Janus kinase 3 (Jak3) is essential for common cytokine receptor
chain (
c)-dependent signaling: comparative analysis of
c, Jak3, and
c and Jak3 double-deficient mice
Kotaro Suzuki,
Hiroshi Nakajima,
Yasushi Saito,
Takashi Saito1,
Warren J. Leonard2 and
Itsuo Iwamoto
Department of Internal Medicine II, Chiba University School of Medicine, Chiba 260-8670, Japan
1 Department of Molecular Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan
2 Laboratory of Molecular Immunology, National Heart, Lung and Blood Institute, Bethesda, MD 20892, USA
Correspondence to:
H. Nakajima, Department of Internal Medicine II, Chiba University School of Medicine, 1-8-1 Inohana, Chiba City, Chiba 260-8670, Japan
 |
Abstract
|
---|
The common cytokine receptor
chain (
c) is an essential receptor component for IL-2, IL-4, IL-7, IL-9 and IL-15, and thereby
c-deficient mice exhibit impaired T cell and B cell development. The Janus family tyrosine kinase 3 (Jak3) is known to be associated with
c, and the reported phenotypes of
c-deficient (
c) and Jak3-deficient (Jak3) mice are similar, indicating that Jak3 is an essential transducer of
c-dependent signals. Nevertheless, certain differences have been suggested related to the range of actions of
c and Jak3. To clarify whether
c-dependent cytokines can partially transduce their signals without Jak3, we compared lymphocyte development in
c, Jak3, and
c and Jak3 double-deficient (
cJak3) mice in the same genetic background. With the exception that T and B cells in Jak3 mice express high levels of
c, the defects in thymocyte and peripheral T cell and B cell development are indistinguishable among
c, Jak3 and
cJak3 mice. Interestingly, although Bcl-2 induction was previously suggested to be Jak3-independent, IL-7 cannot induce Bcl-2 expression in CD4 single-positive (SP) thymocytes in either
c or Jak3 mice nor can IL-7 rescue CD4 SP thymocytes from dexamethasone-induced cell death in
c or Jak3 mice. These results indicate that Jak3 is absolutely essential for
c-dependent T cell and B cell development, and for
c-dependent prevention of thymocyte apoptosis.
Keywords: apoptosis, Bcl-2, common cytokine receptor
chain, Jak3, knockout mice
 |
Introduction
|
---|
The common cytokine receptor
chain (
c) is a shared component of the receptors for IL-2, IL-4, IL-7, IL-9 and IL-15 (13). The
c gene is located on the X chromosome and when mutated results in X-linked severe combined immunodeficiency (XSCID) (4), a disease in which both T cell and NK cell development is profoundly diminished. This phenotype is consistent with the importance of IL-7 signaling for thymic T cell development (57) and with the importance of IL-15 for NK cell development (8,9). Analyses of mice lacking
c, IL-7 or IL-7R
revealed that
c-dependent signals are involved in a number of important events in T cell development (5,6,1017). First,
c-dependent signals are required for normal cell division of immature thymocytes (13). Second, IL-7 signals are vital for providing anti-apoptotic signals for CD4CD8 double-negative (DN) thymocytes, most likely at least in part by inducing Bcl-2 expression (1417). Third,
c-dependent signals are also required for the normally occurring up-regulation of Bcl-2 expression from CD4+CD8+ double- positive (DP) to CD4 single-positive (SP) thymocytes (13). However,
c appears to play additional important role(s) in thymocyte development beyond the induction of Bcl-2 because thymic cellularity in Bcl-2 transgenic
c-deficient mice is increased, but still is only 1220% of the normal level (13,17). Fourth,
c-dependent signals may play a role in thymic selection since the requirement of
c-dependent signals for T cell development differs depending on the affinity of the TCR for MHC (13). Fifth, IL-7 may function as a co-factor for TCR rearrangement (18,19), although its role in this process is controversial (reviewed in 20).
While
c-dependent signals play multiple roles in lymphocyte development, Janus kinase 3 (Jak3) is the only molecule which is known to transduce
c-dependent signals (reviewed in 21,22). Moreover, mutation of the Jak3 gene results in a form of severe combined immunodeficiency (SCID) that is clinically and immunologically indistinguishable from XSCID, except for the autosomal recessive inheritance pattern in Jak3 SCID (23,24), suggesting the important role of Jak3 in
c-dependent signaling for lymphocyte development. However, certain findings suggested that additional signaling pathway(s) might exist in
c-dependent signaling. For example, a mutant
c lacking the C-terminal 30 amino acids retained the ability to induce c-myc expression, although it did not retain the ability to induce c-fos and c-jun expression (25). In addition, it has been reported that Bcl-2 induction via
c is independent of Jak3 activation, whereas induction of c-fos and c-myc is Jak3 dependent (26). These observations could be consistent with findings that multiple kinases are associated with the IL-2 receptor complex (reviewed in 27,28). Recently, Jak3 has been suggested to function beyond
c-dependent signaling. Hanissian et al. demonstrated that Jak3 was associated with CD40 in B cell lines as well as normal B cells and played a role in CD40-induced ICAM-1 expression presumably by activating Stat3 (29). However, this conclusion was not supported by experiments performed using peripheral B cells from Jak3-deficient SCID patients (30).
