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*
Howard Hughes Medical Institute, Department of Medicine,
Department of Dermatology, and
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143; and
§
Division of Rheumatology, Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells, whereas ZAP-70 is essential
for the normal development of T cells and TCR signaling. In this study,
we show that although development of the 
ß lineage was arrested in
the thymus, CD3-positive T cells, primarily of the lineage, were
present in the lymph nodes of mice lacking ZAP-70. Moreover, in the
absence of ZAP-70, dendritic epidermal T cells were fewer in number and
of abnormal morphology, and intestinal intraepithelial lymphocytes,
normally containing a large proportion of T cells, were markedly
reduced. These data suggest that T cells show a variable
dependence upon ZAP-70 for their development. Biochemical analyses of
thymocytes revealed a lack of basal 
-chain tyrosine phosphorylation.
However, several other substrates were inducibly tyrosine
phosphorylated following TCR stimulation. Thus, TCR-mediated signaling
in ZAP-70-deficient thymocytes is only partially impaired. These
studies suggest that Syk compensates only partially for the loss of
ZAP-70, and that there is an absolute requirement of ZAP-70 for ß
T cells and epithelial T cells, but not for some T cells
in peripheral lymphoid tissues. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (3, 4). These chains
noncovalently associate with the TCR -chain and CD3 subunits, and
the resultant expressed pre-TCR complex delivers a signal to allow for
passage beyond the first checkpoint. This leads to cellular
proliferation and progression to the DP stage, where TCR gene
rearrangement is induced (2, 5). The product of the successfully
rearranged TCR -chain gene replaces the pre-T -chain and
completes the formation of the mature TCR ß heterodimer. Signals
through this TCR are then essential for the passage through the second
checkpoint to the more mature single-positive (SP) CD4+ or
CD8+ stage (2). These SP thymocytes are the immediate
precursors of the mature peripheral TCR ß-expressing T cells.
Another lineage of T cells, the TCR -expressing cells, can also
develop in the thymus (6). However, although ordered rearrangement of
TCR genes and preferential association with distinct -chains
occur during ontogeny of this lineage (7), distinct ordered
developmental checkpoints and phenotypic transitions have not been well
defined. Moreover, some T cells expressing the TCR may develop
extrathymically (8). Like the TCR ß subunit, the TCR
subunit associates with the CD3 and or Fc
RI subunits, which
mediate the signals leading to cell activation. The TCR T cells
are different from TCR ß T cells in that they may have less
dependence upon coreceptor (CD4 or CD8) function and may recognize
distinct types of Ags (6). Whereas the functions of these cells still
remain enigmatic, they often appear to localize preferentially in
epithelial tissues.
Biochemical and genetic evidence has demonstrated the requirement for
two families of PTK, Src and Syk/ZAP-70, in TCR signaling (reviewed in
Refs. 9 and 10). The Syk and ZAP-70 family of PTK share structural
features that include tandem SH2 domains and a C-terminal kinase
domain. Via their two SH2 domains, ZAP-70 and Syk associate with the
constitutively tyrosine-phosphorylated -chain in normal murine
thymocytes and with inducibly phosphorylated - or CD3 chains in T
cell lines and clones (11, 12). The Src-family PTK Lck is required for
the constitutive and inducible TCR -chain phosphorylation of
tyrosine residues within the immunoreceptor tyrosine-based activation
motif (ITAM) (11, 13). The phosphorylation of ZAP-70 as well as its
catalytic activation is dependent upon TCR stimulation and upon the
function of the Src kinase Lck (11, 14, 15, 16). The phosphorylation and
activation of Syk may be less dependent upon Src kinase function,
suggesting this kinase may be less coreceptor dependent (17, 18).
Both families of PTK play critical roles in lymphocyte development. In
Lck-deficient mice, there is a substantial block in TCR ß T cell
development, although DP and SP thymocytes as well as mature T cells do
develop in reduced numbers (19). In contrast, mice deficient in the Src
kinase Fyn have no apparent developmental phenotypic abnormalities,
although some functional defects are present in mature thymocytes and
peripheral T cells (17, 18, 20, 21). In mice lacking both Lck and Fyn,
there is a complete block in thymic development at the first
developmental checkpoint, the transition from the DN stage to the DP
stage, suggesting a defect in pre-TCR signaling function (22, 23).
