By
From the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
The Jak family tyrosine kinase, Jak3, is involved in signaling through cytokine receptors that
utilize the common chain (
c), such as those for IL-2, IL-4, IL-7, IL-9, and IL-15. Recent studies of Jak3-deficient mice and humans have demonstrated that Jak3 plays a critical role in B
and T lymphocyte maturation and function. The T lymphocyte defects in Jak3-deficient mice
include a small thymus, a decrease in peripheral CD8+ cells, an increase in the surface expression of activation markers, and a severe reduction in proliferative and cytokine secretion responses to mitogenic stimuli. To determine whether the peripheral T lymphocyte defects result
from aberrant maturation in the thymus or from the absence of Jak3 protein in peripheral T cells,
we generated reconstituted mice that express normal levels of Jak3 protein in the thymus but
lose Jak3 expression in peripheral T cells. Jak3 expression in the thymus restores normal T cell
development, including CD8+,
, and natural killer cells. However, the loss of Jak3 protein in
peripheral T cells leads to the Jak3
/
phenotype, demonstrating that Jak3 is constitutively required to maintain T cell function.
T lymphocyte maturation and function are regulated by
a number of signal transduction pathways. During T cell
development in the thymus, the progressive maturation of
T cells in large part is mediated by the TCR. First, the preTCR is required to induce the differentiation of CD4 Recent studies utilizing gene targeting in mice have revealed the importance of the B7 ligand, CTLA-4, and the
IL-2 signaling pathway in preserving the balance between
resting and activated T cells. For instance, loss of expression
of the costimulatory molecule CTLA-4 (15, 16), or of the
Recently, we and others have generated mice lacking expression of the These data provide an interesting contrast to the phenotype of T cells in mice lacking other components of the IL-2
signaling pathway, such as IL-2 itself, or the IL-2 receptor
Transgenic Mice and Southern Blot Analysis.
Both wild-type and kinase-dead Jak3 cDNAs (33) were introduced into the Lck proximal promoter vector (34), a gift from R. Perlmutter. Lck-Jak3
sequences were removed from the bacterial vector DNA by
cleavage with NotI, and prepared for microinjection. DNA was
injected into (C57Bl/6 × C3H)F2 fertilized eggs by standard procedures (35). Pups were screened for the transgene by Southern blot analysis of EcoRI digested tail DNA probed with an 0.35-kb EcoRI-HindIII fragment of the Jak3 cDNA clone. Founders were
backcrossed to C57Bl/10 mice; transgenic progeny were then
crossed to Jak3 Western Blot Analyses.
Lysates from individual thymi or spleens
were prepared by generating a cell suspension, counting the cells,
and lysing them at 108/ml in buffer containing 1% Triton X-100.
Jak3 was immune precipitated from lysate of 1 × 107 thymocyte-
cell equivalents or 2 × 107 splenocyte-cell equivalents with an
anti-Jak3 monoclonal antibody specific to the carboxy-terminal
25 amino acids of murine Jak3 (33). Washed immune precipitates
were fractionated by SDS-PAGE, transferred to nylon membranes, and probed with an anti-Jak3 rabbit antiserum, as described previously (26).
Flow Cytometry Analysis.
Bone marrow, thymocyte, and splenocyte cell suspensions were prepared and counted for total cellularity. For flow cytometry, 5 × 105 cells were stained with directly
conjugated antibodies to CD45R (B220), CD4, CD8 (GIBCOBRL, Gaithersburg, MD) or biotinylated anti-IgM (PharMingen)
and streptavidin-Cy-Chrome (PharMingen, San Diego, CA). For
activation marker analyses, 5 × 105 splenocytes were stained with
antibodies to CD4, CD8 (GIBCO-BRL), and biotinylated antiCD44 (PharMingen) or biotinylated anti-CD62L (MEL-14) (PharMingen), followed by streptavidin-FITC (PharMingen or Southern Biotech, Birmingham, AL).
Intracellular IL-2 Assays.
