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Chiroscience R&D, Inc., Seattle, WA 98021; and
Oak Ridge National Laboratory, Oak Ridge, TN 37830
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Males hemizygous (Xsf/Y) for the scurfy mutation develop a progressive lymphocytic infiltration of the lymph nodes, spleen, liver, and skin, resulting in gross morphologic symptoms that include splenomegaly, hepatomegaly, greatly enlarged lymph nodes, runting, exfoliative dermatitis, and thickened malformed ears 7, 8 . Other clinical symptoms include elevated leukocyte counts, hypergammaglobulinemia, and severe anemia 6 ; the death of affected males usually occurs by 3 wk of age. The sf locus has been mapped to the extreme proximal region of the X chromosome, approximately 0.7 cM from the locus for sparse-fur (spf) 6, 10 , itself a point mutation within the ornithine transcarbamylase gene (Otc) 11 . The sf locus is also tightly linked to the murine Gata1, Tcfe3, and Wasp loci 10, 12 . Similarities between scurfy and human Wiskott-Aldrich syndrome have been noted 6 , and the mouse Wasp gene has been proposed as a candidate for scurfy 6, 12 . Closer biologic examination reveals significant differences between Wiskott-Aldrich syndrome and scurfy, however, and the two loci have been demonstrated to be nonallelic (Jeffery & Brunkow, unpublished data). Thus, the identity of the scurfy gene remains to be determined.
Disease in scurfy mice has been shown to be primarily mediated by
CD4+CD8- T lymphocytes 8, 9, 13 , suggesting
that the sf gene plays an important role in regulating T
cell function. This T cell defect is primarily manifested as a
generalized overproduction of cytokines. Dysregulated expression of a
variety of cytokine genes, including IL-2, IL-4, IL-5, IL-6, IL-10,
IFN-
, and TNF-
, has been demonstrated in scurfy mice at the
levels of mRNA and protein expression 13, 14 . Excessive production of
these cytokines correlates well with the range of pathologic changes
observed in scurfy mice and may be the proximal cause of scurfy
immunopathology, yet the cellular and molecular mechanism(s) leading to
cytokine dysregulation and ultimately to pathology in scurfy mice
remain unknown.
The following functional and phenotypic studies of scurfy T cells were undertaken to further our understanding of the disease mechanism(s) underlying scurfy syndrome. Using flow-cytometric analysis, we demonstrate that the onset of scurfy disease is characterized by an increased relative abundance of Mac-1+ cells and a corresponding drop in the relative abundance of B220+ cells in lymphoid tissues. We also show that scurfy T cells produce extremely high levels of the cytokine GM-CSF,3 a differentiation factor for granulocytic and monocytic cells that is produced by activated T cells and is known to suppress B lymphopoiesis 15 . Cell surface staining of CD4+ T cells from scurfy mice and normal controls shows that scurfy T cells express elevated levels of activation-related Ags, including CD69, CD25 (IL-2R), and B7-1/B7-2.
Although CD4+ T lymphocytes from scurfy mice exhibit an activated surface phenotype, they are not constitutively activated, but rather are hyperresponsive to TCR stimulation. In this study, we demonstrate that scurfy CD4+ cells exhibit the same requirement as normal T cells for two activation signals, yet differ markedly from normal cells in the magnitude of responsiveness to these signals. The heightened expression of B7 on scurfy cells may contribute to the hypersensitivity of scurfy T cells to activation in vitro. Our data are consistent with a model in which scurfy syndrome results from a biochemical defect that interferes with the normal down-regulation of T cell activation responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The original scurfy mutation arose spontaneously in the partially inbred MR stock at Oak Ridge National Laboratory (Oak Ridge, TN) in 1949. A strain doubly mutant for sf and the closely-linked sparse-fur (spf) mutation was generated 7 and has been maintained by mating sf spf/++ females to (C3Hf/Rl x 101/Rl)F1 or (101/Rl x C3Hf/Rl)F1 males. The spf mutation appears to have no effect on the scurfy phenotype. Doubly mutant carrier females were obtained from Oak Ridge National Laboratory, and the stock continued to be maintained by crossing to (101/Rl x C3Hf/Rl)F1 males. Animals were housed in a conventional environment with a standard pelleted diet and reverse osmosis water fed ad libitum. All animals used in these functional studies were obtained from doubly mutant progeny-tested females. Carriers of spf were identified directly by PCR amplification of a 171-bp fragment from the Otc gene 11 . Upon digestion of the PCR products with MseI, nonmutant DNA produced fragments of 43 and 128 bp, while mutant DNA produced fragments of 43, 93, and 35 bp. The primers used were 5'-TCTGCTGGGAGGACACCC-3' and 5'-GGCATTATCTAAGGAGAAGCATCA. Mice aged 10–15 days were used for all experiments, except where noted.
