR -chain DNA sequences failed to
produce PCR products due to the large distances between forward and
reverse primers. Primers to IL-2R-chain gene (forward,
5'-CAAGGTCTCTCACTACATTG; reverse, 5'-TGGCCTTGTCCGAAAGGTCA) were
used as a control in PCR with equivalent amounts of DNA for 27 cycles
of 30 s at 94°C, 1 min at 60°C, and 90 s at 72°C.
Statistical analysis
The Statistical Package for the Social Sciences v. 8.0 was used to conduct parametric comparative analyses using the t test for independent samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-transgenic mice
We developed a series of IL-7R-transgenic mice regulated by the
proximal lck promoter, which preferentially targets
transgene expression to the thymus (25, 28, 29). In
addition to a WT construct, two mutated forms of IL-7R were
generated, such that the expressed protein lacked either the T domain
(T) or both the T and S domains (ST) (Fig. 1A). Two transgenic founders
for each IL-7R construct were identified and backcrossed to
IL-7R-/- mice for three or more generations
to obtain progeny mice for the analyses performed in this study. Thymic
transgene expression and function of each founder pair were essentially
identical. All founders on either the IL-7R+/+
or IL-7R-/- genetic backgrounds were
healthy, long-lived, and vigorous breeders. No phenotypic differences
were observed in any of these transgenic mice on
IL-7R+/+ or IL-7R+/-
genetic backgrounds, indicating that transgenic IL-7R expression did
not obviously influence T cell development. Data presented for
transgenic mice in all subsequent analyses represent transgenic mice on
an IL-7R-/- background, which for simplicity
are referred to as WT, T, and ST mice.
CD4/CD8 subset distribution of thymocytes from all the transgenic lines
was generally comparable to IL-7R+/+ and
IL-7R-/- mice (Table I, and Fig. 1B). When compared
with background IL-7R staining using control
IL-7R-/- thymocytes, the transgenic IL-7R
chain was detected on all thymic subsets (Fig. 1B).
Importantly, IL-7R expression in the CD4-
CD8- double-negative (DN) thymocyte compartment
of IL-7R-/- mice was largely restored by the
transgene (Fig. 1B). In addition, transgenic IL-7R was
also expressed on CD4+ CD8+
double-positive (DP) thymocytes, on which it is normally
down-regulated, and was generally higher on CD8+
single-positive (SP) thymocytes when compared with thymocytes from
normal mice. 125I-labeled ligand-binding studies
revealed that whole thymocytes from all transgenic lines specifically
bound IL-7 at equal or greater levels than those detected for cells
from normal mice (Fig. 1C), confirming functional expression
of transgenic IL-7R protein.
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expression was assessed on
subsets of
CD3-CD4-CD8-
TN thymocytes (Fig. 2A). TN T
cell precursors may be progressively staged based on their
expression of CD25 (IL-2R) and CD44 (pgp-1) as follows: stage 1
(CD44+CD25-), stage 2
(CD44+CD25+), stage 3
(CD44-CD25+), and stage 4
(CD44-CD25-)
(30). Both transgenic and endogenous IL-7R were
expressed on each of these TN subsets, albeit at different levels. The
expression of T- and ST-transgenic IL-7R on TN cells was
generally equal to or higher than endogenous IL-7R expression in
normal mice, while the expression of WT transgenic IL-7R on stage
1–3 TN cells was reduced when compared with nontransgenic normal mice
(Fig. 2A).
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expression in all transgenic lines was limited to T cells and
thymocytes, with no detectable expression on B cells, NK cells, or
monocytes (data not shown). As expected, the proximal lck promoter did
not mediate WT IL-7R expression in bone marrow lymphocytes (Fig. 2B) and did not restore
CD43-B220+ B lineage cells
in IL-7R-/- mice (Fig. 2C), thus
indicating an absence of functional IL-7R signaling in the bone marrow
of transgenic mice.