These results also suggest that Fyn may play a compensatory role if Lck
function is limiting. In contrast to the profound effects on the TCR
ß lineage, T cells expressing TCR still develop in the
Lck/Fyn doubly-deficient mice (22, 23). ZAP-70 and Syk PTK also play
critical roles in lymphocyte development. In mice deficient in ZAP-70
or carrying a mutation that inactivates ZAP-70 kinase function, there
is an arrest at the DP to SP stage in the thymus, representing a block
at the second developmental checkpoint (24, 25). No defect in TCR
ß thymocyte development is evident in Syk-deficient mice, although
B cell development is arrested, and certain TCR T cells in
epithelial tissues are absent (26, 27, 28). However, a complete arrest in
the progression from the DN stage to DP stage occurs in mice deficient
in both Syk and ZAP-70 (29). This indicates that Syk most likely can
only play a compensatory role at the DN to DP developmental checkpoint
in mice. When overexpressed, under the influence of the Lck proximal
promoter, Syk can rescue the development of SP cells (30). Since the
expression of Syk decreases after the DP stage (31), these results
suggest that the expression of Syk limits thymic development of the
ß lineage of T cells in ZAP-70-deficient mice. Syk appears to play
a critical ZAP-70-independent role in the development of the TCR
cells present in epithelial tissues.
An apparent paradox arises from studies of patients harboring mutations in the ZAP-70 gene. There is an absence of CD8+ T cells in their peripheral blood, and the CD4+ T cells present are unable to transduce signals via their TCRs (32, 33, 34). Although the TCRs on the peripheral T cells fail to signal, TCRs in at least some DP thymocytes do signal in the one patient studied (35). It is possible that Syk may compensate for ZAP-70 in the signaling functions of this patient’s thymocytes, but not in the periphery. As Syk is not preferentially expressed in CD4 or CD8 lineage T cells, differences in Syk expression in thymic subsets could account for differences between the development of T cells in ZAP-70-deficient mice versus humans. A comparative analysis of the Syk expression and TCR signal transduction in ZAP-70-deficient mice and humans, which may help explain the distinct developmental phenotypes, remains to be completed.
A detailed analysis of signaling in thymocytes from ZAP-70-deficient
mice has not yet been performed. Therefore, ZAP-70-deficient mice were
used to address the residual signaling capacity of murine thymocytes
and to perform a more comprehensive analysis of the development of both
ß and T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 
GT11 library was screened with a cDNA probe to obtain a
genomic clone of ZAP-70. ZAP-70-deficient mice were generated by
homologous recombination of a targeting vector into a 129 ES cell line,
clone JM1 (obtained from Roger Pedersen, University of California, San
Francisco). The targeting vector contained the TK gene at the 5' end
followed by 
2 kb of ZAP-70 genomic sequence, the neomycin resistance
gene replacing the ScaI-AatII sequence, followed
by another 2 kb of homologous genomic sequence. ES cells were
selected in growth media containing 2 µM gancyclovir (Cytovene;
Syntex, Palo Alto, CA) and 200 µg/ml G418 (Geneticin; Life
Technologies, Grand Island, NY). Clones were screened by Southern blot
analysis of PstI-digested genomic DNA using a 300-bp
probe comprised of ZAP-70 genomic sequence immediately 5' of the
targeting construct. Hybridization of this probe to wild-type DNA in
Southern blots yielded a 10-kb fragment, in which DNA from a properly
targeted locus yielded a 6-kb fragment. Mutant cells were injected into
wild-type C57BL/6 blastocysts. Chimeric mice were backcrossed to the
C57BL/6 strain of mice (The Jackson Laboratory, Bar Harbor, ME), and
wild-type and heterozygous littermates were used for analysis. Mice
were phenotyped by PCR analysis for the wild-type ZAP-70 gene using
primers a (5'-gcacatatgcactgtccctggtcta-3') and c
(5'-gggtcgctgtagggactctcgtaca-3'), and for the mutant ZAP-70 gene using
primers a and b (5'-tggctacccgtgatattgctgaaga-3'). Mice were bred
and maintained in Transgenic Animal Care Facility at University of
California, San Francisco. MHC class I (ß2-microglobulin
gene)/MHC class II (Ab ß gene) doubly-deficient mice were
obtained from a commercial source (Taconic, Germantown, NY).