T cell activation cultures
Lymph node, spleen, and thymic tissues removed from scurfy mice and normal littermate control (NLC) animals were macerated in culture media between the ground glass ends of sterile microscope slides, filtered through a sterile 70-µm nylon mesh, collected by centrifugation, and cultured at 37°C in complete RPMI (cRPMI) (10% FBS, 0.05 mM 2-ME, 15 mM HEPES, 100 U/ml penicillin, and 100 µg/ml each streptomycin and glutamine) in 96-well round-bottom tissue culture plates. Culture wells were prepared for T cell activation cultures by preincubation with purified anti-TCRß Ab (clone H57-597) in sterile PBS for 2 h at 37°C. Each well was rinsed twice with sterile PBS to remove nonimmobilized Ab before the initiation of T cell cultures. Purified anti-mouse CD28 (clone 37.51) and hamster IgG isotype control (anti-mouse keyhole limpet hemacyanin) (PharMingen, San Diego, CA) were coimmobilized on culture wells when used in functional assays.
Proliferation assays
T cells were cultured for proliferation assays at a cell density of 5 x 104 cells/well (sort purified) or 105 cells/well (whole tissue suspensions) in 200 µl of cRPMI and incubated at 37°C for 72 h. Individual wells were pulsed with 1 µCi/well of [3H]thymidine (Amersham Life Science, Arlington Heights, IL) for the last 8–12 h of culture. Proliferation data reported are based upon mean value of triplicate wells and represent a minimum of three experiments.
Cytokine assays
Supernatants (100 µl) were collected from activation cultures at 48 h poststimulation, and their cytokine levels were determined by the following assays:
IL-2
IL-2 levels were determined using a Cytoscreen murine IL-2 immunoassay kit from BioSource International (Camarillo, CA), according to the manufacturer’s directions. The minimum detection limit for this assay is 8 pg/ml IL-2.
IL-2/IL-4
The combined production of IL-2 and IL-4 was measured using a cellular proliferation assay utilizing the IL-2-dependent cell line HT-2 (clone A5E; American Type Culture Collection, Manassas, VA), which also proliferates in response to IL-4. A total of 104 HT-2 cells/well was cultured in 96-well round-bottom plates together with 100 µl of conditioned supernatants removed from 3-day scurfy or NLC T cell cultures. HT-2 assays were incubated at 37°C for 72 h in 200 µl final volume cRPMI and pulsed with [3H]thymidine for the last 8–12 h of culture. The minimum detection limit for this assay is 0.2 ng/ml (IL-2).
GM-CSF
GM-CSF levels were determined using a Quantikine murine GM-CSF immunoassay kit from R&D Systems (Minneapolis, MN), according to the manufacturer’s instructions. The minimum detection limit for this assay is 4 pg/ml.
Cytofluorometric analysis
Thymus, lymph node, and splenic tissues collected as described
above were resuspended for fluorescence staining in staining buffer
(1% BSA, 0.1% sodium azide in PBS) at a cell density of 20 x
106/ml. Cell aliquots were treated with 2% normal mouse
serum (Sigma, St. Louis, MO) to block nonspecific binding, then stained
by incubation on ice for 30 min with combinations of the following
fluorochrome-conjugated anti-mouse mAbs: B220/Ly-5 (clone RA3-6B2,
rat IgG2a), CD3 (clone 500-A2, hamster IgG), CD8ß (clone CT-CD8b, rat
IgG2a), CD4 (clone CT-CD4, rat IgG2a), CD11b/Mac-1 (clone M1/70.15, rat
IgG2a), CD25 (clone PC61 5.3, rat IgG1), IgG2a control (Caltag
Laboratories, Burlingame, CA); CD28 (clone 37.51, hamster IgG), CD45RB
(clone 23G2, rat IgG2a,
), CD69 (clone H1.2F3, hamster IgG),
CD80/B7-1 (clone 16-10A1, hamster IgG), CD86/B7-2 (clone GL1, rat
IgG2a,), and CTLA-4 (clone UC10, 4F10, hamster IgG) (PharMingen).