5 × 105 splenocytes were plated in
96-well microtiter plates previously coated with goat anti-hamster antibody (5 µg/ml) followed by anti-CD3 antibody (5 µg/ml),
and cultured for 5 h in the presence of a 1/8 dilution of antiCD28 antibody hybridoma supernatant (determined to be saturating by cell surface staining). As a control, cells were cultured in
media alone. To inhibit secretion of newly synthesized IL-2,
stimulations were carried out in the presence of 10 µM monensin and 5 µg/ml brefeldin A (Sigma Chemical Co., St. Louis,
MO). After stimulation, the cells from 4 wells were pooled and
stained with PE-conjugated anti-CD4 antibody and biotinylated
anti-CD44 antibodies followed by streptavidin-Cy-Chrome (PharMingen). Antibody stains were carried out in staining buffer containing 10 µM monensin. Cells were fixed in 4% paraformaldehyde
for 20 min on ice, washed, and permeabilized with 0.5% saponin
(Sigma) in PBS, 1% BSA, 0.05% NaN3. Cells were then stained
with FITC-conjugated anti-IL-2 antibody (PharMingen) for 30 min on ice, washed twice with 0.5% saponin buffer, and analyzed
by flow cytometry.
T Cell Functional Assays.
T cells were stimulated by culturing
in wells coated with goat anti-hamster antibody followed by antiCD3 antibody, in the presence of anti-CD28 antibody hybridoma
supernatant as described above. As a control, cells were cultured
in media alone. For proliferation assays, 1 × 105 total thymocytes
or total splenocytes adjusted to contain 1 × 104 CD4+ T cells per
well were cultured for 48 h, then pulsed overnight with [3H]thymidine and counted. For cytokine assays, 1 × 106 total thymocytes or total splenocytes adjusted to contain 1 × 105 CD4+ T cells
per well were stimulated. Supernatants from duplicate cultures
were harvested at 24 h, and IL-2 and IL-3 levels were quantitated
by titration on indicator cells. For IL-2, 1 U/ml corresponds to
1/50 maximal proliferation of the HT-2 indicator cells (Fig. 4 A)
or 1/10 maximal proliferation of the indicator cells (Fig. 4 B). For
IL-3, 1 U/ml corresponds to 1/10 maximal proliferation of the
DA-1 indicator cells.
We considered two possible explanations for the phenotypic and functional defects in peripheral Jak3 Both transgenic lines were crossed to the Jak3
As a negative control, Jak3 Jak3
Both transgenic lines expressing wild type Jak3 in the
thymus were capable of completely reconstituting the defects in T cell maturation. Thymi from Jak3 The reconstituted Jak3
To address whether expression of the wild-type or kinase-dead Jak3 transgenes had reconstituted the function of
Jak3 Splenic T cells from the reconstituted Jak3 As expected, reconstitution of the Jak3 The above data suggested that the loss of Jak3 protein
from peripheral T cells of Jak3
These studies of Jak3 Second, the simultaneous reconstitution of normal numbers of The tgthy-reconstituted Jak3 In addition to the disappearance of proliferative ability,
the loss of Jak3 protein in peripheral T cells leads to the
acquisition of a memory cell phenotype and the loss of
cytokine secretion capacity. Interestingly, this phenotype
strongly resembles that observed in patients with a moderate combined immunodeficiency disease (XCID). The
XCID defect is a point mutation in the cytoplasmic tail of
Currently, we have not yet identified the biochemical
mechanism responsible for the aquisition of a memory cell
phenotype and the loss of cytokine secretion capacity by
the Jak3
CD8
into CD4+CD8+ thymocytes and their subsequent
expansion (1); at the CD4+CD8+ stage, the mature
TCR is required to mediate both positive and negative selection of the TCR repertoire, as well as CD4 versus CD8
lineage commitment (for review see reference 2). In addition to the TCR, signals induced by interactions of thymocytes with thymic stromal cells are critical for T cell
maturation (3), as are signaling pathways induced by soluble growth factors or cytokines (4, 5). Once T cells are mature, their ability to function properly is again dependent
on a number of different signaling pathways. Most notably,
T cell responses are contingent on an intact TCR signaling
pathway involving molecules such as Zap-70 (6), Lck
(9), Fyn (10, 11), and Vav (12). Until recently, significantly less was known about the role of other signaling
pathways, such as those initiated by costimulatory molecules or cytokines, in the maintenance of a functional and
responsive immune system.
(17) or
(18) chain of the IL-2 receptor, results in dramatic imbalances in T cell function, leading to accumulation of activated T cells and, in two of the cases, to lymphocyte infiltration into many organs and premature death of
the animals. In contrast with this, mice and humans lacking
the IL-2 receptor
chain (
c)1 (19), or the
c-associated
signaling protein, Jak3 (22), have severe combined immunodeficiency (SCID), with defects in lymphocyte maturation as well as function. The extremely severe phenotype
of
c-deficient and Jak3-deficient individuals is due to the
fact that
c is also a component of the receptors for IL-4, IL-7, IL-9, and IL-15 (27); thus, individuals deficient in
c or Jak3 have pleiotropic defects resulting from the loss of
multiple cytokine signaling pathways. Therefore, it has
been impossible to assess the role of
c or Jak3 in mature
peripheral T cell function, as a consequence of the fact that
T cell development is defective in the absence of each of
these important proteins.
c-associated signaling protein, Jak3 (24).