Stimulation, immunoprecipitation, and Western blotting
Single cell suspensions of lymph node, spleen, and thymus were
prepared. Thymocytes were stimulated with purified anti-CD3 mAb
(2C11) for 3 min at 37°C. Pervanadate stimulation of thymocytes was
performed at 37°C for 3 min, as described (13). Cells were lysed in
1% Nonidet P-40 lysis buffer containing protease and phosphatase
inhibitors. Lysates were centrifuged at 10,000 x g,
and supernatants were immunoprecipitated with anti-Syk (5F5 mAb, to
be described in detail elsewhere (D. Chu, N. van Oers, and A. Weiss,
manuscript in preparation)), anti-Cbl (Santa Cruz Biotechnology,
Santa Cruz, CA), anti-PLC-1 mAb (Upstate
Biotechnology, Lake Placid, NY), anti-Slp-76 (sheep antisera, a
gift from G. Koretzky, University of Iowa, Iowa City, Iowa),
anti-TCR (6B10.2 mAb), and anti-Vav (Upstate Biotechnology)
antisera using either protein A- or protein G-Sepharose beads
(Pharmacia Biotech, Piscataway, NJ). Immunoprecipitates were subjected
to SDS-PAGE, and proteins were transferred to polyvinyulidene
difluoride (PVDF) membrane. Membranes were blotted with
anti-phosphotyrosine mAb (4G10 mAb; Upstate Biotechnology),
followed by horseradish peroxidase-conjugated secondary antisera, and
developed using the ECL (Amersham, Arlington Heights, IL) detection
system. The blots were then stripped and reblotted with
immunoprecipitating Ab. Lysates were diluted with SDS sample buffer and
resolved by SDS-PAGE, transferred to PVDF membrane, and immunoblotted
with anti-ZAP-70 (1G7 mAb) or anti-phosphotyrosine Ab (4G10).
FACS analysis
Single cell suspensions of lymph nodes, spleen, and thymus were
incubated with FITC-, PE-, and tri-color-conjugated
anti-CD3, anti-CD4, anti-CD8, anti- TCR,
anti-TCR-ß, and anti-B220 Abs (PharMingen, San Diego, CA).
Cells were analyzed on a FACScan (Becton Dickinson, San Jose, CA) using
Cellquest software. DP thymocytes were sorted on a dual argon laser
FACStar (Becton Dickinson, Milpitas, CA).
Dendritic epidermal T cell staining
Mouse ears were amputated at the base immediately after
sacrifice. Hair removal lotion (Nair; Carter-Wallace, New York, NY) was
used to depilate the skin, which was then separated from the underlying
cartilage using a dissection microscope. The dissected skin was pressed
dermal side down onto an adhesive tape window (Instrumedics,
Hackensack, NJ), and the epidermis was lightly coated with cyanoacrylic
glue (Superglue; Super Glue, Hollis, NY) to provide a structural
support for the fragile epidermal layer. The skin was then placed
dermal side up on an adhesive-coated microscope slide (Instrumedics)
and incubated with 20 mM EDTA in PBS for 3 h at 37°C. Finally,
the dermis was carefully pulled off the epidermis with the aid of fine
forceps and a dissecting microscope. Dendritic epidermal T cells (DETC)
were stained with a 1/100 dilution of FITC-conjugated pan TCR Ab
(PharMingen).
