By
From the * Division of Molecular Genetics, Center for Biomedical Science, Chiba University School of
Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260, Department of Surgical Pathology, Teikyo University
School of Medicine, Ichinara Hospital, 3426-3 Anegasaki, Ichihara, Chiba 299-01, Japan
Jak3 mediates growth signals through cytokine receptors such as interleukin-2 (IL-2), IL-4, and IL-7, and its deficiency results in autosomal recessive SCID in mice and humans. In spite of the severely reduced number of lymphocytes in Jak3-deficient mice, the differentiation profile of thymocytes was normal and mature T cells accumulated in the periphery with age. However, we found that self-reactive T cells were not deleted in the thymus and the peripheral tissues in Jak3-deficient mice. All peripheral T cells were in the activation state and thus were unable to be activated further, as demonstrated by the failure of eliciting Ca2+ response upon T cell receptor (TCR) stimulation. From the analysis of TCR-transgenic Jak3-deficient mice, only self-reactive T cells appeared to be in the activated state and anergic. These findings demonstrate a crucial function of Jak3 in the negative selection of autoreactive T cells and the maintenance of functional peripheral T cells.
Jak3 is associated constitutively with the common SCID is a disease based on several different genetic abnormalities, one of them being X-linked SCID (XSCID)
caused by functional mutations of the Mice.
C57BL/6 mice were purchased from Japan SLC, Inc.
(Hamamatsu, Japan). DO11.10 TCR-transgenic mice (DO-Tg)
were provided by Dr. D. Loh (Nippon Roche Research Center,
Kamakura, Japan) (17). Jak3-deficient mice were previously described (15).
Analysis of Cell Proliferation and IL-2 Secretion.
For proliferation
assay, splenocytes (2 × 105) from 8-wk-old mice were stimulated
with Con A (2.5 µg/ml), PMA (5 ng/ml) plus A23187 (100 ng/
ml), and anti-CD3 Measurement of Intracellular Ca2+ Response.
Intracellular Ca2+
mobilization was measured using Epics Elite ER-3 (Coulter
Corp., Hialeah, FL) with a Cell Quest analyzing program. 2 × 107 cells were incubated with 4 µM Indo-1 (Molecular Probes,
Inc., Eugene, OR) for 30 min at 37°C for loading. For TCR
stimulation, cells were incubated with anti-CD3 Flow Cytometric Analysis.
Splenocytes and lymph node T cells
were incubated with fluorescence- or biotin-conjugated antibodies and analyzed with a FACScan® (Becton Dickinson, Mountain
View, CA) using Cell Quest software. 5 × 105 cells were analyzed
for each sample. For multicolor analysis, cells were first incubated
with anti-FcR When the V
In addition to the existence of autoreactive T cells in thymus, spleen, and lymph node, all peripheral T cells from
Jak3
To analyze further the origin of the preactivated T cells
in the periphery of Jak3 Since splenic T cells in Jak3
To investigate the defects in TCR activation, we analyzed intracellular Ca2+ mobilization as an indicator of the
early signal transduction pathway upon TCR stimulation.
As shown in Fig. 4 A, thymocytes from Jak3
Mutations in How Jak3 is involved in negative selection is unknown
at present. One possibility is that Jak3-mediated growth
signal is crucial for the subsequent deletion of self-reactive
thymocytes in addition to signals through TCR. The other
intriguing possibility is that Jak3 is directly involved in T cell
activation. Our observation that PMA plus Ca2+ ionophore
did not stimulate IL-2 production in splenic T cells from
Jak3 Signaling defects in splenic T cells from Jak3 Further analysis will be required to elucidate the molecular basis of defects of thymic negative selection and signaling in peripheral lymphocytes from Jak3-deficient mice.
chain
(
c), a subunit of the cytokine receptors of IL-2, IL-4,
IL-7, IL-9, and IL-15, and has been shown in vitro to be
essential for their signal transduction (1). In the case of
IL-2 signaling, Jak3 is activated as one of the earliest events
upon IL-2 binding to IL-2 receptor (2, 8), and then activates Stat5 to translocate into the nucleus for mediating
growth signals. In addition, the constitutive activation of
the Jak3-Stat5 signaling pathway causes IL-2-independent
growth for HTLV-1-transformed T cells (10). These in
vitro analyses have demonstrated that the signal through Jak3 is essential for and regulates cell growth of lymphocytes.
