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,§
*
Howard Hughes Medical Institute and the Departments of
Immunology,
Biochemistry, and
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Medicine (Medical Genetics), University of Washington, Seattle, WA 98195;
¶
University of Minnesota Medical School, Roseville, MN 55113;
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University of Tokyo, Institute of Medical Sciences, Tokyo, Japan; and
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Howard Hughes Medical Institute and Department of Biochemistry, St. Judes Children’s Research Hospital, Memphis, TN 38105
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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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Jak3 is associated with the common 
-chain (c), a
signaling component shared by the IL-2, -4, -7, -9, and -15 receptors
(3, 4, 5). In each case, receptor-ligand interaction, or crosslinking with
anti-cytokine receptor Abs, induces phosphorylation of Jak3 on
tyrosine residues (3, 4, 6, 7, 8, 9) and activates its catalytic activity (6, 8). Recent reports have further confirmed that Jak3 plays an
indispensable role in relaying the signals generated by these
cytokines, some of which are absolutely necessary for the normal
development and function of lymphocytes. The human immunodeficiencies
XSCID (X-linked severe combined immunodeficiency) and XCID (X-linked
combined immunodeficiency), both marked by a dramatic reduction in the
number of circulating functional lymphocytes, are associated with
mutations in c that diminish its interaction with Jak3
(4, 10, 11). Mutations that result in reduced Jak3 expression have also
been associated with the SCID syndrome (12, 13). In mice, targeted
disruption of the gene encoding c results in a severe
deficiency of B cells and a hypoplastic thymus. Moreover, peripheral
lymphocytes in these animals do not respond to cytokines that stimulate
c, and these cells bear high levels of surface markers
that normally appear following Ag receptor-induced activation (14, 15).
These features are duplicated in mice that lack Jak3, strongly
suggesting that Jak3 directly mediates signals from c
(16, 17, 18).
Targeted disruption of the genes encoding unique chains of
cytokine receptors, such as the IL-2 receptor 
- (19) and ß-chains
(20) or the IL-7 receptor (21), has demonstrated that these cytokine
receptors play distinct roles during lymphocyte development. Disruption
of the unique IL-7R chain results in a profound reduction in the number
of immature and mature T and B cells, the effect of an early block in
lymphocyte development (21). In contrast, IL-2R - or ß-chain
deficiency results in the polyclonal expansion of lymphocytes that are
nonresponsive to IL-2, with increased surface expression of activation
markers such as CD69 or CD25 (19, 20). Jak3-deficient mice exhibit
characteristics similar to both the early, IL-7 receptor deficiency and
the late, IL-2 receptor deficiency. However, since an early
developmental block may alter the functional capacity of mature
lymphocytes, defining the role of Jak3 in the late stages of lymphocyte
development has been difficult.
In a reconstitution system, Thomis and Berg (22) recently showed that catalytically active Jak3 is required for early cellular expansion in the thymus and that Jak3 is required for proliferation of mature T cells stimulated in vitro. We have used a similar approach to restore Jak3 expression selectively in immature thymocytes or in peripheral T cells of mice that lack endogenous Jak3. Analyses of T cell development and function in these reconstituted mice confirm the findings reported earlier by Thomis and Berg (22). More importantly, we demonstrate that restoration of Jak3 expression in mature T cells, driven by the lck distal promoter, corrects peripheral T cell defects without increasing thymic cellularity significantly. Our results demonstrate that continuous expression of Jak3 is absolutely required for maintenance of T cell number and function. Moreover, we provide evidence supporting the view that Jak3, perhaps by regulating Fas expression, controls apoptotic responses in T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jak3 transgenes were constructed by juxtaposing a 3.7-kb NotI/RcaI fragment of the mouse Jak3 cDNA (6) with the lck proximal promoter in the p1017 vector (23) or the lck distal promoter vector pW120 (24). The cloned fragment includes the Jak3 coding sequences, the initiation and termination codons, and approximately 60 bp of 5' untranslated region and 360 bp of 3' untranslated region (6). Transgenes encoding a catalytically inactive version of Jak3 were generated by introducing a Lys-to-Glu point mutation at codon 851 in the cDNA, thereby disrupting a pivotal residue for phosphate transfer. This mutant form of Jak3 failed to autophosphorylate in response to IL-2 treatment when expressed in COS cell transfectants (data not shown).