The phenotype of these mice strongly resembles that seen
in
c-deficient mice (19, 20). For instance, both B and T cell
development is aberrant in Jak3 mutant mice. In the bone
marrow, B cell maturation is blocked at the pre-B stage, resulting in few IgM+ B cells in adult Jak3-negative mice. In
the thymus, Jak3-deficient mice have an unusual defect in
T cell development. The thymi of the mice are extremely
small (~1-10% of normal); yet, T cell maturation appears
to progress relatively normally. In spite of these small thymi, adult Jak3
/
mice have nearly normal numbers of
CD4+ (but not CD8+) T cells in their spleens (24), although the Jak3
/
peripheral T cells are phenotypically
and functionally abnormal (26). By surface phenotype, virtually all of the Jak3
/
T cells resemble activated or memory cells, rather than naive, resting cells. Functionally, the
Jak3
/
T cells fail to proliferate in response to mitogenic
stimuli. The most unexpected finding was the severe deficiency in IL-2 secretion by Jak3
/
T cells stimulated
through their TCR plus CD28 (26).
or
chains (17, 18, 32). Instead of being hyper activated
and prone to causing autoimmune syndromes, Jak3
/
T cells
appear anergic (26). From our initial studies, the reason for
the loss of T cell function in the Jak3-deficient mice was
unclear. Since T cells in these mutant mice lack Jak3 protein at all stages of development, it was impossible to distinguish defects due solely to the absence of Jak3 in mature
T cells from defects acquired earlier during T cell maturation. Specifically, aberrant T cell development in the thymus might be responsible for the abnormal phenotype and
responsiveness of the peripheral Jak3
/
T cells. Alternatively, T cells might be functionally normal as they develop, and may acquire the unresponsive phenotype due to
the absence of specific cytokine receptor signals as mature T cells. To address this issue, we have reconstituted Jak3deficient mice with wild type Jak3 under conditions in
which Jak3 is expressed in thymocytes and then lost from
peripheral T cells. These studies demonstrate that all phenotypic and functional defects of Jak3
/
T cells result
from the absence or decreased expression of Jak3 in mature
peripheral T cells.
/
mice to generate homozygous Jak3-deficient
mice heterozygous for each Lck-Jak3 transgene.
Fig. 4.
Functional responses of Jak3/
(tgthy) T cells are restored in the thymus, but lost in the periphery. (A) Thymocytes and splenocytes from
Jak3+/
, Jak3
/
, Jak3
/
(tgthy+spl), and Jak3
/
(tgthy) mice were analyzed for proliferation (top) and IL-2 (middle) or IL-3 (bottom) secretion in response to stimulation with anti-CD3 plus anti-CD28 antibodies. All data shown are from one 35-d-old Jak3
/
(tgthy+spl) mouse and one 35-d-old Jak3
/
(tgthy) mouse (designated B). IL-2 secretion data from splenocytes of two additional Jak3
/
(tgthy) mice, one 25 d of age (designated A) and one 42 d of
age (designated C), are also shown. All values are mean ± SD. Overall, no statistically significant differences between Jak3+/
, Jak3
/
(tgthy+spl), and
Jak3
/
(tgthy) thymocytes were observed for IL-2 or IL-3 secretion responses. (B) Thymocytes and splenocytes from Jak3+/
, Jak3
/
, and Jak3
/
(tgkd)
mice were stimulated with antibodies to CD3 plus CD28. Proliferative, IL-2 secretion, and IL-3 secretion responses are shown. All values are mean ± SD.
For proliferation assays, all cell populations cultured in media alone gave responses of <500 cpm. For cytokine assays, cells cultured in media alone secreted undetectable levels of IL-2 (<0.02 U/ml) and IL-3 (<0.01 U/ml). Data are representative of 2-9 independent experiments.
[View Larger Version of this Image (38K GIF file)]
/
T cells.