Isolation of IEL
IEL were isolated essentially as described (36). Briefly, the small intestines of individual mice were cut into 5-mm pieces and washed twice with medium. The washed intestinal pieces were stirred at 37°C for 20 min in medium with addition of 1 mM dithioerythritol. This step was repeated, and the resultant supernatants were rapidly filtered through nylon wool and the filtrate centrifuged through a 44/67% Percoll step gradient. The cells at the interface of the Percoll gradient were collected and prepared for flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ZAP-70 mice were generated by standard gene-targeting technology.
The targeting vector deleted a segment of the ZAP-70 gene (encoding
amino acids 221 to 520) that included the C-terminal SH2 domain and
replaced it with the neomycin resistance gene (Fig. 1
A). Mutant ES cells and the
mice derived from them were identified by Southern blot and PCR
analysis. PCR analysis of genomic DNA was employed to phenotype mice.
One set of PCR primers detected the wild-type genomic sequence, and the
other set of primers detected the mutant ZAP-70 sequence containing the
neomycin resistance gene. This PCR strategy allowed for the
identification of wild-type, heterozygous, and homozygous deficient
mice (Fig. 1, A and B). Unlike the wild-type or
heterozygotes, no ZAP-70 protein was detected in thymocytes from
ZAP-70-/- mice by Western blot analysis (Fig. 1C). The heterozygous mice expressed an intermediate level
of ZAP-70 protein in the thymus.
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Thymocytes from ZAP-70+/+ and ZAP-70-/-
littermate mice were analyzed for expression of CD4 and CD8 (Fig. 2). As previously reported (24, 25), an
arrest at the DP stage of development was observed in the mutant mice.
There were few, if any, CD3high-positive cells found in the
thymuses of the ZAP-70-/- mice. However, the levels of
CD3 on wild-type and ZAP-70-/- DP thymocytes were
comparable when analyzed by FACS. The spleens of the mutant mice were
primarily composed of B cells, since approximately 90% of splenocytes
stained positively for B220, and very few CD3-positive cells were
present. The lymph nodes in the mutant mice were smaller than wild type
and were predominantly composed of B cells. Surprisingly, unlike the
thymus and spleen, lymph nodes of the ZAP-70-deficient mice contained a
significant percentage of CD3-positive cells (5–10%), but few of
these cells expressed CD4 or CD8. The CD3-positive T cells found in the
spleen and lymph nodes were primarily T cells (Fig. 3). In contrast to wild-type littermates,
in which less than 1% of CD3+ T cells were of the
lineage, the majority of CD3+ cells in lymph nodes of the
ZAP-70-deficient mice were of the TCR lineage. This did not
appear to reflect a peripheral expansion of TCR T cells, since
the absolute number of these cells was comparable with that seen in the
wild-type mice.
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DETC are characterized by the dendritic morphology of their
extensions and their surface expression of a V3/V2 monomorphic
TCR (6). Epidermal sheets were stained with a FITC-conjugated pan TCR
Ab to assess the role of ZAP-70 in the development of this cell
population. In ZAP-70-deficient mice, there were somewhat fewer DETC as
compared with wild-type littermates (Fig. 4). Moreover, the positively staining
cells from the mutant mice had an abnormal morphology that lacked the
characteristic dendritic extensions seen in wild-type cells.
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intestinal intraepithelial
lymphocytes (IEL) development
The origin of IEL is a subject of considerable controversy (8),
but it is likely that a significant proportion of TCR IEL do not
develop within the thymus, while most TCR ß IEL require a thymus
for maturation (37). Unlike peripheral TCR T cells, which are
largely CD4-/CD8-, nearly all IEL TCR
T cells express a CD8 homodimer (38). In contrast to results
obtained with lymph nodes that contained substantial numbers of TCR
T cells (Fig. 3
), few IEL expressed or ß TCRs (Fig. 5). The small percentage of IEL TCR
T cells detected expressed lower levels of TCR than their normal
counterparts.
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The presence of CD3+ T cells in lymph nodes suggests
that T cell development may not be blocked completely in the absence of
ZAP-70. Moreover, the ability of thymocytes to pass through the first
developmental checkpoint (DN to DP stages) suggests that some
ZAP-70-independent TCR signal transduction can occur. To investigate
this possibility, thymocytes from ZAP-70-/- mice and
littermate controls were stimulated in vitro with anti-CD3 Ab.