Although the reported phenotype of Jak3-deficient (Jak3) mice (3133) is similar to that found in
c-deficient (
c) mice (1012), some differences have been suggested. For example, Jak3 mice exhibited more distinct hypoplasia in the thymic medulla than
c mice and Hassall's corpuscles were found in thymic medulla of
c mice but not of Jak3 mice (11,33). Therefore, it seemed possible that Jak3- independent
c signals and/or
c-independent Jak3 activation could play a role in lymphocyte development in vivo. However, these experiments used mice of different genetic backgrounds and ages. We have now compared lymphocyte development in
c, Jak3, and
c and Jak3 double-deficient (
cJak3) mice in the same genetic background. We found that T cell development was similarly impaired in Jak3,
c and
cJak3 mice, and that the defects were comparable even when a rearranged TCR transgene was expressed in Jak3 and
c mice. In addition, the expression level of Bcl-2 in CD4 SP thymocytes was diminished in Jak3 mice at a similar level to that found in
c mice and IL-7 could not induce Bcl-2 expression in Jak3 thymocytes.
 |
Methods
|
---|
Mice and genetic analysis
c-deficient mice (11) and Jak3 mice (33) were back- crossed to BALB/c mice (Jackson Laboratory, Bar Harbor, ME) for at least four generations. As the
c gene is located on chromosome X, mating of
c-deficient Jak3 heterozygous male (
c/YJak3+/) mice (H-2d/d) to
c-heterozygous Jak3 heterozygous female (
c+/Jak3+/) mice (H-2d/d) yielded wild-type, Jak3,
c and
cJak3 double-deficient mice within individual litters. Ovalbumin-specific DO11.10 (DO10) TCR transgenic mice (34) were provided by Dr K. Murphy (Washington University). The mice were genotyped by PCR using the following primer pairs. To detect the wild-type
c allele: 5'-AATAATACATTCCAGGAGYGCAGTCAC-3' and 5'-CCATGG- CCAACAATCTATAAACTCCAG-3'. To detect the
c knockout allele: 5'-ATTCGCAGCGCATCGCCTTCTACTG-3' and 5'-TTGTGCAGGGAAAGAGGGCAAGGG-3'. To detect the wild-type Jak3 allele: 5'-CGCGCACCCAGGTACTCCATGCCCT-3' and 5'-CCAGACCAGCAGAGGGACTT-3'. To detect the Jak3 knockout allele: 5'-CGCGCACCCAGGTACTCCATGCCCT-3' and 5'-CGACCACCAAGCGAAACATCGCATC-3'. To detect the DO10 transgene: 5'-CAGGAGGGATCCAGTGCCAGC-3' and 5'-TGGCTCTACAGTGAGTTTGGT-3'.
Mice were housed in microisolator cages under pathogen-free conditions. All experiments were done on 4- to 5-week-old mice and followed the guidelines of Chiba University.
Flow cytometric analysis
Cells from thymus, spleen, and bone marrow were stained and analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA) using CellQuest software (13). For direct staining, all conjugated antibodies were from PharMingen (San Diego, CA): anti-CD4FITC, phycoerythrin (PE), PerCP and allophycocyanin (APC) (H129.19), anti-CD8FITC, PE and APC (53-6.7), anti-CD25PE (3C7), anti-CD44FITC and CyChrome (IM7), anti-CD45R (B220)PE and APC (RA3-6B2), anti-CD62LFITC and PE (Mel-14), anti-CD69PE (H1.2F3), anti-I-AbFITC (AF6-120.1), anti-I-AdPE (AMS-32.1), anti-IgMFITC (R6-60.2), anti-CD54 (ICAM-1)PE (3E2), anti-CD80 (B7-1)PE (16-10A1), and anti-
cPE (4G2). KJ1-26 mAb, anti-idiotype for DO10 TCR (35) (provided by Dr B. J. Fowlkes, NIH, Bethesda, Maryland), was purified from supernatants of hybridoma cells using Protein G columns (Pharmacia, Uppsala, Sweden) and conjugated to FITC. Prior to staining, Fc receptors were blocked with anti-CD16/32 antibody (2.4G2; PharMingen).
Intracellular staining of Bcl-2
Bcl-2 levels in thymocytes and CD4+ T cells were analyzed on a FACSCalibur as previously described (36) using anti-murine Bcl-2 mAb (clone 3F11, 2 µg/ml final concentration; PharMingen), or purified hamster IgG (anti-TNP; PharMingen) as a negative control.
Histological analysis
After the tissue samples were fixed in buffered 10% formalin, the specimens were embedded in paraffin and 3 µm thick sections were stained with hematoxylin & eosin solution.
Dexamethasone-induced cell death assay of thymocytes
An in vitro cell death assay of thymocytes was performed as previously described (37). In brief, thymocytes (2x105) were cultured in RPMI 1640 media supplemented with 10% FBS, 2 mM glutamine and antibiotics. Where indicated, murine recombinant IL-7 (R & D Systems) was added at 5 ng/ml (330 pM) and dexamethasone was used at 1x108 M. Eighteen hours later, cells were harvested, stained with anti-CD4FITC and anti-CD8PE, and cell viability was determined using propidium iodide (5 µg/ml, Sigma) by a flow cytometer without gating. The percent survival of CD4+CD8 (CD4 SP) cells was calculated as: (number of viable CD4 SP cells cultured with each additive/number of viable CD4 SP cells cultured in medium alone)x100.
IL-7-induced Bcl-2 expression in thymocytes
Thymocytes were cultured in the presence or absence of IL-7 (5 ng/ml) in RPMI 1640 media supplemented with 10% FBS, 2 mM glutamine and antibiotics for 18 h. One aliquot of cells was analyzed for expression of Bcl-2 by FACS as described above. The remaining cells were washed twice with PBS and RNA was extracted using Isogen reagent (Nippon Gene, Tokyo, Japan). The first strand complementary DNA (cDNA) was synthesized from total RNA using Ready-To-Go You-Prime First-Strand Beads (Pharmacia) according to the manufacturer's instructions. cDNAs encoding Bcl-2 and ß-actin (as a control) were amplified by PCR as previously described (38).
