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Departments of
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Microbiology and Immunology,
Pediatrics, and
Medicine and Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; and
§
Centre d’Immunologie Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Marseille-Luminy, Marseille, France
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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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ß lineage of T cells proceeds through an ordered series
of stages defined by the expression of various cell surface markers
(1). Immature thymocytes enter the thymus as
CD4-CD8- cells and
develop into CD4+CD8+
thymocytes. These cells then undergo TCR-dependent positive and
negative selection and become CD4+ or
CD8+ thymocytes that form the mature ß T
cell repertoire in the periphery.
In mice, CD4-CD8-
thymocytes can be further subdivided according to the cell surface
expression of two other proteins, CD44 (Pgp-1) and CD25 (-chain of
the IL-2 receptor) (2, 3). The earliest pro-thymocytes are
CD44+CD25-; these cells
subsequently up-regulate CD25. Next, these thymocytes become
CD44-CD25+, during which
time the ß-chain of the TCR is rearranged and expressed on the cell
surface as a heterodimer with the pre-T
(pT)4 chain. The
CD4-CD8-/CD44-CD25+
thymocytes that receive a pre-TCR signal progress through an
intermediate CD4-CD8+
stage before becoming
CD4+CD8+ thymocytes
(4, 5, 6, 7, 8).
Productive ß-chain rearrangement and membrane expression of the
pre-TCR is detected via a signal that is transduced through the pre-TCR
complex; the nature of this signaling is poorly understood but probably
involves the Ras pathway (9). However, the signal appears
to be independent of a pT or ß-chain ligand (10).
This critical checkpoint in thymocyte development requires two families
of protein tyrosine kinases (PTKs), the Src family of PTKs, including
Lck and Fyn in T cells, and the Syk family of PTKs, comprising Syk and
ZAP-70. These kinases act in a sequential manner to transmit signals
from the pre-TCR and TCR complexes (11).
The TCR and pre-TCR heterodimers are components of multimeric complexes
containing noncovalently associated signal transducing CD3 subunits
, 
, and 
, as well as a 
-chain dimer (11). The
signal transducing subunits recruit cytosolic tyrosine kinases for
their signaling function. This function is initiated by a Src family
kinase that phosphorylates the immunoreceptor tyrosine-based activation
motif which is present as a single copy within each of the CD3 chains
and which is present as three copies within the -chain. These
phosphorylated tyrosines subsequently serve as docking sites for the
tandem SH2 domains of both Syk and ZAP-70. In ex vivo isolated
thymocytes, ZAP-70 or Syk are pre-bound to the -chain because it is
constitutively phosphorylated (12). The Syk family kinases
are activated by tyrosine phosphorylation of their activation loops and
transduce further signals for T cell development, activation, and
differentiation.
Genetic studies have demonstrated the importance of both families of
tyrosine kinases for the development of murine thymocytes. Mice doubly
deficient for Lck and Fyn exhibit a profound block in development at
the CD4-CD8- stage, the
time at which pre-TCR signals are required (13, 14).
ZAP-70-deficient mice have thymocytes that are arrested at the
CD4+CD8+ stage
(15, 16, 17). Syk-deficient mice have no reported ß T
cell defect, although a subset of T cells, contained in
intraepithelial lymphocytes, is impaired in their development
(18). However, Syk has been shown to be expressed at
higher levels in the thymus and is down-regulated in the periphery,
suggesting that it might play an unrecognized role at some point during
thymocyte development (19). Moreover, thymocytes doubly
deficient in Syk and ZAP-70 are arrested at the
CD4-CD8- stage
(20), probably reflecting a partial ability of Syk to
compensate for ZAP-70, but only at the first thymic developmental
checkpoint. Despite this suggestive genetic evidence that Syk plays a
role during thymocyte development, a definitive expression analysis of
Syk in thymocytes has not been performed.
Syk has been shown to be able to compensate for ZAP-70 in many
situations, including ß T cell development and signaling
(21, 22). However, recent evidence has indicated in vitro
and in cell lines that Syk and ZAP-70 have different requirements for
activation. For instance, Syk has less dependence on Src kinases for
its activation (23, 24) and, as mentioned above, has a
distinct distribution within the T cell lineage. Thus, Syk may have
roles distinct from ZAP-70 in certain in vivo situations.