First, Jak3
/
T cells might be abnormal due to defects resulting from aberrant T cell development in the thymus; alternatively, Jak3
/
T cells might be functionally and phenotypically normal when they leave the thymus, and might
acquire their defects due to the absence of Jak3 in the periphery. To investigate the stage of development at which
Jak3
/
T cells acquired their phenotypic and functional
defects, we utilized a transgenic reconstitution system. The
wild-type Jak3 cDNA (33) was placed under control of the
Lck proximal promoter (34). This vector has been used in
numerous transgenic lines to express both cell surface and
signal transduction proteins in thymocytes; in some cases,
the transgene-encoded protein is also expressed in peripheral T cells, and in other cases, transgene expression is restricted to thymocytes (36). One of our transgenic lines
expressed the Jak3 protein in both thymocytes and peripheral T cells, and therefore can serve as a positive control
(hereafter referred to as tgthy+spl). A second line was also
identified in which Jak3 was expressed in thymocytes, but
was lost in peripheral cells over time (hereafter referred to
as tgthy).
/
mice,
to generate mice homozygous for the Jak3 mutation and
heterozygous for one of the transgenes (Fig. 1 A). Analysis
of Jak3 protein levels indicated that both transgenic lines
expressed substantial amounts of Jak3 in thymocytes, while
only the Jak3
/
(tgthy+spl) line expressed levels of Jak3 comparable to wild type in the spleen (Fig. 1 B). In the Jak3
/
(tgthy) line, Jak3 protein is barely detectable in spleen cells
of a very young mouse (25 d) and becomes undetectable in
older mice (Fig. 1 B and C); in contrast, no decrease in the
levels of Jak3 protein was observed in the spleens of Jak3
/
(tgthy+spl) mice up to 120 d of age (Fig. 1 D). These findings
strongly suggest that Jak3 is constitutively expressed in peripheral T cells in the Jak3
/
(tgthy+spl) mice, as the majority of T cells in a 4-mo-old mouse are not recent thymic
emigrants, but cells that have been out of the thymus for
several months. In contrast, the loss of detectable Jak3 protein from the spleen cells of older Jak3
/
(tgthy) mice suggests that, in this transgenic line, the peripheral Jak3 protein
observed in young animals is the residue of thymic Jak3 expression. However, we cannot rule out the possibility that the difference in detectable Jak3 expression between these
two transgenic lines results from a difference in the dose of
expression of the transgenes.
Fig. 1.
Reconstituted expression of Jak3 in Jak3/
mice
with Lck-Jak3 transgenes. (A)
Southern blot of EcoRI-digested
tail DNA from mouse pups indicating the genotyping of the endogenous Jak3 locus (Jak3+/
,
lanes 1, 2, 4; and Jak3
/
, lanes 3 and 5), as well as the wild-type
Jak3 transgenes (tgthy+spl, lanes 3 and 4; and tgthy, lanes 1 and 5).
The blot was probed with an
0.35-kb EcoRI-HindIII fragment of the Jak3 cDNA clone
(33). (B, C, D) Protein immunoblots of Jak3 immunoprecipitates from thymocytes and splenocytes of the indicated mice.
Lysates were from Jak3
/
(tgthy+spl) and Jak3
/
(tgthy) mice
at 25 d of age (B) or 33 d of age
(C). In (D), lysates were from a
Jak3
/
(tgthy+spl) mouse at 120 d
of age and a Jak3
/
(tgkd) mouse
at 49 days of age. Jak3 was immunoprecipitated using an antiJak3 monoclonal antibody (33)
from lysate of 1 × 107 thymocytes or 2 × 107 splenocytes.
The membranes were probed with
anti-Jak3 rabbit antiserum (26).
[View Larger Version of this Image (44K GIF file)]
/
mice were also reconstituted with a kinase-dead Jak3 construct driven by the Lck
proximal promoter (hereafter referred to as tgkd) (Fig. 1 D).
For these experiments, we utilized a Jak3 cDNA carrying a mutation in the codon for the conserved lysine residue
present in all protein kinase domains (43). Substitution of
Arg for Lys at this position (residue 851) eliminates all detectable tyrosine kinase activity of Jak3 (33). The kinasedead Jak3 protein was expressed in both thymocytes and
peripheral T cells at levels comparable to those found in the
Jak3+/
control (Fig. 1 D).
/
(tgthy+spl), Jak3
/
(tgthy), and Jak3
/
(tgkd) mice
were analyzed to determine the reconstitution of both the
B and T cell lineages. Flow cytometry analysis of bone
marrow cells indicated that no reconstitution of B cell development had occurred in any of these lines, as assessed by
the lack of CD45R (B220)+ IgM+ cells (Fig. 2 A). Staining
of bone marrow cells with antibodies to CD43 and CD45R
(B220) also indicated that the block in B cell development
observed in the Jak3
/
mice is not corrected with any of
the Lck promoter-driven Jak3 transgenes (data not shown).