Whole cell lysates from thymocytes of mutant mice blotted with
anti-phosphotyrosine mAb showed anti-CD3-dependent induction of
some phosphoproteins, albeit fewer than heterozygote or wild-type
control littermates (Fig. 6). Note that
the greater induction of tyrosine phosphorylation in the heterozygote
was not reproducibly seen.
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-chain is required
for ZAP-70 binding. In resting thymocytes, the -chain is
constitutively phosphorylated, and receptor stimulation leads to
phosphorylation of the bound ZAP-70. However, in examining long
exposures of Western blots of whole cell lysates obtained from
ZAP-70-/- mice, there was no evidence for constitutive or
inducible -chain tyrosine phosphorylation (data not shown). Further
studies were performed to analyze the constitutive phospho- levels
in cell lysates of total thymocytes and of sorted DP thymocytes from
ZAP-70-/- and wild-type mice. There was no detectable
phospho- present in either total thymocytes or sorted DP thymocytes
from the ZAP-70 mutant mice, in contrast to the constitutive
phospho- present in wild-type thymocytes (Fig. 7). Levels of total as well as
Slp-76, a control for protein loading, were equivalent in all lanes.
Thymocytes were also treated with the phosphatase inhibitor pervanadate
to determine whether the -chain was capable of being phosphorylated
in the absence of ZAP-70 protein. Pervanadate treatment of
ZAP-70-deficient thymocytes allowed for tyrosine phosphorylation of the
-chain, although at lower levels than that of wild-type cells
(data not shown). The difference in basal phosphorylation could
reflect the absence of ZAP-70 protein and our previously reported (39)
function of its SH2 domains in protecting the phosphorylated ITAM
tyrosines, or may be due to another yet to be identified function
provided by the ZAP-70 protein.
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). Several other
proteins known to be important in TCR signaling were also
immunoprecipitated and blotted with anti-phosphotyrosine Abs before
and after CD3 stimulation. Slp-76, Vav, Cbl, and PLC-1 proteins were
immunoprecipitated to analyze their state of tyrosine phosphorylation.
In all cases, the proteins were tyrosine phosphorylated, but at lower
(modestly lower in some cases) levels than wild-type (Fig. 8). As a
control, thymocytes from MHC class I/MHC class II doubly-deficient mice
were analyzed since they are blocked at the same stage of thymic
development as are the ZAP-70-deficient mice. PLC-1, Slp-76, and Syk
were all tyrosine phosphorylated in these MHC-deficient thymocytes
following stimulation with anti-CD3 Abs. Since there was not a
complete blockade of tyrosine-phosphorylated substrates downstream of
ZAP-70, an attempt was made to identify any missing
tyrosine-phosphorylated proteins. An anti-phosphotyrosine Western
blot of anti-phosphotyrosine immunoprecipitates from unstimulated
and stimulated thymocyte lysates failed to show any obvious missing
bands, except for bands at the apparent mobility of ZAP-70 and of TCR
(data not shown). These data suggest that the TCRs or pre-TCRs in
DP and/or DN ZAP-70 thymocytes can couple to Syk and to several other
downstream substrates. However, it is possible that the inducibly
phosphorylated substrates observed when whole thymocytes were
stimulated reflect those present only in a subset of cells, for
instance in the few less mature DN cells present in the unfractionated
thymocyte populations.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cells, but is variably required for
T cells. Although few or no CD3+ T cells were
present in the thymuses or spleens of the mutant mice, a distinct
population of CD3+ cells was reproducibly present in the
lymph nodes. In the mutant mice, these cells were primarily TCR
lineage T cells. This observation also held true for the few
CD3+ cells that were present in the spleens. In contrast,
the absence of ZAP-70 profoundly affected the development of IEL and
DETC. A similar requirement for ZAP-70 in the development of IEL was
reported recently for another ZAP-70-deficient mouse (30).