c (11). It has recently been shown that Jak3 deficiency caused autosomal
recessive SCID (AR-SCID) in humans, with symptoms
similar to XSCID (12). Recently, we and others established Jak3-deficient mice (Jak3
/
) as an animal model of
human AR-SCID (14). Jak3
/
mice revealed severe
developmental inhibition of B cells, NK cells, and
T cells
in the skin and small intestine. These results demonstrated the
important roles of Jak3 in the development of lymphocytes
and NK cells in vivo. Although thymocytes and peripheral T cells were drastically reduced in young Jak3-deficient
mice, T cells gradually accumulated in the peripheral organs
with age in the absence of functional growth signals mediated by Jak3. We analyzed the repertoire and function of
these thymocytes and peripheral T cells in Jak3
/
mice
and found defects of negative selection of self-reactive T cells
in the thymus and the periphery, demonstrating a novel function of Jak3 in mediating negative selection and in
maintaining functional T cells.
mAb (145-2C11) cross-linking for 48 h,
pulsed with 37 kBq [3H]thymidine (Amersham Corp., Arlington
Heights, IL) for the last 8 h of culture, and harvested. [3H]thymidine uptake was measured with a MicroBetaTM liquid scintillation counter (Pharmacia, Uppsala, Sweden). For IL-2 production, proliferation of the IL-2-dependent cell line CTLL-2 was measured.
CTLL-2 cells (6 × 103) were cultured with supernatants from the
proliferation assay after stimulation for 48 h. Cells were pulsed
with [3H]thymidine and harvested as described above.
mAb (1452C11) for 30 min on ice and washed. After measuring the basal
level, 10 µg goat anti-hamster Ig was added for cross-linking. For
the response by Ca2+ ionophore, 1 µg A23187 (Sigma) was
added after measurement of the basal level.
receptor mAb 2.4G2 to prevent nonspecific staining. The following antibodies were used: CD4 (RM4-4), CD8
(53-6.7), CD69 (H1.2F3), CD44 (1M7), CD25 (7D4), and MEL14. These Abs, as well as all anti-V
mAbs (V
2-V
14), were purchased from PharMingen (San Diego, CA). Anti-clonotypic mAb
against DO11.10 TCR, KJ1-26, was provided by Dr. P. Marrack (Denver, CO).
Failure of Deletion of Self-reactive T Cells in the Thymus and
Periphery of Jak3-deficient Mice.
repertoire of
splenic T cells from Jak3-deficient mice with C57BL/6
background was analyzed, there was no difference in the
frequency of V
usage between Jak3
/
mice and wildtype littermates (data not shown). To analyze the repertoire
of T cells reactive to endogenous MMTV products presented on I-E molecule, we backcrossed with BALB/c and
analyzed the V
usage. As shown in Fig. 1 A, the frequency of mature T cells expressing V
5 and V
11, but
not others, was significantly increased in Jak3
/
mice as
compared with heterozygous mice. These V
-expressing T cells are known to be deleted in BALB/c mice, whereas
C57BL/6 mice fail to delete them due to the lack of MHC
class II I-E molecules. Although extensive analysis could
not be performed on thymocytes due to their limited number in Jak3
/
mice, similar increases of CD4 single-positive thymocytes expressing V
5 and V
11 were observed
(Fig. 1 B). It is noteworthy that, while the percentage of
V
11+ T cells in Jak3
/
mice with H-2d background was
almost restored to the level of C57BL/6 mice, the frequency of V
5 was only partly recovered. These data demonstrate that Jak3-deficient mice failed to delete self-reactive
T cells. Furthermore, the observation that the percentage
of V
10+ T cells was reduced in Jak3
/
mice suggests additional defects.
Fig. 1.