Analysis of B cells in the spleen demonstrated a reduced
level of CD45R (B220)+ IgM+ cells in the Jak3
/
mice expressing either wild-type or kinase-dead Jak3 transgenes compared with the Jak3+/
control (Fig. 2 A).
Fig. 2.
Both wild-type Jak3
transgenes reconstitute T cell,
but not B cell, development in
Jak3/
mice. (A) The bone
marrow, thymus, and spleen cells
of Jak3+/
, Jak3
/
, Jak3
/
(tgkd), and 35-d-old Jak3
/
(tgthy+spl) and Jak3
/
(tgthy) mice
were stained with the indicated
antibodies and analyzed by flow
cytometry. Staining is shown on
a logarithmic scale of fluorescence intensity. Numbers in the
quadrants indicate subpopulation percentages. The dot plots
are representative of average
staining profiles, although some
Jak3
/
individuals had greatly
increased CD4+/CD8+ ratios in
the thymus and spleen. (B) The
total cellularity of bone marrow,
thymus, and spleen of mice analyzed in these experiments is indicated. For each organ, Jak3+/
,
lane 1; Jak3
/
, lane 2; Jak3
/
(tgthy+spl), lane 3; Jak3
/
(tgthy),
lane 4; and Jak3
/
(tgkd), lane 5 are shown. Note the reconstitution of normal thymocyte cellularity by both wild-type, but not
the kinase-dead, Jak3 transgenes. Data shown are representative of
greater than six independent experiments.
[View Larger Version of this Image (47K GIF file)]
/
(tgthy+spl)
and Jak3
/
(tgthy) mice were reconstituted to the normal
number of cells (Fig. 2 B). In addition, the increased CD4+/
CD8+ ratio often observed in the thymi of Jak3
/
mice
was not observed in thymuses of either line of Jak3
/
mice reconstituted with wild-type Jak3. Staining of spleen
cells from the Jak3
/
(tgthy+spl) and Jak3
/
(tgthy) mice also
demonstrated the recovery of normal T cell maturation (Fig. 2 A). In particular, the Jak3
/
mice generally lack
peripheral CD8+ T cells, a defect that is corrected in both
of these reconstituted lines. In contrast, the kinase-dead
Jak3 could not reconstitute thymus cellularity or CD8+ cell
development (Fig. 2 A and B). Another feature of the
Jak3
/
mice is that they lack
T cells and NK cells (25);
in addition, peripheral lymph nodes are nearly undetectable
(24). The Jak3
/
mice reconstituted with wild type
Jak3 have normal numbers of CD4
CD8
TCR+ cells in
their thymus and normal numbers of CD4
TCR
NK1.1+
cells in their spleen; however, they still lack lymph nodes
(data not shown).
/
mice were examined for the
surface phenotype and function of their T cells. Splenic
T cells were stained with antibodies to CD4, CD8, and a
panel of activation markers. Analysis of CD44 and CD62L
(MEL-14) levels on gated CD4+ T cells indicate that Jak3
/
T cells resemble activated or memory T cells, expressing
high levels of CD44 and low levels of CD62L (Fig. 3). The
T cells in the Jak3
/
(tgthy+spl) mice are completely restored to normal, appearing indistinguishable from control
(Jak3+/
) T cells at all ages analyzed (Fig. 3; data not shown).
In contrast, the splenic CD4+ T cells from the Jak3
/
(tgkd) mice are indistinguishable from Jak3
/
T cells (Fig.
3). Most interestingly, CD4+ T cells from Jak3
/
(tgthy)
mice have a cell surface phenotype that appears to correlate with peripheral Jak3 protein expression (Fig. 3). In the
youngest Jak3
/
(tgthy) mouse shown (25 d of age), where
Jak3 protein is still detectable in the spleen (see Fig. 1 B),
the majority of CD4+ T cells are CD44lo and CD62Lhi. In
an older mouse (35 d of age), where Jak3 protein is no
longer detectable in the spleen (see Fig. 1 C), two populations of T cells can be seen. In an even older mouse (42 d
of age), most of the T cells in the Jak3
/
(tgthy) mouse are
CD44hi and CD62Llo, and resemble the Jak3
/
T cells.