In humans, ZAP-70-deficient patients have been described (25, 26, 27). However, unlike in mice, in which thymocytes are completely arrested at the CD4+CD8+ stage, human patients with a ZAP-70 deficiency have T cells that are exclusively CD4+ in their peripheral blood. Studies with HTLV-1-transformed thymocyte lines from these patients indicate that Syk is increased in expression in those cells when compared with lines made from normal thymocytes, suggesting that Syk can compensate for the loss of ZAP-70 function in these patients (28). Nevertheless, the mature CD4+ cells that populate the periphery in these patients are unable to signal.
In this paper we use an intracellular staining method for detecting Syk expression in subpopulations of cells. We have analyzed the expression of Syk in thymocytes of both mice and humans. These results suggest that Syk is expressed at the highest levels during the pre-TCR signaling stage and is down-regulated quickly thereafter, suggesting that Syk may play a role in propagation of the pre-TCR signal. These studies also suggest that observed differences in Syk expression in murine and human thymocytes may contribute to the distinct phenotypes seen in ZAP-70-deficient mice and patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mAb 5F5.2 was generated by standard protocols (29). Briefly, BALB/c mice were immunized i.p. with TiterMax adjuvant (CytRx, Norcross, GA) and keyhole limpet hemocyanin (KLH)-conjugated anti-Syk peptide corresponding to aa 306–333 in murine Syk. Mice were boosted with KLH-Syk peptide three times over 2 mo. A final boost was given i.v. via tail vein injection and spleens harvested 3 days later. Fusions were performed with the Ag8.653 murine hybridoma fusion partner using polyethlyene glycol, grown in selective medium (hypoxanthine/aminopterin/thymidine), and screened by ELISA. Positive clones were then subcloned by limiting dilution and rescreened for stable, high-level secretion.
The 4D10.1 mAb was generated by similar methods. In this case, the Ag was a human Syk-derived peptide corresponding to aa 314–339 (30).
Mice, tissues, and Abs
Mice were maintained at the University of California, San Francisco, Animal Care Facility. Wild-type C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). lck-/fyn- mice (13) and RAG-1- mice (generous gifts of Dr. Nigel Killeen, University of California, San Francisco, CA) have been described. Human thymic tissue was obtained from pediatric cardiac surgery patients between 1 wk and 8 years of age.
The following Abs with the following specificities were used: rabbit heterosera, 1598, anti-ZAP-70; anti-Fgr; anti-Hck (anti-Fgr and anti-Hck were gifts of Dr. Clifford Lowell, University of California); mAbs 1F6, anti-Lck; 5F5.2, anti-murine Syk; 4D10.1, anti-human Syk. Abs against cell surface markers were obtained from PharMingen (San Diego, CA), anti-CD44, anti-mCD4; from Becton Dickinson (San Jose, CA), anti-hCD3, anti-hCD4, anti-hCD8; and from Caltag (Burlingame, CA), anti-mCD4, anti-mCD8, anti-hCD19, anti-mCD25, anti-mB220. Streptavidin conjugates were obtained from PharMingen (streptavidin-allophycocyanin, APC) and from Caltag (streptavidin-tricolor, TC).
Ab iodination and FITC conjugation
5F5.2 Ab was dialyzed against iodination buffer and reacted with IodoBeads (Pierce, Rockford, IL) and 1 mCi carrier-free [125I]sodium iodide according to manufacturer’s instructions. Reactions were stopped upon removal of the IodoBead reagent. Free radioisotope was removed using a dextran matrix desalting column (D-Salt Columns, Pierce). Conjugation of affinity purified 5F5.2 and 4D10.1 mAbs to FITC (Molecular Probes, Eugene, OR) was performed as described (31).
Cell lysates and immunoblotting
Cell were lysed in 1% Nonidet P-40 lysis buffer containing 10 mM Tris (pH 7.6), 150 mM NaCl, and protease and phosphatase inhibitors as previously described (12). Protein content of lysates was determined using the Bio-Rad (Hercules, CA) protein assay reagent; values were determined using a spectrophotometer. Lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF; Immobilon-P, Amersham, Arlington Heights, IL). Blots were blocked with nonfat milk powder and BSA and incubated with the appropriate primary Ab. Following washes in Tris-buffered saline plus 0.05% Tween-20, blots were incubated with HRP-conjugated secondary Abs. Bands were visualized using the enhanced chemiluminescence system and autoradiographic film (ECL, Amersham).