Interestingly, in contrast to the absence of IEL, DETC were reduced in
number, but more strikingly exhibited a morphologic change in which
dendritic processes were absent. Thus, ZAP-70 is not only required for
the development of conventional TCR ß-expressing cells, but also
for the development, proliferation, and/or maturation of cells
that reside in epithelial tissues.
The development of the TCR lineage was variably affected by the
absence of ZAP-70. This suggests that development of distinct subsets
of TCR -expressing cells may not have the same TCR signaling
requirements. However, it is important to note, there is no evidence
that the lymph node TCR T cells in the mutant mice are
functionally normal.
The requirement for ZAP-70 in TCR ß T cell development is not
surprising and is consistent with observations made in other
ZAP-70-deficient or ZAP-70 kinase mutant mice (24, 25). However, its
relative requirement for development of TCR T cells in
epithelial tissues is somewhat surprising. A similar requirement for
Syk has previously been reported for the development of these
epithelial T cells (28). The requirement for Syk is consistent with the
notion that this kinase has less dependence upon Src kinases (17, 18),
and, by inference, coreceptor function. Since most TCR T cells
lack the CD4 and CD8 coreceptors, it was not surprising to find that
Syk is important for the development of these T cells in epithelial
tissues. By contrast, a similar requirement for ZAP-70 in the
development of these same cells is surprising. These unexpected
findings suggest that ZAP-70 and Syk may not play completely
overlapping functions in the development of these cells. It is unclear
whether this reflects distinct functions of these PTK or distinct
temporal expression of these PTK during the development of TCR
epithelial T cells. Perhaps the presence of one of the family members
is sufficient for immature cells to migrate to the appropriate tissue,
such as the skin, but differentiation cannot occur without both ZAP-70
and Syk present. An alternative, but less likely possibility is that
these PTK simply have additive identical functions.
Previous studies have suggested the absence of TCR signal transduction
in thymocytes deficient in ZAP-70 or mice expressing a kinase-deficient
mutant of ZAP-70 (24, 25). However, Syk appears to compensate partially
for ZAP-70 function in pre-TCR signaling during developmental events to
allow transition through the first developmental checkpoint, the DN to
DP transition (30). Syk can functionally couple to the mature TCR or
the pre-TCR (13, 40). Since Syk is expressed in thymocytes (31), it was
somewhat surprising that no signaling has previously been detected in
ZAP-70-deficient thymocytes. This is particularly relevant since
thymocytes from ZAP-70-deficient humans can signal in response to TCR
stimulation (35). However, a detailed analysis of tyrosine
phosphoproteins has not previously been performed. In this study, we
show that a number of substrates, including Syk, Vav, PLC-1, Slp-76,
and Cbl, are tyrosine phosphorylated following CD3 stimulation in the
thymocytes from ZAP-70-deficient mice. Syk may compensate for the loss
of ZAP-70 in the DP and/or DN thymocytes and phosphorylate these
downstream proteins. Indeed, we could detect Syk-inducible
phosphorylation following CD3 stimulation. However, the proteins that
are phosphorylated may not be phosphorylated to the same extent or on
the same tyrosine residues that are normally phosphorylated by ZAP-70,
thus explaining the blockade in the signaling events necessary for the
DP to SP transition. However, relevant to these observations are recent
studies in our laboratory of mouse thymocytes that demonstrate a
down-regulation of Syk as DN thymocytes transit to DP thymocytes (Chu
et al., manuscript in preparation). Thus, the observed Syk
phosphorylation may have occurred in response to CD3 stimulation in the
DN thymocytes, and this activation led to the induced tyrosine
phosphorylation of downstream signaling proteins. Such signaling by Syk
is likely to be responsible for thymocytes to pass the checkpoint from
DN to DP cells in these mutant mice (29).