Normal T lymphocyte cellularity requires signaling from the
IL-7R T domain
Thymic cellularity of WT IL-7R-transgenic mice on the
IL-7R-/- background was largely restored and
on average was 74% that of nontransgenic normal littermate mice (Table I, and Fig. 3A). This somewhat
suboptimal restoration of thymic cellularity correlates with the
aforementioned lower level of WT IL-7R transgene expression on stage
1–3 pro-T cells. In contrast, the highly expressed T and ST
IL-7R transgenes both failed to restore thymocyte numbers
substantially, although the cellularity of T and ST mice was
somewhat greater than that of IL-7R-/- mice,
but not statistically different. Although transgenic IL-7R
expression was evident throughout thymic development (see Figs. 1B and 2A), reconstitution of thymic cellularity
was directly related to the absolute number of TN cells, indicating
that the biological consequences of transgene expression occurred at
the level of the pro-/pre-T cell.
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B) and CD8 (Fig. 3C) T cells revealed that with time, T cell numbers in WT
transgenic mice generally accumulated to levels equivalent to those of
normal mice. When compared with IL-7R-/-
mice, splenic T cells of mutant IL-7R-transgenic mice were detected
at higher numbers, a trend that was particularly evident in
CD4+ splenic T cells from older (>8 wk) T
mice. Thus, these data demonstrate a critical role for the cytoplasmic
T region of IL-7R in the production of thymocytes, although the
slight increases in thymic and peripheral T cell numbers in T and
ST mice suggest a minimal degree of signaling from the remaining
portions of IL-7R.
The IL-7R T domain mediates signals for survival and progression
of pro-/pre-T cells
To better understand the mechanism underlying the near failure of
the T- and ST-transgenic lines to promote T cell development and
the somewhat suboptimal repopulation of the thymus by WT transgenic
mice, we explored the survival characteristics of TN cells from these
mice. Attention was focused on stage 2–3 pro-T cells that actively
rearrange TCR - and -chains, because previous work demonstrated
that IL-7R signaling is especially critical for these cells
(9, 10, 11, 12). Commitment to apoptosis was examined ex vivo by
staining these cells with PE-annexin. When compared with cells from
normal mice, stage 2–3 pro-T cells from both T
(p = 0.001) and ST
(p < 0.001) mice displayed a significantly
increased fraction of PE-annexin+ cells, while
the fraction from WT transgenic mice was only slightly higher and did
not statistically differ from normal mice (Fig. 4A). In these analyses, the
PE-annexin dull to intermediate cells excluded the viability dye
7-amino-actinomycin D, and the PE-annexin bright cells also stained
with 7-amino-actinomycin D, a pattern typical of cells undergoing
apoptosis (data not shown). These data indicate that in the context of
the thymic microenvironment, stage 2–3 pro-T cells from WT transgenic
mice display near-normal survival characteristics, whereas cells from
T and ST mice exhibit enhanced apoptosis despite their
higher-than-normal expression of transgenic IL-7R.
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-/- mice (Fig. 4B). Notably,
cultured pro-T cells from WT transgenic mice now demonstrated
significantly greater susceptibility to apoptosis than cells from
normal mice (p = 0.02). This finding suggests
some impairment in survival signals in pro-T cells from WT
transgenic mice.
To explore the molecular basis for these differences in apoptosis, we
examined expression levels of anti-apoptotic bcl-2 and
pro-apoptotic bax molecules in these thymic precursors. Surprisingly,
in addition to a lack of bcl-2 expression in pro-T cells from T and
ST mice, as in IL-7R-/- mice, bcl-2 was
also not detected in the DN precursor cells of WT mice (Fig. 4C). In contrast, bax was present at nearly equivalent
levels in all groups (Fig. 4D). These data imply that a lack
of IL-7-mediated bcl-2 expression leads to enhanced apoptosis of pro-T
cells in vitro. However, other anti-apoptotic signals may also be
transmitted through the T region of IL-7R that promote the
near-normal survival of pro-T cells in WT transgenic mice when examined
ex vivo.