Intracellular staining and flow cytometry
Tissues were strained through a wire mesh to generate a
single-cell suspension. Cells were washed multiple times with PBS,
resuspended in staining buffer, and stained with the appropriate
conjugated Abs in the presence of normal mouse serum (10 µg/ml) or Fc
blocking Ab, 2.4G3. Cells were then fixed in 4% paraformaldehyde in
PBS and washed. Ab for intracellular staining was added in staining
buffer + 0.1% saponin in the presence of normal mouse serum or Fc
blocking Ab. Cells were washed in permeabilization buffer and analyzed
by flow cytometry. Four-color analysis was performed on a
FACStarPlus (Becton Dickinson). All other samples
were collected on a FACScan (Becton Dickinson). Results were analyzed
using CellQuest software (Becton Dickinson). A total of 250 µg of
purified anti-CD3 Abs (2C11) was injected i.p. into
lck-/fyn- or
RAG-1- mice in PBS. After 7 days, thymi
were removed from the mice and stained for Syk expression as outlined
above. Results were analyzed by flow cytometry on a FACScan.
Gene rearrangement assays
DNA samples were extracted from whole preparations of adult murine thymocytes. PCRs were performed in a final volume of 30 µl and included a maximum of 50 ng of template DNA, 1 mM of each primer, 200 µM of each deoxynucleotide triphosphate, and 0.2 U of Taq DNA polymerase (Expand PCR Kit, Boehringer Mannheim, Indianapolis, IN). Reaction mixtures were run on a 1.5% agarose gel, blotted to nylon membranes (GeneScreenPlus, New England Nuclear, Boston, MA), and hybridized with 32P-labeled oligonucleotide probes.
The oligonucleotides and PCR conditions used for the analysis of
TCR-ß rearrangements and TCR- rearrangements were performed
essentially as described (32, 33). Before the analysis of
the relative levels of TCR gene rearrangements, the quality and the
quantity of DNA present in each sample were checked by amplifying the
nonrearranging trithorax gene (MTrx1; see Ref. 34).
Previous experiments have shown that, for the concentration of template
and the PCR conditions used, the product yield is directly proportional
to the input of target sequences. At least two sets of independent
experiments were performed for each sample. Hybridizing bands were
quantitated using a PhosphorImager (BAS1000; Raytest, Courbevoie,
France). Results of each mouse are expressed as the percentage of
rearrangements measured in wild-type mice after normalization for input
DNA. The results presented correspond at the mean value of
rearrangements for two independent Vß-Jß amplifications (Vß5,
Vß8) and three independent V-J amplifications (V2, V5,
and V8).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because Src and Syk family members are critical in the normal
development of thymocytes (13, 14, 20), we were interested
in determining the relative expression levels in thymocytes of a number
of hematopoietic lineage-specific Src family kinases, as well as the
expression of the Syk family kinases Syk and ZAP-70 in these
thymocytes. To analyze the expression of the Syk PTK in thymocyte
development with greater sensitivity and specificity, we generated a
mAb, 5F5.2, against the murine protein. This Ab is specific for a
peptide sequence in interdomain B of murine Syk, aa 306–333.
Immunoblot analysis of whole cell lysates using
[125I]iodinated 5F5.2 Ab revealed that it
reacts primarily with a 72-kDa protein present in large amounts in
murine spleen and lymph nodes and, to a much lesser extent, in the
thymus (Fig. 1
A). This band
was not detected in 3T3 cells, a murine fibroblast line (Fig. 1A). The reactivity against the 72-kDa band is specific, as
it was competed away with peptide against which the Ab was generated
(Fig. 1A). The minor reactivity to a 40-kDa band that was
competed completely with peptide may represent a degradation of product
of Syk has been observed by others, including in the initial
description of Syk (35). A faint nonspecific band in the 45-kDa range
was not reproducibly observed. Using this Ab, we were also able to
detect Syk expression by immunoblotting in a murine B cell line,
Bal-17, and a murine macrophage line, RAW 264.7 (data not shown).
Furthermore, the 5F5.2 Ab is specific for murine Syk, as it did not
detect Syk in human or avian cells (data not shown).
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Thymocytes in RAG-deficient and Lck/Fyn-doubly deficient mice are
arrested at the same
CD44-CD25+
CD4-CD8- stage of
development (Refs. 13, 14 ; also see below). During
studies of thymocytes of developmentally arrested Lck/Fyn doubly
deficient mice, we used immunoblotting to examine the expression of
other Src family as well as Syk family PTKs in the
CD4-CD8- thymocytes of
these mice (Fig. 1
B). Strikingly, in whole thymic
preparations, we detected high levels of expression of the Src family
kinase Fgr, as well as high levels of expression of the Syk PTK (Fig. 1B). The same pattern of expression was observed in thymic
preparations from RAG-deficient mice (Fig. 1B). It is
possible that the high levels of expression of these kinases reflects
the increased proportion of dendritic cells, macrophages, and stromal
cells in the lysates due to the decreased number of thymocytes in the
developmentally arrested thymi. However, the high level of Syk might
reflect the level of expression of Syk in the developmentally arrested
thymocytes. A detailed analysis of Syk expression in the thymocyte
subsets has not been performed, with the exception of one study from
our laboratory which relied on a heterologous antiserum and
immunoblotting of bulk populations (18).