Mice
Transgenic animals were generated by injecting Jak3 constructs into (C57BL/6 x DBA2)F2 embryos as previously described (25). The resulting pups were screened for integrated transgene DNA by Southern blot analysis of tail DNA using a radiolabeled hGH gene fragment (24). Transgene-positive founders were backcrossed with C57BL/6 mice through multiple generations to establish transgenic lines.
For reconstitution experiments, two p1017 wild-type transgenic lines with differing levels of transgene expression (Tglow and Tghigh), one pW120 wild-type Jak3 line, and one mutant Jak3 transgenic line (TgKE) were crossed with Jak3-/- mice (17). Progeny from these crosses were analyzed by Southern blotting or PCR for genotype at the endogenous Jak3 locus and for inheritance of the hGH-containing transgene. Southern blot analysis was performed as described (17). The PCR screen was designed to amplify the HygR cassette and to distinguish untargeted genomic sequences from targeted sequences. Two independent sets of primers were used for this assay: Hyg 5' primer, 5'-CGA CGT CTG TCG AGA AGT TTC TGA, and Hyg 3' primer, 5'-CAC GCC ATG TAG TGT ATT GAC CGA, were derived from HygR gene sequences; 5' Jak3 genomic primer derived from the coding sequences 5' of the HygR insertion site, 5'-AGG AGC CTT GCA TGT GCT CCT TCC, and 3' Jak3 genomic primer, which extends across an exon-intron boundary 3' of the HygR insertion site, 5'-AAC AGG CAG GAT GCC TGA AGG GAT. Transgene-positive Jak3 heterozygotes (Jak3+/-Tg+) were further crossed to yield progeny on a Jak3 +/+, +/-, or -/- background.
Analysis of Jak3 transgene expression
Total cellular lysates were prepared from thymocytes and splenocytes in lysis buffer (1% Triton X-100/50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin). 107 cell equivalent lysates were resolved using 7.5% SDS-PAGE, transferred onto nitrocellulose (Hybond-C, Amersham, Arlington Heights, IL), and blocked with 1% BSA (Tris-saline buffer, pH 7.5, 0.1% Tween-20). The membrane was incubated at room temperature in polyclonal rabbit antisera generated against a Jak3 polypeptide (6) at a 1:2000 dilution, followed by horseradish peroxidase-conjugated donkey anti-rabbit Ig (at 1:3000) as the secondary detection reagent (Amersham). Horseradish peroxidase activity was visualized by chemiluminescence according to the manufacturer’s instruction (Dupont, Boston, MA).
Flow cytometry
Single-cell suspensions of thymocytes, splenocytes, and lymph
node cells were prepared in RPMI 1640 containing 3% FCS. Cells were
treated in hypotonic saline solution to lyse RBCs, then stained as
described previously (25). The following conjugated mAbs and secondary
reagents were purchased from Caltag, San Francisco, CA: phycoerythrin
(PE)-conjugated anti-CD4 (CT-CD4), FITC-conjugated anti-CD8
(CT-CD8), PE-conjugated streptavidin, and tricolor-conjugated
streptavidin. Biotinylated anti-CD3 (2C11), biotinylated
anti-CD69 (H1.2F3), and PE-conjugated anti-Fas (Jo2) were
purchased from PharMingen, San Diego, CA. Biotinylated anti-human IgG
F(ab')2 was purchased from Jackson ImmunoResearch
Laboratories, West Grove, PA. Murine Fas-hFc reagent was a
generous gift from the Immunex Corporation (Seattle, WA) and was used
as described (26). For apoptosis assays, cells were incubated in PBS
containing 20 ng/ml of 7-amino actinomycin D (7AAD; Molecular Probes,
Eugene, OR), washed, fixed in 1% paraformaldehyde, and analyzed as
described (27). Data were collected in list mode files on a FACScan
flow cytometer (Becton Dickinson, Mountain View, CA) using Lysis II
software (Becton Dickinson) and analyzed using Reproman software
(TrueFacts Software, Seattle, WA).
In vivo BrdU uptake and staining
Mice were injected i.p. with 1.6 mg of BrdU (Sigma, St. Louis, MO) in 200 µl of PBS twice over a 4-h interval. At 42 h after the second injection, spleens were harvested and single-cell suspensions were prepared. Cells were stained with biotinylated anti-CD4 and PE-conjugated anti-CD8 mAbs followed by streptavidin-tricolor as the secondary detection reagent. Cells were then fixed and stained with FITC-conjugated anti-BrdU reagent (Becton Dickinson) according to the protocol described by Tough and Sprent (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of Jak3 deficiency are apparent at early stages of T
cell development. To restore Jak3 expression in developing thymocytes,
we used the lck proximal promoter to drive the expression of
transgenes encoding either wild-type or catalytically inactive Jak3.