Data analysis
Data are summarized as mean ± SD. The statistical analysis of the results was performed by the unpaired t-test. P < 0.05 was considered significant.
 |
Results
|
---|
T cell development is similarly impaired in
c, Jak3 and
cJak3 mice
c-dependent signals play multiple roles in T cell development. To determine whether Jak3 activation is required for transducing all
c-dependent signals, first we compared thymocyte development in
c, Jak3 and
cJak3 mouse littermates. Although there was considerable variability,
c, Jak3 and
cJak3 mice had very small thymuses, and we could not find significant differences in thymic cellularities [the mean number of thymocytes ± SD (x106) were as follows; wild-type mice 249.9 ± 42.9,
c mice 1.7 ± 1.2, Jak3 mice 1.9 ± 1.6,
cJak3 mice 1.7 ± 1.4, n = 917 mice, each] (Fig. 1A
). FACS analysis revealed that an increased CD4+CD8/CD4CD8+ ratio was similarly observed in
c, Jak3 and
cJak3 mice (11,33 and data not shown). As previously reported, in
c mice, the thymic cortex was reduced in size and the cortico-medullary junction was indistinct as compared with wild-type thymus. However, consistent with considerable variability of the cellularity of thymus in
c mice, their thymuses exhibited microscopic differences when sectioned and stained with hematoxylin & eosin. In the smallest thymus, the cortical area was almost absent and the cortico-medullary junction was more indistinct. In addition, it was hard to find Hassall's corpuscles (data not shown), similar to the reported characteristics found in Jak3 thymuses (33). Similar percentages of mice with very small thymuses were found among
c, Jak3 and
cJak3 mice (data not shown). Taken together, the impairment of thymocyte development was indistinguishable among
c, Jak3 and
cJak3 mice. Consistent with their impaired thymic development (Fig. 1A
), the number of splenic CD4+ T cells was diminished in these mice (wild-type mice 8.24 ± 3.26,
c mice 2.89 ± 1.83, Jak3 mice 3.57 ± 2.47,
cJak3 mice 2.71 ± 1.57,x106/spleen, mean ± SD, n + 68 mice) (Fig. 1B
). Splenic CD8+ T cells were more severely diminished in these mice (wild-type mice 3.72 ± 13.1,
c mice 0.17 ± 0.25, Jak3 mice 0.16 ± 0.22,
cJak3 mice 0.13 ± 0.15, x106/spleen, n + 68 mice) (Fig. 1C
).
Greatly diminished CD25+CD4+ T cells in
c, Jak3 and
cJak3 mice
As previously described in
c and Jak3 mice (31,3941), in
cJak3 mice, the majority of CD4+ T cells exhibited an activated/memory phenotype (CD62LlowCD44high) (data not shown), whereas CD4+ T cells in wild-type mice exhibited a naive phenotype (CD62LhighCD44low). Interestingly, whereas ~710% of splenic CD4+ T cells expressed the IL-2R
chain (CD25) in wild-type mice, no CD25+CD4+ T cells were found in
c, Jak3 and
cJak3 mice (Fig. 2
).
Jak3 and
c B cells express higher levels of ICAM-1 than wild-type B cells
We next analyzed the development of B cells in
c, Jak3 and
cJak3 mice. The number of B cells in the spleen was severely and similarly diminished in
c, Jak3 and
cJak3 mice, as compared to wild-type mice (wild-type mice 19.08 ± 7.17,
c mice 2.27 ± 1.76, Jak3 mice 3.22 ± 2.55,
cJak3 mice 2.30 ± 1.36, x106/spleen, n = 68 mice) (Fig. 3A
). Because Jak3 had been suggested to play a role in ICAM-1 expression on B cells, resulting from a possible role for Jak3/Stat3 activation in CD40 signaling (29), we investigated whether ICAM-1 expression on B cells differed in
c mice and Jak3 mice. Unexpectedly, ICAM-1 expression was modestly increased rather than decreased in Jak3 B cells as compared to wild-type B cells (Fig. 3B
). Moreover, ICAM-1 expression was also increased in
c-deficient B cells to an extent similar to its increase in Jak3 B cells (Fig. 3B
). B7-1 was also highly expressed on
c B cells and Jak3 B cells (Fig. 3B
). Interestingly,
c B cells as well as Jak3 B cells were increased in size (data not shown), suggesting that these B cells were activated. Thus,
c and Jak3 had apparently similar functions for B cell development and ICAM-1 expression on B cells, consistent with Jak3 not being involved in CD40 signaling (30).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3. Jak3 B cells express high levels of ICAM-1 and B7-1. (A) Shown is the cellularity of splenic B220+ cells (x106) from 4- to 5- week-old wild-type (WT), c, Jak3 and cJak3 mice. The B220+ cells were negative for CD3, CD4, CD8, TCR ß, TCR , Gr-1 and TER119 stainings, and the majority of B220+ cells expressed IgM in WT, c, Jak3 and cJak3 mice (data not shown). (B) Splenocytes from WT, c and Jak3 mice were stained with anti-IgMFITC, anti-B220APC and either anti-ICAM-1PE or anti-B7-1PE, and histograms for ICAM-1 or B7-1 expression were analyzed on B220+ IgM+ cells. Shown are representative histograms (bold lines) and isotype control stainings (dashed lines) from four independent experiments. Figures indicate mean fluorescent intensities (ICAM-1) and percent positive cells (B7-1).
|
|
Jak3 lymphocytes express higher levels of
c than wild-type lymphocytes
As demonstrated above, T cell and B cell development are similarly impaired in
c mice and Jak3 mice. However, it was possible that Jak3 might be essential for
c expression, thereby resulting in similar phenotypes in
c mice and Jak3 mice. Therefore, we analyzed the expression of
c in T cells and B cells in Jak3 mice. Interestingly, the expression of
c on CD4+ T cells and B cells was significantly higher in Jak3 mice than those in wild-type mice (Fig. 4C
versus A for CD4+ T cells and G versus E for B220+ cells). As expected,
c was undetectable in
c mice and
cJak3 mice (Fig. 4
), confirming the correct recognition by anti-
c mAb. From these results, we can consider several hypotheses. First,
c-dependent cytokines can transduce certain signals without Jak3; thus, cells expressing high levels of
c exhibit a growth advantage even in the absence of Jak3. Second, cytokine-induced internalization of
c may be impaired in the absence of Jak3. Third, because
c has been shown to be up-regulated after activation (42,43), high levels of
c expression in Jak3 mice are consistent with the activated phenotype of Jak3 T cells and B cells. Fourth,
c levels can be negatively regulated by Jak3 activation so that in the absence of Jak3,
c levels increase. In order to determine which of these possibilities might be correct, we analyzed the possible Jak3-independent
c signals in lymphocyte development in more detail.