Syk expression can be detected by intracellular staining in murine cells
To examine the expression of Syk with greater sensitivity and to
detect Syk expression specifically at a single cell level within
discrete subsets of thymocytes from a whole thymic preparation, we
decided to try intracellular staining with the 5F5.2 mAb. This
technique has been used extensively for the detection of cytokine
expression. Briefly, cells were stained for surface markers and were
subsequently fixed, permeabilized with saponin, and stained
intracellularly with the anti-Syk Ab. Cells were then analyzed by
flow cytometry. Syk expression could easily be detected in
B220+ splenic B cells (Fig. 2), which express high levels of Syk.
Consistent with the immunoblotting results, intracellular staining for
Syk revealed high levels in macrophages and low levels in peripheral T
cells (data not shown). The staining observed with this mAb is
specific, as we have shown with several controls. First, mAb staining
could be competed away with the Syk-derived peptide against which the
Ab was generated. Furthermore, an isotype-matched Ab control overlapped
with the negative peak of the peptide competition control (Fig. 2).
Similar to the reactivity observed in immunoblotting, the anti-Syk
reagent did not recognize Syk from human or avian cells when used for
intracellular staining (data not shown). Finally, no reactivity was
seen when nonpermeabilized cells were stained with the anti-Syk
reagent, indicating that the Ag recognized by the Ab is an
intracellular protein (data not shown).
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Based on experiments using rabbit heterosera, we previously
reported that in the T cell lineage, Syk is expressed at higher levels
in thymocytes than in peripheral T cells (19). To
investigate Syk expression in thymocyte subsets and to try to
understand the unique functions of Syk in the thymus and its ability to
only partially compensate for the loss of ZAP-70, we stained for
intracellular levels of Syk in murine thymocytes. We costained
thymocytes from wild-type mice for CD4, CD8, and Syk and used flow
cytometry to define the subsets of cells expressing Syk (Fig. 3A). Interestingly, Syk was
expressed at variable levels in
CD4-CD8- thymocytes, with
a subpopulation of
CD4-CD8- cells expressing
relatively high levels of Syk (Fig. 3A). These cells were
not contaminating B cells, NK cells, or macrophages, since we excluded
those B220+, NK1.1, or
Mac-1+ populations, respectively, by fluorescent
gating. Furthermore, the majority of cells that expressed high levels
of Syk were not exclusively cells. Although T cells do
express significant levels of Syk in the thymus, after gating on
CD3- populations, as well as after directly
excluding TCR-expressing cells, we could still detect a
Syk-expressing population in the
CD4-CD8- cells (data not
shown).
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A). These levels
remain low in the progression to more mature CD4+
and CD8+ thymocytes as well as later when these
cells emerge as mature T cells in the periphery (Fig. 3A).
The above results contrast with our previously published data using a
rabbit heteroserum in which immunoblotting experiments suggested that
Syk is expressed at highest levels in the
CD4+CD8+ population
(19). To address this issue further, we sorted
CD4-CD8- thymocytes and
CD4+CD8+ thymocytes for
immunoblotting whole cell lysates with this new anti-Syk mAb and
anti-ZAP-70 antisera for comparison. Whole cell lysates were
generated from FACS-sorted cells that were 99% pure. (Fig. 3B). The results we obtained with these Abs are consistent
with the intracellular staining results: Syk levels are higher in the
CD4-CD8- population and
decrease in the CD4+CD8+
population (Fig. 3B). In contrast, ZAP-70 is expressed
throughout thymic development (Fig. 3B; see also Ref.
19). The immunoblotting experiment (Fig. 3B)
also controls for differences in kinase expression due to differences
in thymocyte size, as the lanes were normalized for protein content,
not cell number. The reasons for the discrepancies in Syk expression
between our previous results (19) and our current results
are not clear. It is possible that in the previous experiments,
contaminating Syk-expressing cells such as B cells were present in the
enriched populations or, more likely, that the Syk heteroserum that had
been previously used cross-reacted with a protein of similar size,
perhaps ZAP-70.