This gradual appearance of phenotypically aberrant peripheral T cells, which correlates with the age of the mice, indicates that Jak3 protein is constitutively required to maintain
a normal population of resting T cells. In total, nine Jak3
/
(tgthy) mice and eleven Jak3
/
(tgthy+spl) mice have been
analyzed; in all cases, the peripheral T cells from the Jak3
/
(tgthy+spl) mice resembled wild-type T cells, whereas the
peripheral T cells from the Jak3
/
(tgthy) mice had a surface phenotype that roughly correlated with the age of the
mice. However, some variation in the precise age at which
the vast majority of Jak3
/
(tgthy) peripheral T cells acquired the Jak3
/
surface phenotype was observed, most
likely owing to variations in the loss of Jak3 protein from
these cells. Overall, a comparable pattern of activation
marker expression is observed on splenic CD8+ T cells in
all mice analyzed (data not shown).
Fig. 3.
Splenic CD4+ T
cells from Jak3/
(tgthy) mice
acquire increasing numbers of
phenotypically activated T cells
as the mice age. Splenocytes from Jak3+/
, Jak3
/
, Jak3
/
(tgthy+spl), Jak3
/
(tgkd), and
three Jak3
/
(tgthy) mice of different ages were stained with antibodies to CD4, CD8, and
CD44 or CD62L (MEL-14).
The staining of CD44 (top) and CD62L (bottom) on gated
CD4+ T cells is shown on a logarithmic scale of fluorescence intensity. All histograms are directly comparable except the
staining of the 25-d-old Jak3
/
(tgthy) mouse, which was performed with a different lot of
streptavidin-FITC, resulting in a
brighter overall level of CD44
and CD62L fluorescence. Data
shown are representative of
greater than six independent experiments.
[View Larger Version of this Image (36K GIF file)]
/
T cells, thymocytes and splenic T cells from the
Jak3
/
(tgthy+spl), the Jak3
/
(tgthy), and the Jak3
/
(tgkd)
mice were assessed for proliferation and cytokine production in response to TCR and CD28 costimulation. A set of
data, representative of a total of nine independent experiments performed with different sets of mice, is shown in
Fig. 4. Jak3
/
thymocytes are substantially reduced in
their proliferative response to CD3 plus CD28 stimulation.
As expected, thymocytes from both lines reconstituted
with wild type Jak3, but not with kinase-dead Jak3, have
restored proliferative capacity (Fig. 4, A and B). The incomplete reconstitution of proliferative capacity with wild type Jak3 most likely results from the failure of the transgenes to be induced following activation. The massive induction of the endogenous Jak3 gene observed after T cell
activation (44) may be essential to sustain a vigorous proliferative response. To test the thymocytes for their cytokine
secretion responses, supernatants from anti-CD3 plus antiCD28-stimulated thymocytes were harvested and assayed
for the presence of IL-2 and IL-3. Jak3
/
thymocytes
make substantially less IL-2 and IL-3 than the control (Jak3+/
) thymocytes (Fig. 4). As expected, thymocytes
from both Jak3
/
(tgthy+spl) and Jak3
/
(tgthy) mice (Fig.
4 A), but not the Jak3
/
(thykd) mice (Fig. 4 B), are restored in their ability to produce IL-2 and IL-3 in response
to stimulation. These data demonstrate that expression of
wild type Jak3 in thymocytes restores the functional capacity of Jak3
/
cells to secrete cytokines when stimulated.
/
mice were
also assessed for their ability to respond to TCR plus CD28
stimulation. Proliferative responses of Jak3
/
splenic T cells
are virtually absent compared with Jak3+/
control cells; in
addition, IL-2 secretion by Jak3
/
T cells is also substantially reduced (Fig. 4, A and B). T cells from Jak3
/
(tgkd)
mice are indistinguishable from Jak3
/
cells for both proliferative and cytokine secretion responses (Fig. 4 B). The
splenic T cells from both the Jak3
/
(tgthy+spl) mice and
the Jak3
/
(tgthy) mice have a limited capacity to proliferate in response to TCR plus CD28 stimulation (Fig. 4 A).
Most likely, this is due to the fact that maximal proliferative
responses depend on both the initial level of Jak3 protein,
as well as the amount of Jak3 protein that can be induced after stimulation. Accordingly, we find that proliferative responses in the two transgenic lines are not consistent between experiments, and do not correlate well with the
observed levels of Jak3 protein in resting peripheral T cells.