Rescue of T cell development by the TCR transgene is similarly observed in Jak3 and
c mice
T cell development was similarly impaired in
c, Jak3 and
cJak3 mice, suggesting that Jak3 is absolutely required for
c-dependent T cell development. However, because IL-7 plays multiple roles in T cell development, it is possible that a single
c-dependent signal at early stage of thymocyte development could mask the possible differences in the later phase of thymocyte development that otherwise would occur between
c and Jak3 mice. TCR rearrangement is an essential step for T cell development and has been suggested to be affected by IL-7 signaling (18,19). To bypass this step, we generated Jak3 mice and
c mice carrying the DO11.10 (DO10) TCR transgene (34). Expression of the DO10 TCR transgene increased the cellularity of
c and Jak3 thymuses, but the cellularity of thymuses in DO10+ Jak3 and DO10+
c remained indistinguishable (DO10+ mice 206.4 ± 45.8, DO10+
c mice 31.0 ± 16.2, DO10+Jak3 mice 31.3 ± 16.5, x106/thymus, n = 78 mice) (Fig. 5A
). In our preliminary experiments, in the two mice examined so far, the expression of DO10 TCR transgene also increased the cellularity of thymuses in Jak3
c mice at a similar level to that found in DO10+
c mice or DO10+Jak3 mice. Interestingly, FACS profiles of CD4 versus CD8 staining revealed that the expression of the DO10 transgene strongly increased the percentage of CD4+ SP thymocytes in Jak3 mice and
c mice (Fig. 5B
) as compared to that in DO10+ mice, suggesting that DO10+ thymocytes may be more efficiently selected in the absence of
c or Jak3. However, the increase of CD4+ SP thymocytes were also indistinguishable between DO10+Jak3 mice and DO10+
c mice, indicating no role for Jak3-independent
c signals in thymocyte development even when successful TCR rearrangement have occurred.
Jak3 is essential for
c-dependent Bcl-2 induction
It was previously shown that Bcl-2 expression was diminished in mature SP thymocytes and peripheral T cells but not in DP thymocytes in
c mice (13,39). Because it has been reported that IL-2-induced Bcl-2 induction via
c is independent of Jak3 activation in a certain cell line (26), we compared the expression levels of Bcl-2 in Jak3 and
c mice. As shown in Fig. 6
, the normally occurring up-regulation of Bcl-2 expression from DP to CD4 SP thymocytes was slightly impaired not only in
c mice but also in Jak3 mice. Bcl-2 expression was also diminished in splenic CD4+ T cells from
c mice and Jak3 mice (Fig. 6
). Bcl-2 expression was lower in splenic CD4+ T cells than in CD4 SP thymocytes in
c mice and Jak3 mice, suggesting that
c-dependent Jak3 activation is more required for maintaining Bcl-2 expression in the periphery. In addition, exogenous IL-7 could not induce Bcl-2 protein (Fig. 7A
) or mRNA (Fig. 7B
) expression in thymocytes in DO10+Jak3 and DO10+
c mice, whereas IL-7 induced Bcl-2 expression in DO10+ mice (Fig. 7A and 7B
). Moreover, similar results were obtained using non-transgenic Jak3 or
c mice (data not shown). These results suggest that induction of Bcl-2 in CD4 SP thymocytes via
c is Jak3-dependent.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 7. IL-7 cannot induce Bcl-2 expression without Jak3 activation. (A) Thymocytes from DO10+, DO10+ c and DO10+Jak3 mice were cultured in the presence (bold line) or absence (dashed line) of IL-7 (at 5 nM) for 18 h and the expression of Bcl-2 was analyzed in CD4 SP thymocytes as described in Methods. (B) Similar to (A), thymocytes were cultured in the presence or absence of IL-7 for 18 h and RT-PCR analysis was performed as described in Methods. Similar results were obtained using CD8-depleted thymocytes (~80% of them were CD4+ SP thymocytes and 20% of them were CD4CD8 DN thymocytes).
|
|
Finally, we analyzed the ability of IL-7 to prevent dexamethasone-induced death of CD4 SP thymocytes. As previously described (37), IL-7 could prevent the dexamethasone-induced cell death in wild-type mice (Fig. 8
). As expected, the prevention of dexamethasone-induced death by IL-7 was not seen in
c mice (Fig. 8
). Similar to that in
c mice, IL-7 could not prevent dexamethasone-induced death of CD4 SP thymocytes in Jak3 mice and
cJak3 mice (Fig. 8
). Analogous results were obtained from the analysis using DO10 transgenic Jak3 mice (data not shown). These results indicate that Jak3 is required for the prevention of dexamethasone-induced death by IL-7.
 |
Discussion
|
---|
We analyzed
c, Jak3 and
cJak3 mice in the same genetic background. Although there was considerable variability of lymphocyte development within each genotype of mice, we could not find any differences in the number of thymocytes and peripheral T cells among
c, Jak3 and
cJak3 mice even when a rearranged TCR transgene was expressed. In addition, B cell development was similarly impaired among these mice. As expected, peripheral lymph nodes and gut-associated lymphoid tissue were absent not only in
c mice and Jak3 mice (11,33) but also in
cJak3 mice (data not shown). Finally, histological analysis revealed that thymus and spleen in
c, Jak3 and
cJak3 mice are indistinguishable. Taken together, these results indicate that Jak3-independent
c signals are not sufficient for T cell and B cell development in vivo. Interestingly, Jak3 but not
c is expressed in thymic epithelial cells (unpublished data), suggesting that Jak3 might function in a
c-independent fashion in these cells. However, because retrovirus-mediated gene transfer of Jak3 to Jak3 bone marrow cells corrected the defect of T cell development (44), the role of Jak3 in thymic epithelial cells, if any, remains unclear. Recently, it has been shown that thymocyte development in
c mice is completely abrogated when
c mice are crossed with either c-Kit-deficient mice (45) or pre-TCR
-deficient mice (46), indicating that c-Kit and pre-TCR signals are essential
c-independent signals which are required for thymocyte development in
c mice. Because no additional impairment of thymocyte development is found in
cJak3 mice as compared to
c mice, the signals which are provided by c-Kit or pre-TCR appear to be Jak3-independent.