Transgene constructs were assembled by inserting wild-type or mutant
forms of the Jak3 cDNA into the p1017 vector (Fig. 1
A, (29)). Transgenic founders
were generated and backcrossed with C57BL6 mice through multiple
generations to establish independent lines.
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We evaluated the levels of Jak3 expression by immunoblotting total
cellular lysates with anti-Jak3 antisera. Figure 1
B
shows the relative levels of transgene expression in
Jak3+/- or Jak3-/- mice. Translational
products derived from the transgene locus increased the observed
abundance of Jak3 in all three genotypic backgrounds, creating a wide
range of Jak3 expression. As expected for transgenes driven by the
thymus-specific lck proximal promoter, the levels of
reconstituted Jak3 were highest in thymocytes and decreased
dramatically in the periphery. This is most evident in
Jak3-/- transgenic mice. While transgene-derived Jak3
protein is detectable in splenocytes of the Tghigh line
(lane 16), it is no longer detectable in
splenocytes of the Tglow line or the TgKE line
(lanes 15 and 20). RNA analysis
indicated that transcripts from the transgene locus were present at a
low level even in splenocytes from
Jak3-/-Tglow and
Jak-/-TgKE mice, suggesting that a diminished
level of transgene product may still be present in the peripheral T
cells of these mice (data not shown).
Wild-type, but not catalytically inactive, Jak3 restores T cell numbers
Jak3-/- mice possess small thymi containing 0.5
to 10% of the normal number of thymocytes (16, 17, 18). This decrease in
cellularity is thought to result from the absence of appropriate
proliferative signals in early thymocytes and is reminiscent of the
phenotype observed in IL-7-null (30), IL-7R-null (21), or
c-null (15) mutant mice. Figure 2A demonstrates that the
wild-type Jak3 transgene restores thymic cellularity in
Jak3-/- mice, resulting in a 10- to 100-fold increase in
cell number. Judged by the average number of total thymocytes, low
level Jak3 transgene expression (Tglow) in
Jak3-/- animals appears as efficient as high level
expression (Tghigh) in driving this early expansion, not a
surprising result given that the level of Jak3 in
Jak3-/-Tglow mice exceeds that in
Jak3+/- thymocytes by 6.4-fold (Fig. 1B,
lanes 1 and 5). Interestingly, thymocyte
number is unaltered in Jak3 heterozygotes by transgene expression,
despite a substantial increase in total Jak3 abundance (data not
shown). These data suggest that expression of wild-type Jak3 at quite
high levels is not injurious. More importantly, the catalytically
inactive version of Jak3 (TgKE) was completely ineffective
in restoring thymus cellularity. Thus, Jak3 catalytic activity is
required to achieve normal thymic cellularity.
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B), as well as
those for CD3, CD69, and CD25 (data not shown). Neither wild-type nor
KE mutant Jak3 proteins affected development of Jak3+/- or
Jak3+/+ thymocytes. However, as previously observed
(16, 17, 18), the ratio of CD4 to CD8 single positive thymocytes increased
in Jak3-/- mice between 4 and 7 wk of age
(lower left), a phenomenon also seen in
c-/- mice (15). This increase was reversed
by expression of the wild-type Jak3 transgene (lower
right). The cause of this skewing in CD4:CD8 ratio is
unknown at present; however, this effect appears to be exaggerated in
the periphery, as will be discussed later.
Splenic and lymph node cellularity of Jak3-/- mice is
reduced, due to a block in early B cell development, in addition to
reduction in the number of mature T cells generated in the thymus
(16, 17, 18). As shown in Figure 2C, total splenic lymphocyte
cellularity is enhanced by expression of the wild-type Jak3 transgene
due to a selective increase in T cell numbers. Flow cytometric analysis
of splenocytes also indicates that B220+ B cells remain
under-represented even in mice with a high level of Jak3 transgene
expression (data not shown).