With respect to promoting pro-/pre-T cell progression, only cells from
WT transgenic mice exhibited a pattern of TN cell progression based on
CD44 and CD25 expression identical with that of normal mice. By
contrast, the pattern of TN cells from T and ST mice was
essentially identical with that seen in
IL-7R-/- mice, with an accumulation of cells
at the stage 2–3 transition and a relatively higher percentage of
cells at stage 1 (Fig. 4E). When compared with TN cells from
+/- littermates or WT mice, TN cells from T, ST, and
IL-7R-/- mice typically contained a 3- to
4-fold higher proportion of stage 2
(CD44+CD25+) pro-T cells,
where TCR rearrangements commence. These data further illustrate the
marked impairment in thymic development by T and ST
mice.
Functional maturation of TCR thymocytes requires IL-7R
signaling, but is partially independent of the T region
Mature peripheral T cells in IL-7R-/-
mice have been shown to be hyporesponsive upon activation through their
TCR (31). We evaluated whether this was due to a potential
failure of thymic T cell maturation by examining the capacity of mature
thymocytes to proliferate to soluble anti-CD3 in the presence of
IL-2 or IL-7. These cytokines are expected to costimulate the
proliferation only of mature thymocytes activated with anti-CD3 mAb
(32, 33, 34). The proliferative response of thymocytes from
nontransgenic normal mice to soluble anti-CD3 was always
suboptimal, but the addition of exogenous IL-2 or IL-7 resulted in
potent costimulation (Fig. 5). Similar
responses were generated by thymocytes from WT transgenic mice,
although the response costimulated by IL-7 was somewhat lower when
compared with normal littermates. However, thymocytes from
IL-7R-/- and ST mice failed to respond to
soluble anti-CD3 and IL-2 or IL-7 (Fig. 5). T mice consistently
responded suboptimally to anti-CD3 and IL-2, but did not
proliferate to anti-CD3 and IL-7. Importantly, these low responses
cannot simply be attributed to reduced thymocyte recovery from these
mice, because the cell number in culture was equivalent for each group
and the CD4/CD8 thymic subset composition was within normal range for
all mice (see Fig. 1B). Therefore, these data indicate that
another role for IL-7R signaling during thymic development is the
maturation of functional T cell responses, which is partially
independent of the T region.
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cells and TCR -chain rearrangement are
partially independent of the T region
The development of TCR lineage cells is strictly dependent
upon IL-7R function (3, 4, 12). As expected, a readily
detectable fraction of DN cells from WT transgenic mice differentiated
into TCR cells, approaching the number seen in normal mice (Fig. 6A). Although variability
between individual mice was high, a significantly greater fraction of
DN cells from T mice expressed TCR (p
= 0.004) in comparison with IL-7R-/- mice,
whereas the number observed in ST mice did not statistically differ
from their gene-deficient littermates (Fig. 6A). Although
the absolute number of TCR cells in T mice was substantially
reduced when compared with normal and WT transgenic mice due to their
low thymic cellularity (see Fig. 3A), the data indicated
that a substantial fraction of DN thymocytes in T mice
differentiated into TCR cells in the absence of IL-7R signaling
associated with the cytoplasmic T region.
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cell development, we assessed
the extent of somatic gene recombination of the TCR -chain locus in
whole thymocyte DNA. TCR -chain V-J gene rearrangement occurs in
CD25+ thymic pro-T cells at the stage 2–3
transition, and IL-7R-mediated signaling has been shown to play a
critical role in this process (23, 35, 36, 37). Using PCR
primers against two specific V and downstream J gene segments,
TCR -chain V-J gene rearrangements were readily detectable in thymic
DNA from both WT and mutant T-transgenic mice, and to a lesser
extent in ST mice (Fig. 6B). These results contrasted
with the absence of TCR -chain V-J rearrangements in
IL-7R-/- mice. Amplification of the
nonrearranged IL-2R gene revealed a similar amount of PCR product
among the groups, indicating that all PCR at the respective dilutions
contained a similar amount of template DNA. Densitometric analysis of
these and other experiments indicated that the extent of TCR -chain
rearrangements by T thymocytes was 
3- to 5-fold lower than that
seen in normal or WT thymocytes, but 20-fold greater than that
detected in thymocytes from IL-7R-/- mice.