A very proximal protein in the TCR signaling pathway that was not
detectably phosphorylated in ZAP-70-deficient thymocytes was the TCR
-chain. In ex vivo murine thymocytes, TCR is constitutively
tyrosine phosphorylated and associated with ZAP-70 via the ZAP-70 SH2
domains (11, 12, 13). Lck is required for this constitutive phosphorylation
of , and it has been proposed that ZAP-70 association can protect
the dephosphorylation of phospho- since overexpression of the tandem
ZAP-70 SH2 domains alone is sufficient to increase -chain
phosphorylation (39). Syk may not be able to sustain this association
or may not be present at sufficient levels in DP thymocytes and,
consequently, would be dephosphorylated in the absence of ZAP-70.
As more detailed analyses of TCR signaling and developmental regulation
are performed, the complexity of the signaling pathways responsible for
developmental decisions is revealed. Through multiple checkpoints in
the developmental process, an intact immune system matures. Kinase
families with multiple members can compensate for the loss of one
another in some, but not all, cases, allowing for normal development
and signaling. These studies show that although Syk may be able to
compensate for the lack of ZAP-70 to some degree in some ß lineage
thymocytes, ZAP-70 and Syk PTK are both required for the normal
development of certain lineage T cells.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, University of Texas, Dallas Southwestern, Dallas, TX 75235.
3 Address correspondence and reprint requests to Dr. Arthur Weiss, Howard Hughes Medical Institute, University of California, 3rd Ave. and Parnassus St., San Francisco, CA 94143-0795. E-mail address:
4 Abbreviations used in this paper: DN, double negative; DETC, dendritic epidermal T cells; DP, double positive; IEL, intraepithelial lymphocyte; ITAM, immunoreceptor tyrosine-based activation motif; PE, phycoerythrin; PLC, phospholipase C; PTK, protein tyrosine kinase; SP, single positive.
Received for publication April 17, 1998. Accepted for publication June 30, 1998.
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  A. Weiss TCR Signal Transduction: Opening the Black Box J. Immunol., October 15, 2009; 183(8): 4821 - 4827. [Full Text] [PDF]
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M. Klinger, J. K. Kim, S. A. Chmura, A. Barczak, D. J. Erle, and N. Killeen Thymic OX40 Expression Discriminates Cells Undergoing Strong Responses to Selection Ligands J. Immunol., April 15, 2009; 182(8): 4581 - 4589. [Abstract] [Full Text] [PDF]
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 Y. J. Chiang, M. S. Jordan, R. Horai, P. L. Schwartzberg, G. A. Koretzky, and R. J. Hodes Cbl Enforces an SLP76-dependent Signaling Pathway for T Cell Differentiation J. Biol. Chem., February 13, 2009; 284(7): 4429 - 4438. [Abstract] [Full Text] [PDF]
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A. M. Becker, L. M. DeFord-Watts, C. Wuelfing, and N. S. C. van Oers The Constitutive Tyrosine Phosphorylation of CD3{zeta} Results from TCR-MHC Interactions That Are Independent of Thymic Selection J. Immunol., April 1, 2007; 178(7): 4120 - 4128. [Abstract] [Full Text] [PDF]
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X. Liu, B. J. Taylor, G. Sun, and R. Bosselut Analyzing Expression of Perforin, Runx3, and Thpok Genes during Positive Selection Reveals Activation of CD8-Differentiation Programs by MHC II-Signaled Thymocytes J. Immunol., October 1, 2005; 175(7): 4465 - 4474. [Abstract] [Full Text] [PDF]
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 O. Adjali, R. R. Vicente, C. Ferrand, C. Jacquet, C. Mongellaz, P. Tiberghien, K. Chebli, V. S. Zimmermann, and N. Taylor Intrathymic administration of hematopoietic progenitor cells enhances T cell reconstitution in ZAP-70 severe combined immunodeficiency PNAS, September 20, 2005; 102(38): 13586 - 13591. [Abstract] [Full Text] [PDF]