Among
c-dependent cytokines, IL-7 appears to be the most important cytokine for murine T and B cell development (5,6). Analogous to IL-2, IL-7 can activate a number of signaling molecules (4750). Like Jak3, Jak1 is also essential for T cell development (51), suggesting that Jak1 is the obligate partner for Jak3 for inducing IL-7 signals. However, Jak1 is well known to play additional roles beyond IL-7 signaling, and Jak1-deficient thymocytes are unresponsive to the combination of phorbol myristate acetate (PMA) and ionomycin (51), whereas thymocytes from
c or Jak3 mice proliferate normally to PMA and ionomycin (11,33). The most well-characterized molecules downstream of Jak1/Jak3 are Stat5a and Stat5b (47), but these STATs are not essential either for T or B cell development (37,52,53), although there is some decrease in the number of T cells in mice lacking Stat5a or Stat5b (37,52). Stat3 is also activated by IL-7 (47,54), but is also not essential for lymphocyte development (54). Other possible downstream molecules of Jak1/Jak3 are phosphoinositide 3-kinase (48,49), STAM (55,56) and Pyk2 (57). Recently, phosphoinositide 3-kinase was shown to be essential for B cell development (58,59) and T cell development (60). The role of STAM and Pyk2 in lymphocyte development is still unknown.
Although splenic T cell numbers are diminished in young
c mice, splenic CD4+ T cells dramatically accumulate in the majority of adult
c mice (39). These CD4+ T cells exhibit a memory/activated (CD62LlowCD69high) phenotype and the acquisition of the phenotype appears to be TCR dependent (39). Analogous to
c mice, for unclear reasons, the majority of peripheral CD4+ T cells are also activated in Jak3 mice (40,41). Interestingly, however, CD4+ T cells bearing CD25 were absent in these mice (Fig. 2
). Approximately 10% of CD4+ T cells express CD25 in unimmunized wild-type mice and these CD25+CD4+ T cells seem to play an important role in the prevention of autoimmune diseases, presumably by suppressing CD25CD4+ T cell activation (6163). Therefore, it is possible that the disappearance of CD25+CD4+ T cells may be involved in the spontaneous activation of CD25CD4+ T cells in
c mice and Jak3 mice. Our results indicate that
c-dependent signals are essential for the development of CD25+CD4+ T cells and that CD25+CD4+ T cells may be a developmentally different lineage of cells from CD25CD4+ T cells. Interestingly, although the absolute number of conventional B cells was diminished in
c, Jak3 and
cJak3 mice, we found that these B cells increased the expression of ICAM-1 and B7-1, suggesting that B cells may be activated in these mice. Since activated B cells are potent antigen-presenting cells, it is also possible that these cells may contribute to the activation of CD4+ T cells.
In addition to cell division of thymocytes,
c is also vital for anti-apoptotic signals for thymocytes (20,64). Because the relative frequency of apoptotic cells is increased in DN and CD4+ or CD8+ SP thymocytes in
c-deficient mice, correlating with diminished Bcl-2 expression in these cells (46 and Nakajima et al., submitted),
c-induced Bcl-2 expression may be important for thymocyte survival. These observations are consistent with observations that thymic cellularity in
c-deficient or IL-7R
deficient mice is increased by Bcl-2 transgene (13,1517). Among
c-dependent signals, induction of Bcl-2 was suggested to be Jak3 independent (26). Specifically, it was reported that over-expression of a dominant negative Jak3 construct inhibited IL-2-induced c-fos and c-myc but not Bcl-2 induction, whereas over-expression of a dominant negative
c construct inhibited all of these responses (26). However, our results demonstrate that IL-7-induced Jak3 activation is required for
c-dependent Bcl-2 induction in CD4 SP thymocytes and that Jak3 is also required for the prevention of dexamethasone-induced cell death of CD4 SP thymocytes by IL-7. However, we could analyze only thymocytes with IL-7 stimulation because peripheral T cells in Jak3 mice exhibit a memory/activated phenotype even without exogenous stimulation and may not be proper for analysis. Therefore, it is still possible that other
c-dependent cytokines besides IL-7 might induce Bcl-2 expression in other cell types. A conditional Jak3 knockout after normal development has occurred could be valuable in further investigating this issue.
Interestingly, using
c/Jak3 chimeric molecules, Nelson et al. demonstrated that a membrane-proximal (PROX) region of
c (39 out of 86 amino acids of the
c cytoplasmic lesion) is essential for Jak3 activation (65). They also found that a truncation mutant of
c containing the PROX domain but lacking the ability to associate with Jak3 could mediate the phosphorylation of IL-2Rß and SHP-2 upon cytokine stimulation (65). Taken together, these results may suggest that an unidentified signaling molecule (perhaps a kinase), which is constitutively associated with the PROX domain of
c, plays an important role in
c-dependent Jak3 activation. Alternatively, although the association of Jak3 with a truncation mutant of
c containing the PROX domain was not detected by Western blotting (65), it is conceivable that a low amount of Jak3 can interact with this region and that this is sufficient for phosphorylation of IL-2Rß and SHP-2.