V repertoire of T cells in the thymus and periphery of Jak3deficient mice. (A) V
repertoire of CD4+ peripheral T cells from Jak3homozygous (
/
) and heterozygous (+/
) mice. The percentages of T
cells expressing representative V
s among CD4+ T cells were shown as
the average ± SD of three experiments. (B) Expression of V
5 and V
11
on CD4+ CD8
thymocytes from Jak3-homozygous (
/
) and heterozygous (+/
) mice. Staining profiles with anti-V
5 and anti-V
11
mAbs (solid line) and control mAb (dotted line) on CD4+CD8
thymocytes. The percentages of V
5- and V
11-expressing T cells among
CD4+CD8
thymocytes were indicated in each panel. For both (A) and
(B), thymocytes or B cell-depleted splenocytes were incubated first with
each anti-V
mAb and then with a mixture of biotin-coupled goat anti-
mouse Ig and rat Ig Abs. After blocking with mouse and rat Ig, the cells
were stained with FITC-anti-CD4 and PE-anti-CD8 and streptavidin-
Quantum Red.
[View Larger Version of this Image (19K GIF file)]
/
mice were activated as determined by surface expression of several markers. These T cells expressed high
levels of CD44 and CD69 (18) and a low level of Mel14, representing the phenotype of activated T cells (Fig. 2 A).
Fig. 2.
Expression of activation markers on splenic T cells
from Jak3-deficient mice (A) and
DO-Tg·Jak3/
mice (B). (A)
Staining profiles of CD69,
CD44, and Mel-14 on splenic T
cells from Jak3-deficient mice
(
/
) and wild-type littermates (+/+). Cells were stained for either CD69, CD44, or Mel-14 in addition to CD4 and CD8, and
the profiles were shown for
CD4+ T cells. The profiles for
CD8+ T cells were almost the
same as for CD4+ T cells. Thick
and thin lines indicated staining
with each Ab and control staining, respectively. (B) Expression
of CD44 (left) and Mel-14 (right)
on CD4+ splenic T cells from
DO-Tg (+/+) and DOTg·Jak3
/
(
/
) mice. (Top
left) Staining profiles with the
clonotypic mAb KJ1-26; cells
were divided into two groups,
KJ1-26 high and low. (Bottom
left) Thin and thick lines indicate
CD44 staining for KJ1-26 high
and low cells, respectively. (Top
right) Staining profiles with antiV
8 mAb; cells were divided
into two groups, V
8 high and
low. (Bottom right) Thin and
thick lines indicate Mel-14 staining for V
8 high and low cells,
respectively.
[View Larger Version of this Image (36K GIF file)]
/
mice, we crossed Jak3
/
mice
with OVA-specific DO-Tg mice whose TCR (DO-TCR)
was detected by staining with anti-clonotypic mAb KJ1-26
for the TCR
dimer and anti-V
8 mAb F23.1 for the
TCR
chain. ~20% of the T cells from DO-Tg mice expressed endogenous TCR, but the rest of the cells were
stained with KJ1-26 and F23.1 (17). Whereas KJ1-26 positive and negative populations from DO-Tg mice did not
show a significant difference in CD44 expression, the DOTCR-expressing (KJ1-26high) T cells from DO-Tg·Jak3
/
mice were CD44low (Fig. 2 B). In contrast, most of the T cells
expressing endogenous TCR (KJ1-26low) were CD44high,
which is the same phenotype as Jak3
/
splenic T cells
(Fig. 2 B). This was also shown by Mel-14 expression. Whereas DO-TCR expressing T cells (V
8high) were composed of both Mel-14high and Mel-14low populations, T cells
expressing endogenous TCR (V
8low) were all Mel-14low
(Fig. 2 B). These data demonstrate that T cells with endogenous TCR but not DO-TCR-expressing T cells were reactivated in DO-Tg·Jak3
/
mice, suggesting that splenic
T cells in Jak3
/
mice may be autoreactive and have been
activated with self-antigens.
/
mice are in
the activated state, we asked whether these T cells might
be functionally unresponsive to further stimulation. Indeed,
splenic T cells from Jak3
/
mice failed to proliferate upon
stimulation with either Con A or anti-CD3
mAb crosslinking, regardless of the presence of exogenous IL-2.