Although Jak3 protein is not detectable in Jak3-/-Tglow splenocytes, increased numbers of mature T cells exit the thymus and appear in the spleen and lymph nodes. Surprisingly, these animals develop splenomegaly by 7 wk of age. In contrast, Jak3-/-Tghigh animals maintain normal splenic cellularity. Further analysis revealed that the splenomegaly in Jak3-/-Tglow mice results from a fivefold increase in the representation of Mac-1+ cells, as well as normalization of T cell numbers. While the mechanism underlying this accumulation of myeloid cells in the spleens of Jak3-/-Tglow mice remains unknown, it appears that the loss of Jak3 expression in otherwise normal T cells can profoundly affect leukocyte homeostasis.
Loss of Jak3 correlates with the development of an activated phenotype among peripheral T cells
Previous analyses of Jak3- or IL-2R-deficient mice indicated that
peripheral lymphocytes in these animals spontaneously develop an
"activated" phenotype in vivo, while mounting minimal proliferative
responses to in vitro stimulations. T cells in these animals manifest
increased surface expression of CD25 (16), CD69 (16, 20), or CD44 (16, 19), markers that typically appear following Ag receptor-mediated
activation, and these activated cells accumulate with age. As shown in
Figure 3, a larger fraction of
CD4+ splenic T cells from Jak3-/- mice
bears high levels of CD69. In
Jak3-/-Tglow mice, the same effect
is observed, albeit with somewhat delayed kinetics. Moreover, T cells
from Jak3-/-Tglow mice failed to proliferate
in response to TCR cross-linking or mitogens (data not shown). In
contrast, peripheral T cells from
Jak3-/-Tghigh animals do not
express high levels of activation markers at age 7 wk or even when
obtained from a 20-week old animal (data not shown), suggesting that
the low level of Jak3 (60% of
Jak3+/-Tg- control; Fig. 1B, lanes 11 and 16) retained
in peripheral T cells in these mice is sufficient to establish and/or
maintain the resting state of circulating T cells. Interestingly, the
ability of T cells to proliferate in response to TCR crosslinking was
only partially restored in Jak3-/-Tghigh
animals (data not shown).
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In mice as in other species, the ratio of CD4 to CD8 T cells is
tightly regulated (31). However, increased CD4:CD8 T cell ratios are
characteristically observed in mice deficient in the
c, IL-2R - and ß-chains or Jak3 (16, 17, 18).
Thus, in Jak3-deficient animals, this ratio often exceeds 10:1 (Fig. 4A, lower
left), a value much higher than the normal 2:1 ratio
(upper left). High level expression of the
Jak3 transgene restores the normal CD4:CD8 ratio in splenic T cells in
Jak3-/- animals (lower right)
but has no effect on Jak3+/- control cells
(upper right and middle). Low level
transgene expression also restores the normal ratio initially (data not
shown); however, the ratio increases significantly in older animals
(lower middle), suggesting that continuous
presence of Jak3 is required to maintain the balance between the CD4
and CD8 compartments.
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The increased CD4:CD8 ratio in Jak3-/- mice may
be caused by preferential expansion of the CD4+ population
or by preferential elimination of the CD8+ population.
Figure 4B indicates that splenic CD4+ T cells
accumulate much more rapidly, compared with CD8+ T cells,
in Jak3-/- mice (filled squares), while the numbers of
CD4+ and CD8+ T cells in Jak3+/-
controls are maintained at relatively constant levels over time
(unfilled circles). Expression of the Jak3 transgene at low levels
augments accumulation of CD8+ T cells (crossed squares),
although it does not prevent accumulation of CD4 T cells. These data
suggest that asymmetric expansion of cells results in the increased
ratio between the CD4 and CD8 compartments. To examine this phenomenon
in more detail, we assessed BrdU incorporation by splenic T cells in
vivo. As shown in Figure 4C, markedly increased
incorporation of BrdU is observed for the Jak3-/-
splenocytes compared with the Jak3+/- controls, suggesting
that Jak3-/- T cells are actively synthesizing DNA and
are proliferating. Furthermore, our data clearly demonstrate that
CD8+ T cells in Jak3-/- mice incorporate BrdU
as well as do their CD4+ counterparts. These data
demonstrate that preferential expansion of CD4+ T cells
cannot by itself explain the observed skewing of the CD4:CD8 ratio.