Syk is expressed at the pre-TCR signaling stage in mice
In mice, CD4-CD8-
thymocytes can be further subdivided into subpopulations based on
expression of the CD44 and CD25 cell surface proteins (2, 3). The expression of Syk within the
CD4-CD8- population was
analyzed. Using four-color analysis, wild-type thymocytes expressing
CD4, CD8, CD3, B220, and Mac-1 were excluded. The remaining thymocytes
were analyzed for CD44, CD25, and Syk expression (Fig. 4A). From these staining
results, it can be seen that Syk expression is elevated from the
CD44+CD25- through the
CD44-CD25+ stage (Fig. 4A). After the
CD44-CD25+ stage, Syk
levels appear to be down-regulated, with a notable decrease in Syk
expression by the
CD44-CD25- stage, and
uniformly low by the time the thymocytes have matured to
CD4+CD8+ cells (Figs. 3A and 4A). Interestingly, the
CD44-CD25+ stage of
thymocyte development corresponds to the stage at which thymocytes
receive signals through the pre-TCR complex (3). Thus, it
appears that Syk expression is elevated until the stage at which the
pre-TCR signal is propagated and then its expression is rapidly
down-regulated.
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, whole-cell lysates of thymocytes from
Lck/Fyn-deficient mice and RAG-deficient mice appeared to express
higher levels of Syk than wild-type mice. Because these mice are
arrested in thymic development at the
CD44-CD25+ stage
(13, 14), we reasoned that the increased Syk expression
seen in whole cell lysates results from the fact that the majority of
thymocytes are arrested at the point when Syk is most highly expressed.
We verified this hypothesis by staining for CD44 and CD25 in thymic
populations from Lck/Fyn-deficient and RAG-deficient mice (Fig. 4B and data not shown). As in the wild-type thymus, Syk was
expressed most highly at the
CD44-CD25+ stage and
decreased in expression by the
CD44-CD25- stage. Note
that the differences in maximal Syk staining intensity between
wild-type (Fig. 4A) and mutant (Fig. 4B) mice are
the result of using different flow cytometers (see legend to Fig. 4). Syk expression is down-regulated from the CD4-CD8- to CD4+CD8+ transition in murine thymocytes
We next sought to determine whether we could actively induce a
pre-TCR signal to down-regulate Syk during the transition from
CD4-CD8- thymocytes to
CD4+CD8+ thymocytes. To do
so, we used RAG- and
lck-/fyn- mice. As
mentioned above, the majority of thymocytes from these mice are
arrested at the pre-TCR checkpoint during
CD4-CD8- thymocyte
development. Previous experiments in RAG-1-deficient mice have
demonstrated that injection of anti-CD3 Abs can induce the arrested
CD44-CD25+ thymocytes to
mature into CD4+CD8+
thymocytes, presumably by cross-linking low levels of CD3 chains that
have reached the surface in the absence of the pre-T and ß-chains
(36). Because Lck/Fyn-deficient mice are arrested at an
identical stage in development, we wished to determine whether
injection of anti-CD3 Abs would have a similar effect. We suspected
that, similar to the case in SLP-76-deficient mice (37),
in the absence of both Lck and Fyn, anti-CD3 treatment of these
developmentally arrested thymocytes would not have an effect, because
these thymocytes are missing critical mediators of the pre-TCR
signaling pathway.
Surprisingly, the
CD4-CD8- cells from
lck-/fyn- mice
could be induced to develop, similar to the results observed in
RAG- mice (Fig. 5 and data not shown). These results
demonstrate that the developmental arrest in these thymocytes can be
overcome by the stimulation of the CD3 complex. In the
Lck/Fyn-deficient thymocytes, the TCR ß-chain was detected by
intracellular staining even before Ab treatment (data not shown),
suggesting that a functional pre-TCR complex can be generated in a
subpopulation of these thymocytes, which may allow for further
development of these cells. Induction of the
CD4+CD8+ cells following
anti-CD3 stimulation also resulted in TCR -chain rearrangements,
as detected by PCR assays (Fig. 6),
suggesting that the signal through the pre-TCR is generating bona fide
CD4+CD8+ thymocytes. As can
be seen from Fig. 6, before anti-CD3 treatment, only TCR ß-chains
are rearranged, whereas after anti-CD3 treatment, -chains are
also rearranged. Note that in RAG-deficient mice, neither - nor
ß-chains are rearranged, consistent with the function of the RAG
genes in TCR gene rearrangement. The ability of anti-CD3-treated
CD4-CD8- thymocytes to
mature in the absence of both Lck and Fyn might reflect Syk functioning
rather than ZAP-70 because of the decreased dependence of Syk on
Lck/Fyn function (24), and because Syk is expressed in
these cells.