Furthermore, these T cells did not proliferate even after stimulation with PMA and Ca2+ ionophore (Fig. 3 A). In
contrast with the splenic T cells, thymocytes from Jak3
/
mice responded to both anti-CD3
cross-linking and stimulation with PMA plus Ca2+ ionophore (15). As shown in
Fig. 3 B, thymocytes from Jak3
/
mice secreted a considerable amount of IL-2 compared with normal thymocytes,
while splenic T cells from Jak3
/
mice produced very little. Cell surface staining revealed no difference in TCR expression between Jak3
/
and wild-type mice (15). These
data demonstrated that splenic T cells in Jak3
/
mice possess defects in the signal transduction pathway leading to
IL-2 production upon TCR stimulation, in addition to
growth signal defects.
Fig. 3.
Functional analysis
of thymocytes and splenic T cells
from Jak3-deficient mice. (A)
Proliferation of splenic T cells
upon mitogenic stimulation.
Splenocytes (2 × 105) from Jak3
homozygous (/
), heterozygous (+/
) mutant mice, and
wild-type littermates (+/+)
were stimulated with anti-CD3
mAb (145-2C11, 10 µg/ml),
Con A (2.5 µg/ml), IL-2 (40 U/
ml), Staphyloccocal enterotoxin
B (10 µg/ml), and the combination of PMA (5 ng/ml) and
A23187 (100 ng/ml). Cells were
cultured for 48 h and pulsed
with [3H]thymidine for 8 h. (B)
IL-2 production of thymocytes
and splenic T cells upon mitogenic stimulation. Thymocytes
and splenic T cells from Jak3homozygous (
/
), and heterozygous (+/
) mutant mice, and wild-type littermates (+/+)
were stimulated with 145-2C11, Con A, and PMA plus A23187
as described in (A). All results
were presented as mean ± SD
from triplicate cultures.
[View Larger Version of this Image (36K GIF file)]
/
mice elicited almost comparable Ca2+ response to that of thymocytes from wild-type mice upon stimulation with both
anti-CD3
mAb cross-linking and Ca2+ ionophore. In
contrast, splenic T cells from Jak3
/
mice failed to elicit
Ca2+ response upon TCR cross-linking in spite of the fact
that these cells showed Ca2+ flux upon stimulation with
Ca2+ ionophore (Fig. 4 B). These data clearly demonstrate
that splenic T cells from Jak3
/
mice have defects in early
Ca2+ signaling upon activation through the TCR complex.
Fig. 4.
Intracellular Ca2+ mobilization of thymocytes (A) and splenic
T cells (B) from Jak3-deficient mice (/
) and wild-type littermates (+/+).
Thymocytes and splenocytes were stained with anti-CD4 and anti-CD8
mAbs, loaded with Indo-1, stimulated with cross-linking with 2C11 or
A23187 (Iono.), and then Ca2+ responses were measured. Data for CD4+
cells were represented. In both A and B, the peak (thick line) and sustained
line (dotted line) Ca2+ responses were shown.
[View Larger Version of this Image (23K GIF file)]
c and Jak3 in the patients of XSCID (11)
and AR-SCID (12), respectively, caused growth defects
in T cells because
c and Jak3 are associated (2, 8) and are
both required for growth signal in T cells (9). The failure of
cell growth has been thought to be due to defective cytokine receptor signaling. In the present study, we have demonstrated that, in addition to the growth defects, AR-SCID
model mice have defects in negative selection of self-reactive T cells. Thus, Jak3-deficient mice possess forbidden autoreactive T cells in the thymus and periphery. The reason
these mice do not develop autoimmune diseases may be because these autoreactive T cells are anergic to further stimulation. Although Jak3 deficiency resulted in a dramatic decrease in the number of precursor cells in the thymus, once
they were seeded in the thymus, thymocyte differentiation
appeared to take place normally (15). However, we found
that Jak3 deficiency resulted in a failure to eliminate self-reactive thymocytes, consequently leading to the accumulation
of autoreactive but anergic peripheral T cells.
/
mice, as well as the previous finding that Jak3 is
crucial for preventing the induction of anergy in T cells
(21), are consistent with this idea.