Collectively, these findings indicate that production of TCR
lineage cells and TCR -chain rearrangements show a greater
independence of the IL-7R T region than other functions mediated by
the IL-7R. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T region-associated signaling pathways are required for
the survival and progression of pro-/pre-T cells and the production of
normal numbers of thymocytes, whereas T region signaling contributes to
the maturation of functional responsiveness to cytokines and the
development of TCR lineage cells. When compared with normal
littermate and WT transgenic mice, the overall thymic cellularity as
well as the survival characteristics and progression of pro-/pre-T
cells were similarly impaired in T and ST mice and nearly
comparable to IL-7R-/- mice. Our findings
indicated that these aspects of T cell development must have input from
signaling associated with the T region of IL-7R. Both
PI3-kinase and STAT5 have been shown to signal
through the T region of the IL-7R in vitro (14, 38). Based
on the ability to reconstitute mouse fetal thymic organ culture with
human precursor cells transduced with constitutively active STAT5,
PI3-kinase, or chimeric mutant IL-7R
constructs (24), as well as mouse precursors transduced
with STAT5-inducible PIM1 (39), it has been suggested that
these signaling proteins function critically as direct targets
physiologically regulated by IL-7R during thymic development. Our data
are thus consistent with important roles for STAT5,
PI3-kinase, and any other T region-associated
signaling molecule for IL-7R-dependent thymic T cell development.
It has been postulated that the primary role of IL-7 in T cell
development is as a survival factor to maintain bcl-2 levels.
Consistent with this idea, IL-7 has been shown to induce increased
bcl-2 levels in murine thymocytes (8). However, with the
exception of one study (7), transgenic bcl-2 expression in
IL-7-/-, IL-7R-/-,
or c-/- mice only partially
restored (
30%) thymic cellularity (6, 23, 37, 38, 40, 41). Our analysis of transgenic IL-7R-deficient WT mice is
consistent with the notion that IL-7R stimulation of cell survival and
bcl-2 expression during pro-/pre-T cell development represents an
important but less critical component of IL-7R signaling in thymic
development. In contrast to normal C57BL/6 mice, bcl-2 expression was
not evident in stage 2–3 pro-T cells from WT transgenic mice.
Moreover, WT pro-T cells were more susceptible to apoptosis upon
culture in media for 6 h, suggesting that other anti-apoptotic
molecules were not redundantly functioning to promote the survival of
these pro-T cells in vitro. Whether other anti-apoptotic molecules
are controlled by IL-7R signaling in vivo remains to be determined.
Despite this failure in pro-/pre-T cell survival, WT transgenic mice
exhibited near-normal thymic cellularity (75%) and pro-/pre-T cell
progression. Thus, these findings suggest that the dominant function of
IL-7R signaling is T lineage progression, with a more modest
contribution to cell survival and growth.
It is unclear why the WT IL-7R transgene did not reconstitute
bcl-2-mediated survival upon expression in the thymi of
IL-7R-/- mice. The simplest explanation is
that the WT transgene did not entirely recapitulate the normal pattern
of IL-7R expression in lymphoid progenitor cells. When compared with
cells from normal mice, the WT IL-7R transgene was not detected in the
bone marrow, and its expression was substantially lower on early pro-T
cells, especially at stage 1. This pattern of transgenic IL-7R
expression appears to be typical for transgenes under the control of
the proximal lck promoter (42). Thus, the slightly
suboptimal thymic cellularity in WT mice may be the result of absent or
suboptimal IL-7R signaling at the level of the common lymphoid
progenitor, which is normally marked by IL-7R expression
(43), or stage 1 pro-T cells. Alternatively, it cannot be
ruled out that the impaired survival and proliferation of WT TN cells
is the result of selective suboptimal IL-7R signaling at all stages of
TN development, resulting from somewhat lower expression levels.