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 M. D. Myers, L. L. Dragone, and A. Weiss Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCR{zeta} for degradation J. Cell Biol., July 18, 2005; 170(2): 285 - 294. [Abstract] [Full Text] [PDF]
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 Y. J. Chiang, C. L. Sommers, M. S. Jordan, H. Gu, L. E. Samelson, G. A. Koretzky, and R. J. Hodes Inactivation of c-Cbl Reverses Neonatal Lethality and T Cell Developmental Arrest of SLP-76-deficient Mice J. Exp. Med., July 6, 2004; 200(1): 25 - 34. [Abstract] [Full Text] [PDF]
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 T. R. Petersen, S. Gulland, E. Bettelli, V. Kuchroo, E. Palmer, and B. T. Backstrom A chimeric T cell receptor with super-signaling properties Int. Immunol., July 1, 2004; 16(7): 889 - 894. [Abstract] [Full Text] [PDF]
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 A. Mocsai, H. Zhang, Z. Jakus, J. Kitaura, T. Kawakami, and C. A. Lowell G-protein-coupled receptor signaling in Syk-deficient neutrophils and mast cells Blood, May 15, 2003; 101(10): 4155 - 4163. [Abstract] [Full Text] [PDF]
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X. Liu, A. Adams, K. F. Wildt, B. Aronow, L. Feigenbaum, and R. Bosselut Restricting Zap70 Expression to CD4+CD8+ Thymocytes Reveals a T Cell Receptor-dependent Proofreading Mechanism Controlling the Completion of Positive Selection J. Exp. Med., February 3, 2003; 197(3): 363 - 373. [Abstract] [Full Text] [PDF]
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Q. Gong, X. Jin, A. M. Akk, N. Foger, M. White, G. Gong, J. B. Wardenburg, and A. C. Chan Requirement for Tyrosine Residues 315 and 319 within {zeta} Chain-Associated Protein 70 for T Cell Development J. Exp. Med., August 20, 2001; 194(4): 507 - 518. [Abstract] [Full Text] [PDF]
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M. A. Basson, T. J. Wilson, G. A. Legname, N. Sarner, P. D. Tomlinson, V. L. J. Tybulewicz, and R. Zamoyska Early Growth Response (Egr)-1 Gene Induction in the Thymus in Response to TCR Ligation During Early Steps in Positive Selection Is Not Required for CD8 Lineage Commitment J. Immunol., September 1, 2000; 165(5): 2444 - 2450. [Abstract] [Full Text] [PDF]
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F. Colucci, D. Guy-Grand, A. Wilson, M. Turner, E. Schweighoffer, V. L. J. Tybulewicz, and J. P. Di Santo A New Look at Syk in {alpha}{beta} and {gamma}{delta} T Cell Development Using Chimeric Mice with a Low Competitive Hematopoietic Environment J. Immunol., May 15, 2000; 164(10): 5140 - 5145. [Abstract] [Full Text] [PDF]
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M. R. Kuhne, G. Ku, and A. Weiss A Guanine Nucleotide Exchange Factor-independent Function of Vav1 in Transcriptional Activation J. Biol. Chem., January 21, 2000; 275(3): 2185 - 2190. [Abstract] [Full Text] [PDF]
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D. H. Chu, N. S. C. van Oers, M. Malissen, J. Harris, M. Elder, and A. Weiss Pre-T Cell Receptor Signals Are Responsible for the Down-Regulation of Syk Protein Tyrosine Kinase Expression J. Immunol., September 1, 1999; 163(5): 2610 - 2620. [Abstract] [Full Text] [PDF]
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F. Colucci, M. Turner, E. Schweighoffer, D. Guy-Grand, V. Di Bartolo, M. Salcedo, V. L. J. Tybulewicz, and J. P. Di Santo Redundant Role of the Syk Protein Tyrosine Kinase in Mouse NK Cell Differentiation J. Immunol., August 15, 1999; 163(4): 1769 - 1774. [Abstract] [Full Text] [PDF]
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C. B. F. Thien, D. D. L. Bowtell, and W. Y. Langdon Perturbed Regulation of ZAP-70 and Sustained Tyrosine Phosphorylation of LAT and SLP-76 in c-Cbl-Deficient Thymocytes J. Immunol., June 15, 1999; 162(12): 7133 - 7139. [Abstract] [Full Text] [PDF]
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