/
mice
were observed in association with reactivated status, namely
the high expression of activation markers such as CD44
and CD69 as well as the downregulation of Mel-14.
Splenic T cells were reactivated in Jak3-deficient mice and
were all refractory to further activation. From the analysis
of DO-Tg·Jak3
/
mice, we showed that the appearance
of reactivated and refractory T cells depended on the specificity of TCR. Because thymocytes from Jak3-deficient mice
do not exhibit the activated phenotype and proliferate, secrete IL-2 and exhibit Ca2+ flux upon TCR stimulation,
preactivation of splenic T cells probably takes place during
immigration after leaving the thymus or within the periphery. Considering that only T cells with endogenous TCRs exhibited the activated phenotype, it is likely that these T cells were activated with self-peptides in the periphery, while
OVA-specific T cells could not be activated in the absence
of OVA peptide. The fact that defects in negative selection
were influenced by TCR specificity is consistent with our
observation that some V
+ T cells were completely restored from deletion, while some others were only partly
recovered by Jak3 deficiency (Fig. 1). Such autoreactive T cells are in an anergic state after activation. Alternatively, provided that all T cells had been activated during immigration from thymus to the periphery and then returned to
the resting state, T cells may fail to return in the absence of
Jak3, although some of them can still return to the resting
state depending on their TCR specificity. In either case,
Jak3 plays a pivotal role in maintaining the normal phenotype and function of peripheral T cells.
Address correspondence to Takashi Saito, Division of Molecular Genetics, Center for Biomedical Science, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan.
Received for publication 23 October 1996
This work was supported by grants to T. Saito from the Ministry for Education, Science, and Culture and from the Agency for Science and Technology, Japan and partly by a grant from Ciba-Geigy Foundation (Japan) for the Promotion of Science.We thank Dr. D. Loh for providing DO11.10 Tg mice, Dr. P. Marrack for mAb, Drs. S. Miyatake and T. Shirasawa for helpful discussions, Ms. M. Sakuma for technical assistance, and Ms. H. Yamaguchi for preparing the manuscript.
1. |
Giri, J.G.,
M. Ahdieh,
J. Eisenman,
K. Shanebeck,
S. Kumaki,
A. Namen,
L.S. Park,
D. Cosman, and
D. Anderson.
1994.
Utilization of ![]() ![]() |
2. | Johnston, J.A., M. Kawamura, R.A. Kirken, Y.Q. Chen, T.B. Blake, K. Shibuya, J.R. Ortaldo, D.W. McVicar, and J.J. O'Shea. 1994. Phosphorylation and activation of the Jak-3 Janus kinase in the response to interleukin-2. Nature (Lond.). 370: 151-153 [Medline] . |
3. | Kawamura, M., D.W. McVicar, J.J. Johnston, T.B. Blake, Y.Q. Chen, B.K. LaL, A.R. Lloyd, D.J. Kelvin, J.E. Staples, J.R. Ortaldo, and J.J. O'Shea. 1994. Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes. Proc. Natl. Acad. Sci. USA. 91: 6374-6378 [Abstract] . |
4. |
Kondo, M.,
T. Takeshita,
N. Ishii,
M. Nakamura,
S. Watanabe,
K. Arai, and
K. Sugamura.
1993.
Sharing of the interleukin-2 (IL-2) receptor ![]() |
5. |
Kondo, M.,
T. Takeshita,
M. Higuchi,
M. Nakamura,
T. Sudo,
S. Nishikawa, and
K. Sugamura.
1994.
Functional participation of the IL-2 receptor ![]() |
6. |
Noguchi, M.,
Y. Nakamura,
S.M. Russell,
S.F. Ziegler,
M. Tsang,
X. Cao, and
W.J. Leonard.
1993.
Interleukin-2 receptor ![]() |
7. |
Takeshita, T.,
H. Asao,
K. Ohtani,
N. Ishii,
S. Kumaki,
N. Tanaka,
H. Munakata,
M. Nakamura, and
K. Sugamura.
1992.