Loss of Jak3 correlates with Fas and FasL up-regulation
Although both CD4+ and CD8+ T cells
proliferate in vivo in Jak3-/- mice, in seemed possible
that these populations might differ with respect to their ability to
undergo apoptosis. To address this possibility, we first compared the
relative levels of Fas expression on splenocytes from
Jak3-/- mice and Jak3+/- controls (Fig. 5A). Both CD4 and CD8 T
cells from Jak3-/- mice express high levels of Fas
relative to cells from Jak3+/- mice. This is not
surprising, given that activated T cells up-regulate Fas (32). When
these cells were subsequently treated with anti-CD3 in an overnight
culture, Jak3+/- controls readily up-regulated Fas, while
Jak3-/- cells, which express maximal levels of Fas before
stimulation, fail to increase Fas expression further (data not shown).
These effects correlate with the relative levels of Jak3 protein, as
Tglow reconstituted cells displayed an intermediate level
of Fas. Similar profiles were seen when p75 TNF receptor levels were
analyzed (data not shown). These findings, in conjunction with results
already discussed, suggest that T cells become activated spontaneously
in vivo following the loss of Jak3 and that these activated T cells
up-regulate CD69, CD44, Fas and p75 TNFR.
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B). In fact, a fraction of CD4+ T cells
in Jak3-/- animals appear to have down-regulated FasL
expression. This distinction between FasL expression in
CD4+ vs CD8+ T cells is consistent with
published data indicating that the level of FasL expression on
activated CD8+ splenic T cells is roughly 10-fold higher
than that found on CD4+ T cells (33). CD8+ T
cells from Jak3-/- mice reconstituted with low levels of
the Jak3 transgene (Jak3-/-Tglow) display an
intermediate level of FasL expression, indicating that regulation of
FasL expression is sensitive to the abundance of Jak3.
Finally, we analyzed ex vivo splenocytes for apoptosis. The percentage
of apoptotic cells was approximately twofold higher among the
CD8+ T cells from Jak3-/-Tg-
mice, an effect that disappeared in the
Jak3-/-Tglow cells (Fig. 5C). Low levels of Jak3 transgene expression
significantly lowered the proportion of apoptotic cells. These data
suggest that CD8+ T cells, while expanding at higher than
normal levels in the Jak3-/- animals, become highly
susceptible to apoptosis in the absence of Jak3. Furthermore, the
increased surface expression of Fas and FasL in vivo suggests that the
ongoing apoptosis may be at least partially mediated via a
Fas-dependent mechanism.
Amelioration of peripheral defects correlates with Jak3 abundance in mature T cells
Even though the peripheral T cell defects in reconstituted animals
correlated roughly with Jak3 protein abundance in those cells, it was
formally possible that recent thymic emigrants (with high levels of
Jak3 protein) displaced any resident peripheral T cells (which have
lost Jak3 protein) that were undergoing phenotypic changes, thereby
masking the accumulation of abnormal cells. To address this question,
we thymectomized Jak3-/-Tghigh mice and
monitored the pool of peripheral T cells over time. Before surgery, at
11 wk of age, T cells from Jak3-/-Tghigh mice
exhibited normal CD4/CD8 ratios and normal profiles of surface CD69
protein (data not shown). After the surgery, we analyzed the PBL of
unthymectomized and thymectomized
Jak3+/-Tghigh,
Jak3-/-Tghigh, and Jak3-/-
animals by flow cytometry at 2-wk intervals. We observed no significant
change of CD4:CD8 T cell ratios (Fig. 6A) or surface
expression of CD69 (Fig. 6B) in thymectomized animals
compared with their unthymectomized littermates, even at 6 wk
post-thymectomy. Immunoblot analysis of splenic T cells indicated that
Jak3 abundance did not decrease in thymectomized animals (Fig. 6C), suggesting that expression from the Jak3
transgene locus persisted in the periphery.
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A). Despite its
inability to reconstitute thymocyte numbers, the distal promoter-driven
expression of Jak3 considerably reduced the accumulation of splenic T
cells with high levels of surface CD69 (Fig. 7B).