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B and data not
shown). Consistent with the results observed in wild-type mice, the
induced CD4+CD8+ thymocytes
showed decreased levels of Syk expression compared with the
CD4-CD8- thymocytes (Fig. 5B and data not shown). Thus, Syk appears to be actively
down-regulated following a pre-TCR signal during the
CD4-CD8- to
CD4+CD8+ thymocyte
transition. The elevated levels of Syk at the
CD44-CD25+ stage may allow
anti-CD3 treatment to overcome the developmental block in
lck-/fyn- and
RAG- thymocytes. This result is especially
noteworthy because Syk activation is less dependent than ZAP-70 on Src
kinases (23, 24). Syk is expressed in human thymocytes
Having determined that Syk is down-regulated during the
CD4-CD8- to
CD4+CD8+ transition in
murine thymocytes, we were next interested in determining the pattern
of Syk expression in the human thymus. We believed that regulation of
Syk family members might differ between humans and mice, as suggested
by the observations of ZAP-70-deficiency in mice and humans
(15, 16, 17, 25, 26, 27, 28). Patients with SCID due to ZAP-70
deficiency have nonfunctional peripheral CD4+ T
cells, whereas in mice, loss of ZAP-70 results in a complete arrest at
the CD4+CD8+ to
CD4+ or CD8+ transition. We
reasoned that one possible explanation for the ability of human but not
murine thymocytes to mature was a differential regulation of Syk
expression in humans. To examine more closely the expression of Syk in
human thymocytes, we used an anti-Syk mAb that we have used for
immunoprecipitation and immunoblotting of human Syk (Ref.
30 and data not shown]. When used for immunoblotting,
this Ab detects Syk in lysates of human thymocytes as well as PBLs
(Fig. 7A). To assess whether
this Ab was also capable of staining Syk intracellularly, we costained
B cells from human PBLs with Abs against surface CD19 and intracellular
Syk. Syk could be stained in this population of cells, and the
specificity of staining once again was demonstrated by the ability of
specific peptide to compete with the staining (Fig. 7B).
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, in the
normal human thymus, similar to the mouse thymus, an elevated level of
Syk expression was present in a subpopulation of
CD4-CD8- cells; in this
case, a smaller subset express high levels of Syk. Syk levels remained
somewhat elevated even in the
CD4+CD8+ until the
transition to either the CD4+ or
CD8+ stage, where a majority of these thymocytes
showed decreased Syk expression. Strikingly, and in dramatic contrast
to the murine thymus, 5–10% of the CD4+
thymocytes expressed substantially elevated levels of Syk (Fig. 8). To
confirm the Sykhigh cells that we observed in the
CD4-CD8- and
CD4+ populations were thymocytes and not other
contaminating cell types, we examined forward and side scatter gating,
indicating that the Sykhigh cells were small
lymphocytes (data not shown). Exclusion of CD19+
and Mac-1+ (CD11b) cells by fluorescence gating
indicated that the
CD4-CD8- and the
CD4+ cells with elevated Syk expression were not
B cells, macrophages, or monocytes (data not shown).
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We hypothesized that this subpopulation of CD4+ thymocytes might represent a subset of cells that become precursors for the human CD4+ peripheral T cells that develop in ZAP-70-deficient patients. Alternatively, we considered the possibility that the CD4+ thymocytes were an immature thymic population that is an intermediate between CD4-CD8- and CD4+CD8+, as has been described in studies of human thymocyte development (38). Unlike the murine system, in which thymocytes develop from CD4-CD8- cells to CD4+CD8+ via a CD8+ intermediate, human thymocyte development proceeds through a CD4+ intermediate during the transition from CD4-CD8- to CD4+CD8+ thymocytes.
To assess whether the small subpopulation of CD4+
cells expressing elevated levels of Syk was immature or mature, we
examined their CD3 expression. Mature CD4+
thymocytes express high levels of CD3, whereas the immature
CD4+ intermediates express low levels of CD3. CD4
vs CD3 staining indicated that, in fact, the CD4+
Sykhigh cells expressed low levels of CD3 and
therefore represent the CD4+ developmental
intermediates between
CD4-CD8- and
CD4+CD8+ populations (Fig. 9). A comparable population of
Sykhigh cells in CD8+
thymocytes was not detected (data not shown). The expression of Syk in
the CD4+CD3low population
corroborates the idea that Syk is expressed in immature cells and
decreases in expression as thymocytes mature.