The fluorescence intensity of approximately 105 cells was examined using a MoFlo flow cytometer (Cytomation, Fort Collins, CO) and analyzed with Cyclops (Cytomation) software. Cell doublets and monocytic cells were eliminated from the analysis on the basis of forward and side light scatter gates, and dead cells were excluded by propidium iodide (10 µg/ml) staining. Data typically are shown for pooled cells from two to four mice per experiment.
Fluorescence-activated cell sorting
CD4+ T lymphocytes were sort purified from lymph nodes for functional assays, as follows: lymph node cells were fluorescence stained by incubation on ice at a density of 20 x 106/ml in sterile staining buffer (no sodium azide) for 20 min with FITC-conjugated anti-mouse B220 and anti-mouse CD8ß (Caltag Laboratories) at a final concentration of 5 µg/ml. The FITC-stained target population consisting of B cells and CD8+ T lymphocytes (approximately 30–40% of the total cell population) was negatively sorted from the desired CD4+ T lymphocyte population by cell sorting using MoFlo and Cyclops (Cytomation) software. Typical sort purities, as determined by postsort analysis, were 97–99% of the target population and approximately 90–95% CD4+.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The gross features of the scurfy mutation have been described
previously 6, 7, 8 . Neonatal scurfy pups are indistinguishable from NLC
until several days of age, but rapidly exhibit runting and failure to
thrive. Scurfy mice subsequently become increasingly moribund until
their deaths at 2–3 wk of age. To define better the changes in the
lymphoid system that accompany this rapid disease progression, we
performed flow cytometric analysis of immunocytes from normal and
scurfy animals. Although disease progression in the scurfy mouse has
been attributed to CD4+ T cells, the disease was most
noticeably manifest by alterations in the relative abundance of B cells
and macrophages between scurfy mice and normal littermates (Fig. 1
).
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). Although monocytic cells
would be expected to be large and granular in nature, CD4+
cells from scurfy mice also display a significant size increase. This
also correlates with the increase in total number of cells in scurfy
mice. By days 5–10, scurfy mice routinely have between 3 to 10 times
as many lymph node cells as their littermate controls, with up to 20
times as many cells by 15 days of age, with virtually all lymph nodes
affected. Thus, as disease progresses, there is a large increase in
Mac-1+ cells and a smaller, but significant increase in the
number of CD4+ cells. CD4+ scurfy T cells produce elevated levels of GM-CSF
GM-CSF, a cytokine produced by activated CD4+ T cells,
is a differentiation factor for granulocytic and monocytic cells and
has been shown experimentally to inhibit B lymphopoiesis in vivo 15 .
Northern blots, bioassays, PCR, and in situ hybridization studies have
shown that scurfy tissues overexpress mRNA from a wide variety of
cytokine genes 13, 14 . In light of these reports and our observations
of altered subset ratios of Mac-1+ and B220+
cells in scurfy lymphoid tissues, levels of GM-CSF produced by cultured
CD4+ T cells from scurfy mice and normal controls were
determined by ELISA. In response to TCR stimulation, CD4+ T
cells freshly explanted from scurfy mice produced levels of GM-CSF more
than 1000-fold greater than normal controls (Table I). In contrast to NLC, scurfy T cells
cultured in vitro without stimulation also produced detectable levels
of GM-CSF (approximately 8 pg/ml). In addition, GM-CSF was also
detected in the sera of several scurfy pups at pg/ml levels, but was
never detected in the sera of NLC pups (data not shown). Hence, the
elevated levels of GM-CSF in scurfy animals most likely account for the
proportional reduction in B cells and elevated percentage of
macrophages reported in this work.