Total Jak3 abundance in splenocytes of
Jak3-/-DTg+ animal was threefold over
Jak3+/-Tg- control, as judged by
immunoblotting (data not shown). These data suggest that developing
thymocytes that lack Jak3 are not automatically committed to express
high levels of CD69 and CD44 after they exit the thymus. Instead, Jak3
expression maintains the quiescent state of peripheral T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of JAKs in response to IFNs and growth factors has been associated with phosphorylation and nuclear translocation of STAT factors (reviewed in Refs. 34–36). The actual sequence of events immediately following receptor engagement remains unresolved at the molecular level; however, a current model supports the view that activated JAKs phosphorylate tyrosine residues in cytokine receptor molecules as well as those in STAT factors themselves. In mutant human cell lines lacking Jak1 or Jak2, catalytically inactive versions of the respective kinase are ineffective at restoring IFN-inducible gene expression or antiviral cytopathic activities (37). Although the kinase-negative mutant Jak1 was shown to induce low levels of gene expression in this system, its ability to complement the overall response was significantly lower than that observed using wild-type Jak1. Our study, like the previous analysis by Thomis and Berg (22), demonstrates that Jak3 catalytic activity is absolutely required for normal thymocyte development. In this regard, it is interesting that high level expression of catalytically inactive Jak3 did not in any way impair lymphocyte development (data not shown). We infer from this that comparatively little Jak3-derived signal is required to sustain normal lymphocyte function. The reconstitution of peripheral T cell function that we observe is consistent with this inference.
Requirement for Jak3 can be correlated with distinct cytokines that regulate T cell development and function
The normal maturational and functional response of a T cell
requires orchestrated cytokine signals (38, 39, 40). Loss of Jak3 leads to
a complex phenotype, presumably reflecting defects in the function of
cytokines that utilize c. The earliest defect,
relating to the severe reduction in lymphocyte numbers, resembles loss
of the IL-7 signal, which is indispensable for generation of
proliferating immature lymphocytes. Jak3 transgene expression under the
control of the lck proximal promoter successfully restores
this early signal, increasing the number of T cells. In this
environment, selective loss of Jak3 in the periphery appears to affect
T cell functions in a manner consistent with the loss of an IL-2
signal. IL-2 is thought to provide a costimulatory signal in the Ag
receptor-mediated response, as well as in regulating the course of
activation responses. This requirement is distinct from the early one,
because restoring Jak3 expression in immature thymocytes does not
prevent peripheral T cells from becoming spontaneously activated.
During maturation, thymocytes undergo multiple rounds of replication as they transit from the CD4-CD8- (double negative) to CD4+CD8+ (double positive) stage (reviewed in 41 . A developmental block at this stage has been correlated with reduction of thymic cellularity and an increase in the proportion of double negative thymocytes, as has been observed in Rag-1- or Rag-2-deficient mice (42, 43), or in mice lacking p56lck and p59fyn function (44, 45, 46). Given that the small number of thymocytes that remains in Jak3-/- mice develops normally from the double negative to the double positive stage, the paucity of thymocytes in these mice almost certainly reflects a requirement for Jak3 at an earlier stage. Indeed the number of Lin-c-kit+CD4-CD8- cells, thought to be thymocyte precursors, was reportedly reduced in Jak3-/- mice (18). Data obtained from the study of IL-7R-null mice, which manifest a similar phenotype, suggest that Jak3 acts during thymocyte development primarily by delivering an IL-7R-derived signal.
In reconstituted Jak3-/- mice, transgene-derived Jak3 protein selectively restores the T cell compartment, while other compartments of the hemopoietic system continue to suffer from Jak3 deficiency. Total numbers of B cells in the spleens and lymph nodes of transgene-positive (both Tghigh and Tglow) Jak3-/- mice are approximately 1 to 2% of the Jak3+/- control and are often lower than those of their transgene-negative Jak3-/- littermates (5–10%). We suspect that in the process of T cell expansion, mature B cells that completely lack Jak3 may suffer a competitive disadvantage. It is also possible that circulating T cells in some way promote B cell death in the periphery. A similar scenario has been described for IL-2R ß-chain-deficient mice (20) in which an age-dependent reduction in B cell numbers was prevented by in vivo administration of anti-CD4 mAb.
Jak3 regulates the activation of mature peripheral T cells
Prior studies demonstrate a pivotal role for Jak3 in delivering IL-2R-derived signals. Therefore, it is not surprising that those T cells that mature successfully in Jak3-/- mice fail to proliferate following TCR stimulation. Our experiments demonstrate that this nonresponsivess does not reflect a developmental anomaly, since levels of Jak3 that are fully sufficient to restore normal thymocyte numbers still cannot entirely correct the proliferative defect in peripheral T cells (data not shown). Thus, the replicative response of peripheral T cells to TCR stimulation is regulated by Jak3 abundance.