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-chain and the pre-TCR are most highly expressed
(39). Thus, the CD4+ intermediate
stage in humans appears to correspond functionally to the
CD44-CD25+ stage of murine
CD4-CD8- cells. It
appears the elevation of Syk expression during pre-TCR signaling has
been preserved between mice and humans, and that Syk is down-regulated
following the pre-TCR signal. Note also that
CD3int cells, contained within the
CD4+CD8+ stage of
development, again have higher levels of Syk than the more mature
subset. This higher level of Syk expression in
CD4+CD8+ human thymocytes
may be important for the ability of CD4+ cells to
develop in ZAP-70-deficient patients. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A role for Syk in pre-TCR signaling
As we have noted, Syk appears to be highly expressed in CD4-CD8- thymocytes, in contrast to our previously published results (19). We have confirmed the elevated expression in CD4-CD8- using our anti-Syk mAb both by intracellular staining and by immunoblot analysis of sorted thymocyte subpopulations. Because the analyses performed in this report utilized mAbs reactive with Syk, rather than a rabbit anti-peptide heteroserum, and analyzed Syk at the single cell level as well as by Western blotting, it is likely that the result contained in our previous report relating to Syk expression in murine thymic subsets was incorrect. Moreover, we have further shown that Syk is expressed at highest levels in the thymus in the CD44-CD25+ stage in the CD4-CD8- thymocyte compartment. The expression of Syk in CD4-CD8- thymocytes raises the possibility that Syk may be involved in pre-TCR signaling. A role for Syk in pre-TCR signaling has been suggested by prior genetic data (20). Although ZAP-70 deficient thymocytes progress to the CD4+CD8+ stage, thymocytes from Syk/ZAP-70 doubly-deficient mice are arrested at the CD44-CD25+ pre-TCR checkpoint in CD4-CD8- thymocytes (15, 16, 17, 20). Thus, expression of Syk in the absence of ZAP-70 appears to be sufficient for progression to the CD4+CD8+ stage. As we show here, Syk is in fact expressed at its highest levels just before the CD4-CD8- to CD4+CD8+ transition, at the CD44-CD25+ checkpoint.
The expression of Syk at the pre-TCR signaling stage has apparently
been preserved between mice and humans. Examination of the expression
of the human pT has revealed that this gene, and also the human
pre-TCR complex, is first expressed at the CD4+
intermediate stage (38). The TCR -chain is not
rearranged until after the CD4+ intermediate
thymocytes have become
CD4+CD8+ thymocytes,
presumably after a pre-TCR signal. We demonstrate that Syk is
strikingly elevated in the immature CD4+
thymocytes in which pre-TCR signaling is thought to occur. The
conservation of Syk expression and the pre-TCR in both mice and humans
suggests that Syk and the pre-TCR may in fact be functionally
linked.
Down-regulation of Syk after pre-TCR signaling
In mice, Syk is down-regulated as
CD4-CD8- cells become
CD4+CD8+ cells. We have
demonstrated this both by our observations of Syk expression in
wild-type thymocytes, as well as by inducing developmentally arrested
CD4-CD8-
Lck-/Fyn- thymocytes to mature with an
artificial stimulus. The fact that anti-CD3 treatment of Lck/Fyn
doubly deficient thymocytes allows for development past the pre-TCR
checkpoint is surprising, given that Src family kinases are thought to
be critical for initiation of the pre-TCR signal. Our results showing
that Syk is expressed at high levels at the pre-TCR thymocyte stage
(Figs. 4 and 5), as well as previous observations from our lab and
others that Syk is less dependent on Src family kinases than ZAP-70
(23, 24), suggest that Syk may be allowing the
anti-CD3 treatment to overcome the block in development in the case
of Lck/Fyn deficiency. Indeed, Syk can be activated in ZAP-70-deficient
thymocytes (17), suggesting that Syk can be activated
following a pre-TCR signal. Interestingly, in vivo treatment of
SLP-76-deficient mice does not induce progression past the
CD4-CD8- stage. This
result would be expected, because SLP-76 is a substrate for both Syk
and ZAP-70, and therefore in the absence of this common critical
downstream substrate, further development cannot occur.
Because of the down-regulation of Syk expression after pre-TCR signaling, only low amounts of Syk remain at the CD4+CD8+ stage, when positive and negative selection occur. Therefore, in ZAP-deficient CD4+CD8+ thymocytes, Syk is not expressed at sufficient levels, so positive and negative selection cannot occur. However, in ZAP-70-deficient mice in which a Syk transgene is constitutively expressed throughout thymic development, complete thymic development is restored, indicating that, if expressed at sufficiently high levels, Syk can compensate for ZAP-70 at later stages of development (22).