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To evaluate the activation status of CD4+ T cells, we
performed three-color flow-cytometric analysis on freshly explanted
lymphoid tissues from scurfy and age-matched NLC. When compared with
NLC, CD4+ T cells from scurfy mice were found to express
increased levels of cell surface activation markers. The expression of
both CD25 (IL-2R) and CD69 was analyzed on unstimulated
CD4+ lymph node cells from scurfy mice and NLC at 12 days
of age (Fig. 2). The percentage of cells
staining positive for CD69 was consistently four- to fivefold greater,
and the percentage of cells staining positive for CD25 was five- to
eightfold greater on scurfy cells relative to NLC. Scurfy cells also
displayed a decreased expression of Mel-14/CD62L and an increased
percentage of CD45RBlow cells (data not shown). Stains of
splenic lymphocytes from scurfy mice and NLC yielded similar results.
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). The percentage of cells
expressing elevated levels of B7.1 is dramatically increased compared
with NLC, whereas the effect on B7.2, while significant, is more
modest. Following optimal induction using LPS, both NLC and scurfy B
cells express comparably high levels of B7 (data not shown).
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). Expression of these ligands was also
significantly higher on TCR-stimulated CD4+ scurfy T cells
relative to NLC after 48 h in culture (Table II). B7.1, in
particular, was very highly expressed on scurfy cells (seven- and
eightfold greater expression than NLC, respectively) on both
nonstimulated and 48-h stimulated CD4+ T cells. These data
demonstrate a heightened state of activation for scurfy cells in vivo
and a phenotype that includes increased levels of costimulatory ligands
on T cells and splenic non-T cells.
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As our data show an activated phenotype for CD4+ T
lymphocytes, cells that are also thought to be the critical effectors
of scurfy disease 13 , we examined the proliferation requirements of
scurfy and normal T cells in vitro. Initially, T cells from whole lymph
node suspensions were used; no effort was made to remove
non-CD4+ cells from the heterogeneous population. In
contrast with normal controls, lymph node cells freshly removed from
scurfy mice underwent low, but significant levels of spontaneous
proliferation in the absence of TCR stimulation (Fig. 4A). When stimulated with an
immobilized mAb directed against TCRß, proliferation was enhanced
approximately sixfold, suggesting that scurfy T cells are not
constitutively activated, but retain a requirement for TCR engagement
for optimal responsiveness. The amount of proliferation of scurfy and
normal lymph node cells was approximately equivalent in the presence of
high dose (10 µg/ml) anti-TCR stimulation.
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B, highly
purified CD4+ cells from scurfy animals still incorporate a
small but reproducible amount of thymidine without exogenous
stimulation. These cells are also highly sensitive to stimulation
through their TCR. Very low doses of Ab to the TCR triggered strong
proliferation by scurfy, but not normal, CD4+ cells. This
proliferative response is also signified by a more rapid response as
well as a maximal response to lower doses of stimulating Ab. We have
purified CD4+ cells by both positive (CD4 selection) and
negative (removal of B220+ and CD8+ cells) cell
sorting and demonstrated similar results. Scurfy cells exhibit a decreased requirement for exogenous costimulation
The B7-CD28/CTLA-4 costimulatory pathways provide signals critical for the modulation of T cell activation and effector function 16, 20, 21 . Following TCR engagement, ligation of CD28 on T cells by B7-1 (CD80) or B7-2 (CD86) is required to costimulate the activation of normal T cells. Because scurfy CD4+ cells express B7 when examined directly ex vivo, these cells may not require the addition of any further source of costimulation. To address this, CD4+ T cells from scurfy and NLC were sort purified by negative selection and compared for their ability to proliferate and produce IL-2 in the absence of exogenous costimulation by APCs or an anti-CD28 mAb.
The proliferation of purified anti-TCR-stimulated CD4+
scurfy T cells was modestly but reproducibly increased by the presence
of a costimulatory anti-CD28 mAb, but not by a control Ig,
suggesting that the CD28 costimulatory pathway is functionally intact
in scurfy cells. However, the observed increases in proliferation were
significantly less for scurfy than for NLC cells; under identical
culture conditions, the addition of anti-CD28 to CD4+
NLC cells resulted in eightfold increases in
[3H]thymidine incorporation (Fig. 5A). In contrast with normal
cells, purified anti-TCR-stimulated CD4+ scurfy T cells
produced high levels (1.5–2 ng/ml) of IL-2 in the absence of exogenous
costimulation and exhibited only a two- to fourfold increase in IL-2
production in the presence of anti-CD28. IL-2 production by NLC was
not detected in the absence of anti-CD28 costimulation, but NLC
produced high levels of IL-2 (approximately 3.5 ng/ml) when
costimulated by anti-CD28 (Fig. 5B). Together, these
data demonstrate that scurfy T cells are hyperresponsive to TCR
stimulation and exhibit a decreased requirement for exogenous
costimulation by anti-CD28 relative to normal controls, but that
the CD28 signaling pathway is intact.