Curiously, T cells that lack Jak3 nevertheless accumulate in vivo and,
by several criteria, appear activated, in that they express high levels
of surface CD69 and CD44 protein. Similar observations have been made
in mice lacking c, or the IL-2R - or ß-chain. In
Jak3-/- mice, the accumulation of these activated cells
can be detected at 3 wk of age, and it increases thereafter. The
accumulation of CD44+CD69+ T cells in
reconstituted mice varies as a function of relative levels of Jak3
protein. While the initial event responsible for activation remains
undefined, our study clearly demonstrates that the continuous presence
of a threshold level of Jak3 is necessary to prevent this accumulation
of activated cells in the periphery.
Several observations support the view that activation of Jak3-deficient
T cells occurs after migration from the thymus. First, augmented
expression of CD69 and CD44 is seen only in peripheral T cells and not
in mature (single-positive) thymocytes. Second, provision of Jak3
intrathymically via the p1017-Jak3 transgene (Tglow)
reconstituted normal thymocyte development, but failed to prevent the
accumulation of activated cells. While it might be imagined that
constant production of normal cells by the
Jak3-/-Tghigh-reconstituted thymus displaced
any abnormal cells that might otherwise accumulate, thymectomized
Jak3-/-Tghigh mice retained normal peripheral
T cell profiles, indicating that reconstitution of Jak3 expression in
the periphery, a result of "leaky" promoter activity in this
compartment, was responsible for the ameliorating effect. Finally,
expression of Jak3 under the control of the distal lck
promoter, which directs expression primarily in peripheral T cells,
suppressed the generation of activated T cells without appreciably
improving thymocyte development (Fig. 7). Therefore, although Jak3
mediates cytokine-induced proliferation, cells lacking Jak3 respond to
a stimulus in the periphery that yields both illegitimate activation
and replicative expansion.
Jak3 regulates T cell homeostasis
Jak3 deficiency in the periphery engenders in T cells several abnormal features that are seemingly paradoxical. As discussed above, loss of Jak3 impairs in vitro proliferation of mature T cells in response to mitogens or TCR stimulation, while it promotes their constitutive activation in vivo. Loss of Jak3 also results in the asymmetric accumulation of CD4+ T cells, while it initiates peripheral proliferation of both CD4+ and CD8+ compartments.
Although the surface expression of many T cell activation markers increases with the loss of Jak3, only FasL expression differed between CD4+ and CD8+ T cells. While CD8+ T cells expressed higher levels of FasL, its receptor molecule, Fas, is expressed at high levels on both CD4+ and CD8+ T cells of Jak3-/- mice, a result consistent with the known increase in Fas expression typically observed following TCR stimulation. FasL expression, however, is especially dramatic in stimulated CD8+ cells (33). Our data both confirm the differential expression of FasL on activated CD4+ vs CD8+ T cells in Jak3-/- mice and suggest a mechanism for the augmented CD4:CD8 ratio in such animals.
Engagement of Fas with FasL triggers an apoptotic signal (47, 48). Moreover, mice lacking either of these genes manifest a lymphoproliferative disorder, suggesting that Fas-mediated cell death marks a normal end point in the course of lymphocyte activation (reviewed in Refs. 49–53). Since Fas can interact with FasL expressed on the same cell to induce apoptosis in a cell-autonomous manner (54, 55), we hypothesize that overabundant expression of FasL on CD8+ T cells in Jak3-/- mice triggers preferential elimination of these cells by a Fas-FasL-mediated suicide mechanism, thereby skewing the CD4-:CD8 ratio in the periphery.
Interestingly, previously published studies have suggested that an IL-2
signal is required to prime lymphocytes for apoptosis (56). A recent
study further suggests that IL-2 is required for cells to undergo
Fas-mediated apoptosis (57). Since apoptosis occurs readily in
Jak3-/- cells (Fig. 5C), we conclude
that alternative mechanisms must exist to sensitize these lymphocytes.
More to the point, Jak3-deficiency (as demonstrated here) encompasses a
broad range of signaling defects, resulting in profound abnormalities
in splenic T cell homeostasis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. R. M. Perlmutter, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065. E-mail address:
3 Abbreviations used in this paper: JAK, Janus kinase; c, -chain; PE, phycoerythrin; FasL, Fas ligand; Tg, transgene; DTg, distal promoter-Jak3 transgene.
Received for publication August 8, 1997. Accepted for publication November 6, 1997.