Thus, we include these proliferative responses to demonstrate that reconstituting Jak3 expression does restore some
proliferative capacity to the Jak3
/
cells, even though quantitative conclusions from these data are not possible.
/
mice with the
(tgthy+spl) transgene restored normal IL-2 secretion from
stimulated splenic T cells (Fig. 4 A). In contrast, IL-2 secretion by the Jak3
/
(tgthy) splenic T cells varied between
individuals, and correlated with the surface phenotype of
the T cells (see Fig. 3). In the Jak3
/
(tgthy) mouse in
which the vast majority of cells resembled normal resting T cells, IL-2 secretion was normal (Fig. 4,
/
tgthy-A). In
comparison, splenic T cells from a Jak3
/
(tgthy) mouse in
which ~40-50% of the splenic T cells were CD44hi and
CD62Llo, secreted substantially less IL-2 when stimulated
(Fig. 4,
/
tgthy-B). Finally, in a Jak3
/
(tgthy) mouse in
which the vast majority of cells were CD44hi and CD62Llo,
splenic T cells were severely defective in secreting IL-2
(Fig. 4,
/
tgthy-C). Analysis of IL-3 secretion supports
these basic conclusions (Fig. 4). These data indicate that
sustained Jak3 expression is required in peripheral T cells to
maintain the resting surface phenotype and T cell function.
/
(tgthy) mice results in the
gradual increase of phenotypically activated CD4+ T cells
and the reduced capacity of these cells to synthesize cytokines when stimulated. To test whether the loss of cytokine production was associated with the change in activation
marker surface expression, we assessed the functional capability of CD44lo and CD44hi CD4+ T cells separately. For
these experiments, T cells were stimulated with anti-CD3
plus anti-CD28 for 5 h and the cytoplasmic IL-2 levels of
CD44lo and CD44hi CD4+ T cells were measured by flow
cytometry. This substantially shorter assay for IL-2 production also addresses the possibility that the decreased cytokine secretion we observed from stimulated Jak3
/
, Jak3
/
(tgthy), and Jak3
/
(tgkd) peripheral T cells might result
from increased apoptosis of these cells compared with
Jak3+/
T cells over the course of the usual 24-h stimulation period. As shown in Fig. 5, 19.0% of CD44lo and
11.0% of CD44hi CD4+ splenic T cells from the Jak3+/
mouse had detectable levels of intracytoplasmic IL-2 after 5 h
of stimulation. In contrast, CD3 plus CD28 stimulation did
not result in any significant IL-2 production by CD44lo
T cells from Jak3
/
mice, whereas 2.9% of CD44hi T cells
stained positive for intracellular IL-2. The IL-2 profiles of
stimulated CD4+ T cells from three different Jak3
/
(tgthy)
mice demonstrated that the percentage of IL-2+ cells decreases as the number of phenotypically activated CD44hi
T cells increases. These data support the conclusion that
peripheral T cells in Jak3
/
(tgthy) mice acquire the activated surface phenotype and lose responsiveness in parallel,
and that the functional defects in these cells are not due to
increased apoptosis.
Fig. 5.
Functional deficiencies of Jak3/
and Jak3
/
(tgthy) T cells are confirmed by
intracytoplasmic IL-2 staining.
Splenocytes from a Jak3+/
, a
Jak3
/
, and three Jak3
/
(tgthy)
mice were cultured for 5 h in
medium alone or with anti-CD3 plus anti-CD28 antibodies. Cells
were harvested, stained with antibodies to CD4, CD44, and
IL-2. CD44 staining on freshly
gated CD4+ cells is shown at left;
dot plots of IL-2 versus CD44
staining of gated CD4+ cells cultured in either medium alone or
stimulated with CD3 plus CD28
are shown at right. CD44 stainings are shown on a logarithmic scale of fluorescence intensity;
IL-2 staining is shown on a linear scale. Data are representative of
three independent experiments.
[View Larger Version of this Image (49K GIF file)]
/
mice expressing wild-type or
kinase-dead Jak3 from the Lck proximal promoter have
demonstrated several important features about the role of
Jak3 in T cell maturation and function. First, the failure of
the kinase-dead Jak3 gene to reconstitute any of the T cell
defects in the Jak3
/
mice indicates that Jak3 kinase activity is essential for all the functions of Jak3 assessed in these
experiments. For instance, the small thymus size in the
Jak3
/
mice is presumed to result from the absence of IL-7
receptor signaling (4, 5). As the kinase-dead Jak3 protein is
likely to be fully functional in binding to
c (45, 46), the
phosphorylation of Jak3 by Jak1 in response to IL-7 binding might have been hypothesized to restore partial function to the IL-7 receptor. A similar situation might also
have occurred in mature thymocytes or peripheral T cells
in response to IL-2 binding. Yet no thymocyte expansion in vivo, or T cell proliferation in response to TCR stimulation in vitro, was observed in tgkd-reconstituted Jak3
/
mice. This requirement for Jak3 kinase activity is in direct contrast with the ability of kinase-dead Jak1 to reconstitute IFN-
-inducible gene expression in Jak1
cells (47).