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In an effort to characterize signal transduction pathway(s) that
may play a role in the hypersensitivity of scurfy T cells to TCR
signaling, the effector function of scurfy and NLC T cells was analyzed
following exposure to metabolic inhibitors of known specificity. The
inhibitors used in these experiments included herbimycin A, a protein
tyrosine kinase (PTK) inhibitor that down-regulates TCR signal
transduction via inhibition of tyrosine phosphorylation of TCR
chain
22 ; genistein, a competitive inhibitor of ATP known to block PTKs
implicated in T cell signal transduction, including
p56lck and
p59fyn 23, 24 ; and cyclosporin A (CsA),
a potent immunosuppressive agent that blocks nuclear translocation of
the transcription factor NF-AT via inactivation of
Ca2+/calmodulin-dependent calcineurin phosphatase 25 . In
these experiments, effector function was measured by the proliferation
of a T cell clone, HT-2, in response to IL-2 and IL-4 released into
conditioned supernatants from scurfy and NLC cultures. Our functional
data revealed that T cells from scurfy mice are refractory to
inhibition by CsA and inhibitors of PTKs. In proliferation assays, the
concentration of CsA for which HT-2 proliferation was inhibited by 50%
of the maximum level (IC50 dose) for scurfy cells
(mean = 106 ng/ml) was approximately 10–15-fold higher than the
IC50 of normal controls (mean = 6.6 ng/ml) (Fig. 6). The IC50 concentrations
of genistein and herbimycin A were approximately three- to fourfold
higher for scurfy mice than for NLC (Table III). These
data suggest that scurfy syndrome may result from a biochemical defect
that either alters the magnitude of the intracellular signaling
cascade, or interferes with the normal down-regulation of T cell
activation responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies show the costimulatory ligands B7.1 and B7.2 to be markedly up-regulated on CD4+ scurfy T cells as well as on scurfy CD4-B220+ APCs both in the presence and absence of stimulation in vitro. We also show that freshly explanted scurfy T cells express elevated levels of the cell surface activation markers CD69 and CD25 (IL-2R). In addition to the presence of activated CD4+ T cells, subset analyses of splenic and lymph node cells show that disease progression is accompanied by striking increases in the percentage of Mac1+ (monocytic) cells and a corresponding drop in the percentage of B220+ cells. Our studies revealed that stimulated CD4+ scurfy T cells produced GM-CSF levels more than 1000-fold greater than normal CD4+ T cells. Since GM-CSF is a differentiation factor for monocytes and inhibits B lymphopoiesis in vivo 15 , excessive GM-CSF production by scurfy T cells most likely accounts for the observed alteration of B220+/Mac-1+ ratios.
We also show that although cultured scurfy T cells are capable of low level proliferation in the absence of TCR stimulation, they retain a requirement for TCR stimulation for optimal proliferation. Scurfy T cells are also hyperresponsive to low amounts of TCR stimulation. These observations suggest that scurfy cells are not constitutively activated, but rather, are activated in vivo by an unknown stimulus, perhaps self Ag. This preactivation of scurfy T cells is further supported by the activated phenotype of lymph node cells and a decreased requirement for costimulation through CD28. In contrast with normal controls, scurfy T cells produce significant levels of IL-2 in the absence of exogenous CD28 stimulation. Despite this decreased dependence on CD28, the CD28/B7 costimulatory pathway remains functionally intact in scurfy T cells and is required for optimal IL-2 production.