In conclusion, our results indicate that Jak3 is essential for
c-dependent lymphocyte development, IL-7-dependent Bcl-2 induction in the thymus and the prevention of dexamethasone-induced cell death. Although we cannot formally exclude the possibility of Jak3-independent
c signals in cell lineages (such as NK cells) that did not develop and thus were not studied, our data are consistent with the requirement of Jak3 in IL-7-dependent signals in the thymus at least for mainstream
ß T cells. Moreover, our data are also consistent with the absence of important roles for Jak3 beyond
c-dependent signals in lymphocytes.
 |
Acknowledgments
|
---|
We thank K. Murphy for DO10 mice and B. J. Fowlkes for KJ1-26 cells. This work was supported in part by grants from the Ministry of Education, Science and Culture and from the Agency for Science and Technology, Japan.
 |
Abbreviations
|
---|
APC allophycocyanin |
c common cytokine receptor chain |
DO10 DO11.10 |
DN double negative |
DP double positive |
Jak3 Janus kinase 3 |
PE phycoerythrin |
PMA phorbol myristate acetate |
SCID severe combined immunodeficiency |
SP single positive |
XSCID X-linked severe combined immunodeficiency |
 |
Notes
|
---|
The first two authors contributed equally to this work
Transmitting editor: S. L. Swain
Received 21 July 1999,
accepted 21 September 1999.
 |
References
|
---|
-
Leonard, W. J. 1996. The molecular basis of X-linked severe combined immunodeficiency: defective cytokine receptor signaling. Annu. Rev. Med. 47:229.[ISI][Medline]
-
Sugamura, K., Asao, H., Kondo, M., Tanaka, N., Ishii, N., Nakamura, M. and Takeshita, T. 1995. The common
-chain for multiple cytokine receptors. Adv. Immunol. 59:225.[ISI][Medline]
-
DiSanto, J. P., Kuhn, R. and Muller, W. 1995. Common cytokine receptor
chain (
c)-dependent cytokines: understanding in vivo function by gene targeting. Immunol. Rev. 148:19.[ISI][Medline]
-
Noguchi, M., Yi, H., Rosenblatt, H. M., Filipovich, A. H., Adelstein, S., Modi, W. S., McBride, O. W. and Leonard, W. J. 1993. Interleukin-2 receptor
chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 73:147.[ISI][Medline]
-
von Freeden-Jeffry, U., Vieira, P., Lucian, L. A., McNeil, T., Burdach, S. E. and Murray, R. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181:1519.[Abstract]
-
Peschon, J. J., Morrissey, P. J., Grabstein, K. H., Ramsdell, F. J., Maraskovsky, E., Gliniak, B. C., Park, L. S., Ziegler, S. F., Williams, D. E., Ware, C., Meyer, J. D. and Davison, B. L. 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180:1955.[Abstract]
-
Puel, A., Ziegler, S. F., Buckley, R. H. and Leonard, W. J. 1998. Defective IL7R expression in T()B(+)NK(+) severe combined immunodeficiency. Nat. Genet. 20:394.[ISI][Medline]
-
Ogasawara, K., Hida, S., Azimi, N., Tagaya, Y., Sato, T., Yokochi-Fukuda, T., Waldmann, T. A., Taniguchi, T. and Taki, S. 1998. Requirement for IRF-1 in the microenvironment supporting development of natural killer cells. Nature 391:700.[ISI][Medline]
-
Lodolce, J. P., Boone, D. L., Chai, S., Swain, R. E., Dassopoulos, T., Trettin, S. and Ma, A. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669.[ISI][Medline]
-
DiSanto, J. P, Müller, W., Guy-Grand, D., Fischer, A. and Rajewsky, K. 1995. Lymphoid development in mice with a targeted deletion of the interleukin-2 receptor
chain. Proc. Natl Acad. Sci. USA 92:377.[Abstract]
-
Cao, X., Shores, E. W., Hu-Li, J., Anver, M. R., Kelsall, B. L., Russell, S. M., Drago, J., Noguchi, M., Grinberg, A., Bloom, E. T., Paul, W. E., Katz, S. I., Love, P. E. and Leonard, W. J. 1995. Defective lymphoid development in mice lacking expression of the common cytokine receptor
chain. Immunity 2:223.[ISI][Medline]
-
Ohbo, K., Suda, T., Hashiyama, M., Mantani, A., Ikebe, M., Miyakawa, K., Moriyama, M., Nakamura, M., Katsuki, M., Takahashi, K., Yamamura, K.-i. and Sugamura, K. 1996. Modulation of hematopoiesis in mice with a truncated mutant of the interleukin-2 receptor
chain. Blood 87:956.[Abstract/Free Full Text]
-
Nakajima, H. and Leonard, W. J. 1999. Role of Bcl-2 in
ß T cell development in mice deficient in the common cytokine receptor
chain (
c): the requirement for Bcl-2 differs depending on the T cell receptor/MHC affinity. J. Immunol. 162:782.[Abstract/Free Full Text]
-
von Freeden-Jeffry, U., Solvason, N., Howard, M. and Murray, R. 1997. The earliest T lineage-committed cells depend on IL-7 for Bcl-2 expression and normal cell cycle progression. Immunity 7:147.[ISI][Medline]
-
Akashi, K., Kondo, M., von Freeden-Jeffry, U., Murray, R. and Weissman, I. L. 1997. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor- deficient mice. Cell 88:1033.