Syk expression and human ZAP-70 immunodeficiency
In humans, the down-regulation of Syk expression is not as
complete following pre-TCR signaling (Fig. 8). The fact that
CD4+CD8+ human thymocytes
still express significant levels of Syk may explain the differences
between the severity of the developmental phenotype of ZAP-70-deficient
mice and humans. Because Syk is more abundant in human than in murine
CD4+CD8+ cells, it may be
able to compensate for the loss of ZAP-70 more effectively in human
than in murine thymocytes, thereby allowing the development of some
human CD4+ thymocytes despite the lack of ZAP-70.
This subpopulation of CD4+ thymocytes would then
become the CD4+ peripheral T cells observed in
ZAP-70-deficient patients. However, because Syk expression is
down-regulated in peripheral T cells, the CD4+ T
cells that develop in ZAP-70-deficient patients remain unable to signal
(25, 26, 27, 28).
The signal that causes Syk to be down-regulated has not been identified. It is possible that the signal is a direct result of pre-TCR signaling, but it may be that another signal, perhaps delivered via a cytokine receptor, is responsible. Analysis of the syk promoter may indicate what types of transcriptional regulation control Syk expression.
Syk and coreceptor-independent signaling
The proposed function of Syk during pre-TCR signaling is consistent with the idea that Syk, when compared with ZAP-70, has a decreased dependence on Lck/Fyn function (23, 24, 40). One in vivo example of a situation in which Lck plays less of a role in TCR signaling is during coreceptor-independent signaling. In such situations, the TCR can signal in the absence of CD4 or CD8 coreceptors and therefore in the absence of the Lck molecules that are associated with the cytoplasmic tails of those coreceptors. Pre-TCR signaling is by definition coreceptor-independent, because it occurs in the absence of expression of either CD4 or CD8, at least in the murine system. Thus, during pre-TCR signaling, because CD4 and CD8 are not expressed, Lck is not recruited to the CD3 signaling complex as efficiently.
Based on the observed differences in regulation of expression and kinase activity of Syk and ZAP-70, we propose the following model. Syk and ZAP-70 both function during thymocyte development, but they are most critical during different stages. Their individual characteristics are appropriate for the delivery of two different types of signals through the thymocyte receptor complex. Conveniently, the expression of these kinases correlates with the time in development when they are proposed to be most useful. The pre-TCR appears to be less discriminating than the mature TCR in terms of signal initiation: a truncated pre-TCR lacking extracellular domains can restore thymic development in a RAG-deficient background, suggesting that no specific ligand-binding is necessary for stimulation of the pre-TCR (10). On the other hand, the TCR/coreceptor complex is responsible for discriminating ligand affinities over a wide concentration range. Syk is less dependent on upstream activators than ZAP-70: it is capable of autophosphorylation on its activation loop tyrosines, and it can function independently of CD45 or Lck (24, 41, 42). ZAP-70, in contrast, relies on transphosphorylation by Lck and Fyn for activation (43). Thus, Syk can more readily act as a signaling molecule during the pre-TCR signaling process and is expressed at the pre-TCR, but not the TCR, signaling stage. Conversely, ZAP-70 is likely to be more important during the processes of positive and negative selection at the CD4+CD8+ stage, when differences in the strength of the TCR signal are important to transmit and therefore tighter regulation of signaling is required. Although Syk-deficient mice have no severe defect in thymocyte development (44, 45), the efficiency of pre-TCR signaling and thymocyte development, as well as potential effects on repertoire development, have not been studied in these mice. Thus, it still remains possible that Syk may play an unrecognized specialized role in more efficient signal transduction by the pre-TCR, but not the mature TCR, in the thymic development. Our observations further demonstrate a sensitive and powerful application of the combination of intracellular staining and flow cytometry for the study of signaling proteins in situations in which cells must be analyzed at high purity or at limiting cell numbers.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75235.
3 Address correspondence and reprint requests to Arthur Weiss, Howard Hughes Medical Institute, Box 0795, 3rd and Parnassus Avenues, University of California, San Francisco, CA 94143. E-mail address:
4 Abbreviations used in this paper: pT, pre-T; PTK, protein tyrosine kinase; TC, tricolor; APC, allophycocyanin; PVDF, polyvinylidene difluoride.
Received for publication March 12, 1999. Accepted for publication June 28, 1999.
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