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M. A. Burchill, C. A. Goetz, M. Prlic, J. J. O'Neil, I. R. Harmon, S. J. Bensinger, L. A. Turka, P. Brennan, S. C. Jameson, and M. A. Farrar Distinct Effects of STAT5 Activation on CD4+ and CD8+ T Cell Homeostasis: Development of CD4+CD25+ Regulatory T Cells versus CD8+ Memory T Cells J. Immunol., December 1, 2003; 171(11): 5853 - 5864. [Abstract] [Full Text] [PDF]
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A. L. Mora, J. Youn, A. D. Keegan, and M. Boothby NF-{{kappa}}B/Rel Participation in the Lymphokine-Dependent Proliferation of T Lymphoid Cells J. Immunol., February 15, 2001; 166(4): 2218 - 2227. [Abstract] [Full Text] [PDF]
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R. Wen, D. Wang, C. McKay, K. D. Bunting, J.-C. Marine, E. F. Vanin, G. P. Zambetti, S. J. Korsmeyer, J. N. Ihle, and J. L. Cleveland Jak3 Selectively Regulates Bax and Bcl-2 Expression To Promote T-Cell Development Mol. Cell. Biol., January 15, 2001; 21(2): 678 - 689. [Abstract] [Full Text]
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B. O. Porter, P. Scibelli, and T. R. Malek Control of T Cell Development In Vivo by Subdomains Within the IL-7 Receptor {{alpha}}-Chain Cytoplasmic Tail J. Immunol., January 1, 2001; 166(1): 262 - 269. [Abstract] [Full Text] [PDF]
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 R. Malaviya, C.-L. Chen, C. Navara, R. Malaviya, X.-P. Liu, M. Keenan, B. Waurzyniak, and F. M. Uckun Treatment of Allergic Asthma by Targeting Janus Kinase 3-Dependent Leukotriene Synthesis in Mast Cells with 4-(3',5'-Dibromo-4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P97) J. Pharmacol. Exp. Ther., December 1, 2000; 295(3): 912 - 926. [Abstract] [Full Text]
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M. Fujimoto, T. Naka, R. Nakagawa, Y. Kawazoe, Y. Morita, A. Tateishi, K. Okumura, M. Narazaki, and T. Kishimoto Defective Thymocyte Development and Perturbed Homeostasis of T cells in STAT-Induced STAT Inhibitor-1/Suppressors of Cytokine Signaling-1 Transgenic Mice J. Immunol., August 15, 2000; 165(4): 1799 - 1806. [Abstract] [Full Text] [PDF]
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T. R. Malek, B. O. Porter, E. K. Codias, P. Scibelli, and A. Yu Normal Lymphoid Homeostasis and Lack of Lethal Autoimmunity in Mice Containing Mature T Cells with Severely Impaired IL-2 Receptors J. Immunol., March 15, 2000; 164(6): 2905 - 2914. [Abstract] [Full Text] [PDF]
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C. Merritt, H. Enslen, N. Diehl, D. Conze, R. J. Davis, and M. Rincón Activation of p38 Mitogen-Activated Protein Kinase In Vivo Selectively Induces Apoptosis of CD8+ but Not CD4+ T Cells Mol. Cell. Biol., February 1, 2000; 20(3): 936 - 946. [Abstract] [Full Text]
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D. C. Thomis, J. Aramburu, and L. J. Berg The Jak Family Tyrosine Kinase Jak3 Is Required for IL-2 Synthesis by Naive/Resting CD4+ T Cells J. Immunol., November 15, 1999; 163(10): 5411 - 5417. [Abstract] [Full Text] [PDF]
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M. P. Brown, T. Nosaka, R. A. Tripp, J. Brooks, J. M.A. van Deursen, M. K. Brenner, P. C. Doherty, and J. N. Ihle Reconstitution of Early Lymphoid Proliferation and Immune Function in Jak3-Deficient Mice by Interleukin-3 Blood, September 15, 1999; 94(6): 1906 - 1914. [Abstract] [Full Text] [PDF]
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) are compared with
Jak3-/-, 
) and reconstituted Jak3-/-
(Jak3-/-Tglo, 
) mice.
C, T cell proliferation in vivo. Following pulse-labeling
with BrdU, splenocytes were triply stained using Abs directed against
CD4, CD8, and BrdU. BrdU incorporation within CD4-- and
CD8--gated populations is represented as a
single-parameter histogram. The shaded graphs represent unstained
control cells in each case.