-
Maraskovsky, E., O'Reilly, L. A., Teepe, M., Corcoran, L. M., Peschon, J. J. and Strasser, A. 1997. Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant Rag-1/ mice. Cell 89:1011.[ISI][Medline]
-
Kondo, M., Akashi, K., Domen, J., Sugamura, K. and Weissman, I. L. 1997. Bcl-2 rescues T lymphopoiesis, but not B or NK cell development, in common
chain-deficient mice. Immunity 7:155.[ISI][Medline]
-
Candeias, S., Muegge, K. and Durum, S. K. 1997. IL-7 receptor and VDJ recombination: trophic versus mechanistic actions. Immunity 6:501.[ISI][Medline]
-
Oosterwegel, M. A., Haks, M. C., Jeffry, U., Murray, R. and Kruisbeek, A. M. 1997. Induction of TCR gene rearrangements in uncommitted stem cells by a subset of IL-7 producing, MHC class-II-expressing thymic stromal cells. Immunity 6:351.[ISI][Medline]
-
Rodewald, H.-R. and Fehling, H. J. 1998. Molecular and cellular events in early thymocyte development. Adv. Immunol. 69:1.[ISI][Medline]
-
Leonard, W. J. and O'Shea, J. J. 1998. Jaks and STATs: biological implications. Annu. Rev. Immunol. 16:293.[ISI][Medline]
-
Sugamura, K., Asao, H., Kondo, M., Tanaka, N., Ishii, N., Ohbo, K., Nakamura, M. and Takeshita, T. 1996. The interleukin-2 receptor
chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu. Rev. Immunol. 14:179.[ISI][Medline]
-
Macchi, P., Villa, A., Gillani, S., Sacco, M. G., Frattini, A., Porta, F., Ugazio, A. G., Johnston, J. A., Candotti, F., O'Shea, J. J., Vezzoni, P. and Notarangelo, L. D. 1995. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature 377:65.[ISI][Medline]
-
Russell, S. M., Tayebi, N., Nakajima, H., Riedy, M. C., Roberts, J. L., Aman, M. J., Migone, T. S., Noguchi, M., Markert, M. L., Buckley, R. H., O'Shea, J. J. and Leonard, W. J. 1995. Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. Science 270:797.[Abstract]
-
Asao, H., Takeshita, T., Ishii, N., Kumaki, S., Nakamura, M. and Sugamura, K. 1993. Reconstitution of functional interleukin 2 receptor complexes on fibroblastoid cells: involvement of the cytoplasmic domain of the
chain in two distinct signaling pathways. Proc. Natl Acad. Sci. USA 90:4127.[Abstract]
-
Kawahara, A., Minami, Y., Miyazaki, T., Ihle, J. N. and Taniguchi, T. 1995. Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction. Proc. Natl Acad. Sci. USA 92:8724.[Abstract]
-
Nelson, B. H. and Willerford, D. M. 1998. Biology of the interleukin-2 receptor. Adv. Immunol. 70:1.[ISI][Medline]
-
Lin, J.-X. and Leonard, W. J. 1997. Signaling from the IL-2 receptor to the nucleus. Cytokine Growth Factor Rev. 8:313.[Medline]
-
Hanissian, S. H. and Geha, R. S. 1997. Jak3 is associated with CD40 and is critical for CD40 induction of gene expression in B cells. Immunity 6:379.[ISI][Medline]
-
Jabara, H. H., Buckley, R. H., Roberts, J. L., Lefranc, G., Loiselet, J., Khalil, G. and Geha, R. S. 1998. Role of JAK3 in CD40-mediated signaling. Blood 92:2435.[Abstract/Free Full Text]
-
Thomis, D. C., Gurniak, C. B., Tivol, E., Sharpe, A. H. and Berg, L. J. 1995. Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science 270:794.[Abstract]
-
Nosaka, T., van Deursen, J. M., Tripp, R. A., Thierfelder, W. E., Witthuhn, B. A., McMickle, A. P., Doherty, P. C., Grosveld, G. C. and Ihle, J. N. 1995. Defective lymphoid development in mice lacking Jak3. Science 270:800.[Abstract]
-
Park, S. Y., Saijo, K., Takahashi, T., Osawa, M., Arase, H., Hirayama, N., Miyake, K., Nakauchi, H., Shirasawa, T. and Saito, T. 1995. Developmental defects of lymphoid cells in Jak3 kinase-deficient mice. Immunity 3:771.[ISI][Medline]
-
Murphy, K. M., Heimberger, A. B. and Loh, D. Y. 1990. Induction by antigen of intrathymic apoptosis of CD4+ CD8+ TCRlo thymocytes in vivo. Science 250:1720.[ISI][Medline]
-
Marrack, P., Shimonkevitz, R., Hannum, C., Haskins, K. and Kappler, J. 1983. The major histocompatibility complex-restricted antigen receptor on T cells. IV. An antiidiotypic antibody predicts both antigen and I-specificity. J. Exp. Med. 158:1635.[Abstract]
-
Veis, D. J., Sentman, C. L., Bach, E. A. and Korsmeye, S. J. 1993. Expression of the Bcl-2 protein in murine and human thymocytes and in peripheral T lymphocytes. J. Immunol. 151:2546.[Abstract/Free Full Text]
-
Nakajima, H., Liu, X. W., Wynshaw-Boris, A., Rosenthal, L. A., Imada, K., Finbloom, D. S., Hennighausen, L. and Leonard, W. J. 1997. An indirect effect of Stat5a in IL-2-induced proliferation: a critical role for Stat5a in IL-2-mediated IL-2 receptor
chain induction. Immunity 7:691.[ISI][Medline]
-
Kihara-Negishi, F., Yamada, T., Kubota, Y., Kondoh, N., Yamamoto, H., Abe, M., Shirai, T., Hashimoto, Y. and Oikawa, T. 1998. Down-regulation of c-myc and bcl-2 gene expression in PU.1-induced apoptosis in murine erythroleukemia cells. Int. J. Cancer 76:523.[ISI][Medline]
-
Nakajima, H., Shores, E. W., Noguchi, M. and Leonard, W. J. 1997. The common cytokine receptor
chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med. 185:189.[Abstract/Free Full Text]
-
Saijo, K., Park, S. Y., Ishida, Y., Arase, H. and Saito, T. 1997. Crucial role of Jak3 in negative selection of self-reactive T cells. J. Exp. Med. 185:351.[Abstract/Free Full Text]
-
Thomis, D. C. and Berg, L J. 1997. Peripheral expression of Jak3 is required to maintain T lymphocyte function. J. Exp. Med. 185:197.[Abstract/Free Full Text]
-
Nakarai, T., Robertson, M. J., Streuli, M., Wu, Z., Ciardelli, T. L., Smith, K. A. and Ritz, J. 1994. Interleukin 2 receptor
chain expression on resting and activated lymphoid cells. J. Exp. Med. 180:241.[Abstract]
-
Kondo, M., Ohashi, Y., Tada, K., Nakamura, M. and Sugamura, K. 1994. Expression of the mouse interleukin-2 receptor
chain in various cell populations of the thymus and spleen. Eur. J. Immunol. 24:2026.[ISI][Medline]
-
Bunting, K. D., Sangster, M. Y., Ihle, J. N. and Sorrentino, B. P. 1998. Restoration of lymphocyte function in Janus kinase 3-deficient mice by retroviral-mediated gene transfer. Nat. Med. 4:58.[ISI][Medline]
-
Rodewald, H.-R., Ogawa, M., Haller, C., Waskow, C. and DiSanto, J. P. 1997. Pro-thymocyte expansion by c-kit and the common cytokine receptor
chain is essential for repertoire formation. Immunity 6:265.[ISI][Medline]
-
DiSanto, J. P., Aifantis, I., Rosmaraki, E., Garcia, C., von Boehmer, H. and Rocha, B. 1999. The common cytokine receptor
chain and the pre-T cell receptor provide independent but critically overlapping signals in early
/ß T cell development. J. Exp. Med. 189:563.[Abstract/Free Full Text]
-
Lin, J.-X., Migone, T. S., Tsang, M., Friedmann, M., Weatherbee, J. A., Zhou, L., Yamauchi, A., Bloom, E. T., Mietz, J., John, S. and Leonard, W. J. 1995. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 2:331.[ISI][Medline]
-
Sharfe, N., Dadi, H. K. and Roifman, C. M. 1995. Jak3 protein tyrosine kinase mediates interleukin-7-induced activation of phosphatidylinositol-3 kinase. Blood 86:2077.[Abstract/Free Full Text]
-
Dadi, H., Ke, S. and Roifman, C. M. 1994. Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity. Blood 84:2579.