Cloning of the ![]() |
8. | Witthuhn, B.A., O. Silvennoinen, O. Miura, K.S. Lai, C. Cwik, E.T. Liu, and J.N. Ihle. 1994. Involvement of the Jak-3 Janus kinase in signaling by interleukin-2 and -4 in lymphoid and myeloid cells. Nature (Lond.). 370: 153-157 [Medline] . |
9. | Miyazaki, T., A. Kawahara, H. Fujii, Y. Nakagawa, Y. Minami, Z.J. Liu, I. Oishi, O. Silvennoinen, B.A. Witthuhn, J.N. Ihle, and T. Taniguchi. 1994. Functional activator of Jak1 and Jak3 by selective association with IL-2 receptor subunit. Science (Wash. DC). 266: 1045-1047 [Medline] . |
10. | Migone, T.S., J.X. Lin, A. Cereseto, J.C. Mulloy, J.J. O'Shea, G. Franchini, and W.J. Leonard. 1995. Constitutively activated Jak-STAT pathway in the T cells transformed with HTLV-1. Science (Wash. DC). 269: 79-81 [Medline] . |
11. |
Noguchi, M.,
H. Yi,
H.M. Rosenblatt,
A.H. Filipovich,
M. Tsang,
X. Cao, and
W.J. Leonard.
1993.
Interleukin-2 receptor ![]() |
12. | Macci, P., A. Villa, S. Giliani, M.G. Sacco, A. Frattini, F. Porta, A.G. Ugazio, J.A. Johnston, F. Candotti, J.J. O'Shea, et al . 1995. Mutation of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature (Lond.). 377: 65-68 [Medline] . |
13. | Russel, S.M., N. Tayebi, H. Nakajima, M.C. Riedy, J.L. Roberts, M.J. Aman, T.-S. Migone, M. Noguchi, M.L. Markert, R.H. Buckley, et al . 1996. Mutation of Jak3 in a patient with SCID: Essential role of Jak3 in lymphoid development. Science (Wash. DC). 270: 797-800 [Abstract] . |
14. | Nosaka, T., J.M.A. van Deursen, R.A. Tripp, W.E. Thierfelder, B.A. Witthuhn, A.P. McMickle, P.C. Doherty, G.C. Grosveld, and J.N. Ihle. 1996. Defective lymphoid development in mice lacking Jak3. Science (Wash. DC). 270: 800-802 [Abstract] . |
15. | Park, S.Y., K. Saijo, T. Takahashi, M. Osawa, H. Arase, N. Hirayama, K. Miyake, H. Nakauchi, T. Shirasawa, and T. Saito. 1995. Developmental defects of lymphoid cells in Jak3 kinase-deficient mice. Immunity. 3: 771-782 [Medline] . |
16. | Thomis, D.C., C.B. Gurniak, E. Tivol, A.H. Sharpe, and L.J. Berg. 1995. Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science (Wash. DC). 270: 794-796 [Abstract] . |
17. | Murphy, K.M., A.B. Heimberger, and D.Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymus in vivo. Science (Wash. DC). 250: 1720-1723 [Medline] . |
18. |
Budd, R.C.,
J.C. Cerottini,
C. Horvath,
C. Bron,
T. Pedreazzini,
R.C. Howe, and
H.R. MacDonald.
1987.
Distinction
of virgin and memory T lymphocytes. Stable acquisition of
the Pgp-1 glycoprotein concomitant with antigenic stimulation.
J. Immunol.
138:
3120-3129
|
19. | Swat, W., M. Dessing, H. von Boehmer, and P. Kisielow. 1993. CD69 expression during selection and maturation of CD4+CD8+ thymocytes. Eur. J. Immunol. 23: 739-746 [Medline] . |
20. | Yamashita, I., T. Nagata, T. Tada, and T. Nakayama. 1993. CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen receptor-mediated positive selection. Int. Immunol. 5: 1139-1150 [Abstract] . |
21. |
Boussiotis, V.A.,
D.L. Barber,
T. Nakarai,
G.J. Freeman,
J.G. Gribben,
G.M. Bernstein,
A.D. D'Andrea,
J. Ritz, and
L.M. Nadler.
1994.
Prevention of T cell anergy by signaling
through the ![]() |