Although still somewhat suboptimal, TCR rearrangements, TCR
expression, and proliferation to anti-CD3 and IL-2 were readily
detected in unfractionated thymocytes from T mice, while these
functions were undetected or minimal in cells obtained from
IL-7R-/- or ST mice. These results indicate
that T region-associated signaling is partially dispensable for these
IL-7R-mediated functions and suggest that S region-associated
signaling primarily induced these activities. It has been suggested
that JAK1 associates with the S region (14) and represents
a candidate signaling molecule mediating this residual thymic function
in the T mice.
It has been previously shown that peripheral T cells from
IL-7R-/- mice displayed intrinsic functional
defects (31). We observed similar defects in mature
thymocytes from IL-7R-/- mice by virtue of
their reduced capacity for IL-2-dependent costimulation of
anti-CD3-induced proliferation. In contrast, thymic expression of
the WT IL-7R transgene was sufficient to largely restore this
thymocyte proliferative response, indicating that the functional defect
is imposed at the level of the thymus rather than in the peripheral
immune compartment. This finding raises the possibility that IL-7R
signaling during thymic development functions in a broader role than
previously appreciated, perhaps by programming developing T cells to
become competent to generate functional T cell responses, either at the
level of SP cells or earlier in the developmental scheme.
Recent studies have demonstrated that IL-7R signaling appears to be
necessary for positive selection and coreceptor reversal during CD4 and
CD8 lineage determination, leading to the production of CD8 SP
thymocytes (44, 45). This conclusion was reached in part
by the capacity of anti-IL-7R and
anti-c mAbs to block the appearance of CD8
SP thymocytes in fetal thymic organ culture (44). However,
in all of our transgenic lines and in transgenic-negative
IL-7R-/- mice, a normal fraction of CD8 SP
thymocytes was detected. Furthermore, the constitutive expression of
the WT IL-7R transgene on DP thymocytes did not favor the development
of CD8+ SP thymocytes. Therefore, there must be
an IL-7R-independent pathway in vivo that maintains a normal proportion
of CD4 and CD8 SP thymocytes.
Although thymic cellularity and pro-T cell progression were impaired in
T mice, we readily detected TCR -chain gene rearrangements and
TCR cells in these mice. These data demonstrate that TCR
-chain rearrangements occur in the absence of the T region of
IL-7R, suggesting that they may occur independently of IL-7R
activation of STAT complexes or PI3-kinase. This
conclusion appears to conflict with a recent report that implicated
STAT5 in locus accessibility and germline transcriptional activity of
the TCR -chain locus (23). In that study, thymic
development was examined in fetal thymic organ culture using fetal
liver precursor cells from IL-7R-/- mice
transduced with either activated STAT5 or IL-7R. Thymocytes derived
from the activated STAT5 precursor cells exhibited enhanced cellularity
and TCR -chain germline transcripts, but decreased TCR -chain
rearrangements when compared with thymocytes derived from
IL-7R-transduced precursor cells. Taken together, these findings
might be interpreted to mean that induction of STAT5 is insufficient
for optimal TCR -chain gene recombination. In any case, it should be
stressed that thymic cellularity, including the absolute number of TCR
cells, and pro-T cell progression were markedly impaired in both
T and ST mice. Therefore, in practical terms, signaling molecules
associated with the carboxyl-terminal tail of IL-7R, such as STAT5,
must play an important role in facilitating the production of normal
numbers of TCR cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas R. Malek, University of Miami School of Medicine, Department of Microbiology and Immunology, P.O. Box 016960 (R-138), Miami, FL 33101.
3 Abbreviations used in this paper: c, common -chain; WT, wild type; DN, double negative; DP, double positive; SP, single positive; TN, triple negative; PI3-kinase, phosphatidylinositol 3-kinase; CY, CyChrome; MFI, mean fluorescence intensity.
Received for publication July 18, 2000. Accepted for publication October 9, 2000.
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