-
Venkitaraman, A. R. and Cowling, R. J. 1992. Interleukin 7 receptor functions by recruiting the tyrosine kinase p59fyn through a segment of its cytoplasmic tail. Proc. Natl Acad. Sci. USA 89:12083.[Abstract]
-
Rodig, S. J., Meraz, M. A., White, J. M., Lampe, P. A., Riley, J. K., Arthur, C. D., King, K. L., Sheehan, K. C., Yin, L., Pennica, D., Johnson, E. M., Jr and Schreiber, R. D. 1998. Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses. Cell 93:373.[ISI][Medline]
-
Imada, K., Bloom, E. T., Nakajima, H., Horvath-Arcidiacono, J. A., Udy, G. B., Davey, H. W. and Leonard, W. J. 1998. Stat5b is essential for natural killer cell-mediated proliferation and cytolytic activity. J. Exp. Med. 188:2067.[Abstract/Free Full Text]
-
Moriggl, R., Topham, D. J., Teglund, S., Sexl, V., McKay, C., Wang, D., Hoffmeyer, A., van Deursen, J., Sangster, M. Y., Bunting, K. D., Grosveld, G. C. and Ihle, J. N. 1999. Stat5 is required for IL-2-induced cell cycle progression of peripheral T cells. Immunity 10:249.[ISI][Medline]
-
Takeda, K., Kaisho, T., Yoshida, N., Takeda, J., Kishimoto, T. and Akira, S. 1998. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J. Immunol. 161:4652.[Abstract/Free Full Text]
-
Takeshita, T., Arita, T., Asao, H., Tanaka, N., Higuchi, M., Kuroda, H., Kaneko, K., Munakata, H., Endo, K., Fujita, T. and Sugamura, K. 1996. Cloning of a novel signal-transducing adaptor molecule containing an SH3 domain and ITAM. Biochem. Biophys. Res. Commun. 225:1035.[ISI][Medline]
-
Takeshita, T., Arita, T., Higuchi, M., Asao, H., Endo, K., Kuroda, H., Tanaka, N., Murata, K., Ishii, N. and Sugamura, K. 1997. STAM, signal transducing adaptor molecule, is associated with Janus kinases and involved in signaling for cell growth and c-myc induction. Immunity 6:449.[ISI][Medline]
-
Miyazaki, T., Takaoka, A., Nogueira, L., Dikic, I., Fujii, H., Tsujino, S., Mitani, Y., Maeda, M., Schlessinger, J. and Taniguchi, T. 1998. Pyk2 is a downstream mediator of the IL-2 receptor-coupled Jak signaling pathway. Genes Dev. 12:770.[Abstract/Free Full Text]
-
Fruman, D. A., Snapper, S. B., Yballe, C. M., Davidson, L., Yu, J. Y., Alt, F. W. and Cantley, L. C. 1999. Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85
. Science 283:393.[Abstract/Free Full Text]
-
Suzuki, H., Terauchi, Y., Fujiwara, M., Aizawa, S., Yazaki, Y., Kadowaki, T. and Koyasu, S. 1999. Xid-like immunodeficiency in mice with disruption of the p85
subunit of phosphoinositide 3-kinase. Science 283:390.[Abstract/Free Full Text]
-
Pallard, C., Stegmann, A. P., van Kleffens, T., Smart, F., Venkitaraman, A. and Spits, H. 1999. Distinct roles of the phosphatidylinositol 3-kinase and STAT5 pathways in IL-7-mediated development of human thymocyte precursors. Immunity 10:525.[ISI][Medline]
-
Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M. 1995. Immonologic self tolerance maintained by activated T cells expressing IL-2 receptor
chain (CD25). J. Immunol. 155:1151.[Abstract]
-
Takahashi, T., Kuniyasu, Y., Toda, M., Sakaguchi, N., Itoh, M., Iwata, M., Shimizu, J. and Sakaguchi, S. 1998. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10:1969.[Abstract]
-
Thornton, A. M. and Shevach, E. M. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
-
Akashi, K., Kondo, M. and Weissman, I. L. 1998. Role of interleukin-7 in T-cell development from hematopoietic stem cells. Immunol Rev 165:13.[ISI][Medline]
-
Nelson, B. H., McIntosh, B. C., Rosencrans, L. L. and Greenberg, P. D. 1997. Requirement for an initial signal from the membrane-proximal region of the interleukin 2 receptor
c chain for Janus kinase activation leading to T cell proliferation. Proc. Natl Acad. Sci. USA 92:1878.