T lymphocyte activation and effector function are dependent upon at
least two signals delivered by APCs, ligation of the TCR by the
antigenic peptide-MHC, and ligation of CD28 on T cells by the
counterreceptors B7.1 (CD80) or B7.2 (CD86) 16 . TCR signaling
triggers a cascade of PTK activation and the phosphorylation of
cellular proteins that couple the TCR to an array of downstream signal
transduction molecules 26 . A critical early event in TCR-mediated
signal transduction is the phosphorylation of TCR by the
src family PTKs p56lck and
p59fyn. Inhibition of either of these
molecules, using genetically modified animals or chemical inhibitors,
impairs signaling from the TCR complex. Scurfy T cells are three- to
fourfold less sensitive than NLC T cells to inhibition by herbimycin A,
a reagent that inhibits tyrosine phosphorylation of TCR 22 , and to
genistein, a competitive inhibitor of ATP that inhibits
p56lck and
p59fyn tyrosine phosphorylation in T cells
23 . We are currently evaluating these biochemical events, but
preliminary data suggest that there is a hyperphosphorylation of
signaling molecules in scurfy T cells (data not shown). Scurfy T cells
are also highly refractile to inhibition by the immunosuppressant CsA,
which, upon binding to its intracellular receptor cyclophilin, blocks
the activity of calcineurin phosphatase. CsA-dependent loss of
calcineurin function blocks the nuclear translocation of NF-AT,
which is required for IL-2 gene transcription 25 . Together with data
showing scurfy T cells to be hyperresponsive to TCR stimulation (and
less dependent on costimulation), these inhibition studies suggest that
the scurfy mutation may result in a proximal alteration of TCR signal
transduction. This may be due to either a greatly augmented stimulation
cascade, or an inability to down-regulate T cell activation processes.
Murine B7.1-transfected EL4 T thymoma cells have been shown to effectively costimulate the proliferation of other T cells in vitro 27 . It is therefore feasible that the high levels of B7 costimulatory ligands present on scurfy T cells may allow them to costimulate one another in vivo, contributing to dysregulated IL-2 production, activation, and expansion. This dysregulation may be further compounded by the high levels of B7 present on scurfy APCs such as B220+ cells, resulting in conditions of maximal T cell activation. Other studies have also linked the CD28/B7 costimulatory pathway to the initiation and propagation of autoimmune disease, for example, increased expression of B7.1 has been reported in early lesions of acute multiple sclerosis 28 , and anti-B7 treatment has been shown to modulate the development of diabetes in the nonobese diabetic mouse 29 . Yet, increased levels of B7 expression are not necessarily linked to autoimmune disease; transgenic mice that constitutively express high levels of B7.1 on mature B cells are markedly depressed in T-dependent Ab responses, suggesting that increased B7.1 expression may also have a negative regulatory function in vivo 30 . Similarly, overexpression of B7.2 on B cells results in a decrease in the number of B cells in vivo, suggesting that B7 (and CD28) may play a homeostatic role in lymphocyte biology 31 . One parameter that may play a role in this process is the amount (or ratio) of B7.1 compared with B7.2 19 . It is thus interesting to note that upon activation, the percentage of NLC T cells expressing B7.2 declines, whereas this is not the case for scurfy T cells. The precise role of the up-regulation of B7 in the pathogenesis of disease in scurfy animals awaits the identification of the specific mutation in scurfy mice.
CTLA-4 (CD152), a molecule homologous to CD28, is rapidly expressed following T cell activation and exhibits a 10–20-fold greater affinity for the ligands B7.1 and B7.2 than CD28, and appears to provide a critical antagonistic signal upon preferential ligation of these molecules 16, 17, 20, 32 . Mice deficient for CTLA-4 expression exhibit striking phenotypic similarities to scurfy animals, including the development of a severe lymphoproliferative disease with multiorgan lymphocytic infiltration, spontaneous proliferation and cytokine production by spleen and lymph node cells, and death by 3 wk of age due to activation of CD4+ T cells 33, 34 . As the gene for CTLA-4 is located on chromosome 1, it does not represent a candidate for the scurfy mutation. Furthermore, in a preliminary analysis, we have found CTLA-4 to also be up-regulated on scurfy T cells relative to NLC T cells, indicating that a lack of expression does not account for the phenotype in scurfy mice. The functional activity of this pathway and its potential role in the development of pathology in scurfy mice are currently under study.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Fred Ramsdell, Chiroscience R&D, Inc., 1631 220th Street S.E., Bothell, WA 98021. E-mail address:
3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage colony-stimulating factor; cRPMI, complete RPMI; CsA, cyclosporin A; NLC, normal littermate control; PTK, protein tyrosine kinase.
Received for publication July 13, 1998. Accepted for publication November 4, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu. Rev. Immunol. 14:179.[Medline]
CD3-induced activated killer cells. Cell. Immunol. 174:138.[Medline]
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