TCR+ thymocytes,
TCR+ thymocytes, and
NK cells indicates that the Lck proximal promoter is active
in these three lineages of lymphocytes or in a common precursor to these three cell types. Because we failed to reconstitute B cell development in the bone marrow in these
mice, we conclude that the wild-type Jak3 transgenes are
not expressed in a bone marrow progenitor cell that gives
rise to both B and T lymphocytes. Therefore, these data
substantiate a close lineage relationship between T lymphocytes and NK cells (48, 49), and suggest the existence of a
common
T cell-
T cell-NK cell progenitor that cannot give rise to B lymphocytes. We do not know whether
the expression of the Lck promoter-driven Jak3 genes initiates in the thymus, or in an earlier bone marrow-derived
progenitor cell, as we cannot detect any expression of the
Jak3 transgenes in bone marrow (data not shown).
/
mice provide a system
for distinguishing the roles of Jak3 during T cell development from Jak3 function in peripheral T cells. The normal
level of Jak3 expression in thymocytes from these mice corrects virtually all the detectable defects of Jak3
/
thymocytes. In Jak3
/
(tgthy) mice of all ages, thymocyte numbers are increased to normal, the production of peripheral
CD4+ and CD8+ T cells is restored to normal, and cytokine secretion by thymocytes in response to stimulation is
restored. Nonetheless, peripheral T cells in these tgthyreconstituted Jak3
/
mice slowly acquire all the defects of
Jak3
/
T cells. Although the precise kinetics of this effect
vary between individual animals, the Jak3
/
(tgthy) splenic
T cells eventually become phenotypically and functionally deficient in an identical manner to the Jak3
/
T cells; in
all mice analyzed this process appears complete by 5-6 wk
of age. These results demonstrate that the maintenance of
normal levels of Jak3 protein in mature peripheral T cells is essential to preserve the continued function of these cells. This requirement is met in the tgthy+spl-reconstituted Jak3
/
mice, which retain normal T cell phenotype and function
at all ages analyzed.
c that diminishes, but does not abolish, Jak3 binding (45).
XCID patients have substantial numbers of T cells, but those T cells are deficient in proliferative responses, have an activated/memory phenotype, and are impaired in IL-2 secretion after stimulation (50). Thus, individuals with an
impaired Jak3-
c interaction have sufficient Jak3 function
to generate T cells in the thymus, unlike the SCID patients
completely lacking Jak3 or
c expression; yet the peripheral
phenotype of these T cells mimics that seen in our Jak3
/
mice reconstituted with the tgthy transgene.
/
(tgthy) peripheral T cells. One possible mechanism is suggested by the studies of Nakajima et al. using
c
mice (51). Their studies indicate that the mutant T cells
may be receiving activation signals from self- or environmental antigens; owing to their loss of IL-2 receptor signaling, the T cells then may be driven into a state of anergy in
response to these activation events (52, 53). However, our
intracellular IL-2 staining data indicate that even phenotypically naive T cells lose the capacity to secrete IL-2 when
they lose Jak3 expression. This impaired ability of phenotypically normal Jak3-deficient cells to secrete cytokines
when stimulated also suggests the possibility of a previously
unsuspected role for Jak3 in TCR or CD28 signaling. In
fact, both of these mechanisms may be involved and may
contribute together to the numerous T cell defects observed in the Jak3-deficient mice.
Address correspondence to Leslie J. Berg, Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138.
Received for publication 5 August 1996
This work was supported by the American Cancer Society (L.J. Berg) and the National Institutes of Health (L.J. Berg). D.C. Thomis is a SmithKline Beecham Pharmaceuticals Fellow of the Life Sciences Research Foundation.We thank Roger Perlmutter for the Lck proximal promoter vector; Karen Sporny for technical assistance; and Charles Sagerström, Deborah Yelon, and Stephanie Heyeck for critical reading of the manuscript.
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