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Departments of
*
Internal Medicine,
Pathology, and
Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242
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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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Given the demonstrated role for SLP-76 in signaling through the TCR in
T cell lines, it is possible that SLP-76 may also function to mediate
TCR signals during thymocyte development. The first level of selection
in the thymus occurs before the CD4-CD8- to
CD4+CD8+ transition and is mediated by a
pre-TCR complex consisting of a TCRß-chain coupled to a surrogate
TCR
chain (pT) (13, 14). Many of the early
biochemical signals generated via pre-TCR ligation are similar to those
initiated following engagement of the mature TCR complex, implying that
the pre-TCR complex may recruit signaling intermediates in a manner
similar to the mature TCR complex (15, 16). Furthermore,
the process of positive selection within the
CD4+CD8+ subset is thought to rely on signals
mediated by the mature TCR complex (reviewed in 17 . Thus, SLP-76
may function to couple the pre-TCR and/or the mature TCR with
downstream signaling events at the appropriate stages of thymocyte
development.
In addition to T cells, SLP-76 mRNA or protein expression has been detected in several B cell lines, monocyte lines, and a rat basophilic leukemia (RBL) cell line (7, 18). To understand better which hemopoeitic cell types naturally express SLP-76 and how SLP-76 expression might be regulated during hemopoietic cell development, we surveyed various murine cell lineages for SLP-76 expression using a fluorochrome-labeled SLP-76 Ab and flow cytometry. Given the observation that SLP-76 functions as a TCR-signaling intermediate, we addressed a potential role for SLP-76 in T cell maturation by assaying various thymocyte developmental intermediates for SLP-76 expression. SLP-76 expression following TCR-dependent stimulation of mature T cells was also determined. The data presented reveal that SLP-76 expression in the adult mouse is restricted to cells of granulocyte, monocyte, and T lymphocyte lineage with no detectable expression in any of the B cell compartments analyzed. SLP-76 expression is regulated during thymocyte development, with highest expression detected early and late during thymocyte maturation at developmental stages that coincide with pre-TCR signaling and exit from the selection process which occurs within the CD4+CD8+ compartment. Finally, SLP-76 expression is enhanced following TCR engagement and remains high in those cells that acquire a memory phenotype. Together, these data suggest that SLP-76 expression is coupled with surface expression of a pre-TCR or mature TCR complex and that TCR-mediated signals augment the existing levels of SLP-76 expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were purchased from Harlan Sprague Dawley (Indianapolis, IN) or from the National Cancer Institute (Frederick, MD) and were housed under viral Ag-free conditions at the University of Iowa Animal Care Facility. Seven- to 10-wk-old female BALB/c mice were used for all experiments.
Abs
Hamster anti-mouse CD3
(145-2C11), biotin-conjugated
hamster anti-murine CD69 (H1.2F3), biotin-conjugated rat
anti-mouse Vß6 TCR (RR4-7), and FITC-conjugated rat
anti-mouse Ly-6G (Gr-1, RB6-8C5) were purchased form PharMingen
(San Diego, CA). Phycoerythrin (PE)-conjugated goat IgG was purchased
from Caltag (Burlingame, CA), Texas Red-conjugated ultra-avidin was
purchased from Leinco Technologies (Ballwin, MO), and Texas
Red-conjugated wheat germ agglutinin (WGA) was purchased from Molecular
Probes (Eugene, OR). Rat anti-mouse CD11b (Mac-1, M1/70), Ly-76
(Ter-119), CD4 (GK1.5), CD8 (53.6.72), CD44 (9F3), Vß8 TCR (F23.1),
Ly-6C (15.1), B220 (6B2), CD25 (7D4), CD62L (Mel-14), CD16/32 (2.4G2),
CD43 (S7), Ly-51 (BP-1), CD24 (J11d/HSA), and hamster anti-mouse
CD3 (145-2C11) were partially purified by 50% saturated ammonium
sulfate precipitation from serum-free supernatants (HB101) and were
conjugated where indicated with FITC, biotin, Cyanine 5-18, or PE using
standard procedures.
Purification of sheep anti-murine SLP-76
Glutathione-S-transferase (GST) and a fusion protein consisting of GST in frame with murine SLP-76 amino acids 136–235 (GST/SLP-76) were expressed in bacteria and purifed from bacterial lysates with glutathione agarose (Sigma, St. Louis, MO). Ten milliliters of sheep serum raised against the GST-murine SLP-76 fusion protein were precleared with Sepharose beads coupled to GST alone to remove Abs directed against the GST component of the immunogen. Precleared serum was then passed through an affinity column prepared by coupling GST-SLP-76 fusion protein to cyanogen bromide (CNBr)-activated Sepharose 4B beads that had been prepared according to manufacturer’s suggestions (Pharmacia Biotech, Piscataway, NJ) and washed with 10% FBS in PBS to block any remaining active sites. Following sample application, the column was washed with PBS, and purified Ab was eluted with 200 mM acetic acid. Eluted fractions were monitored for protein content (OD) and samples with peak activity were dialyzed against PBS and concentrated. The purified SLP-76 specific Ab preparation was then conjugated directly to PE using standard protocols.
Cell preparation and stimulation
Thymus, spleen, or lymph nodes (axillary and inguinal) were
removed and homogenized using frosted glass slides. Bone marrow was
flushed from the femur and a single cell suspension prepared using a
22-gauge needle. Cells were washed once in balanced salt solution (BSS)
and isolated by density gradient centrifugation (Ficoll-Lite, Atlanta,
GA) prior to staining. For in vivo stimulation, 8-wk-old mice were
injected i.p. with 30 µg of staphylococcal enterotoxin B (SEB)
(Sigma) or with an equal volume of PBS. The spleen was harvested and
cells analyzed at days 2, 5, and 7 posttreatment. For in vitro
stimulation, cells were stimulated with plate-bound CD3-specific Ab
(145-2C11, preincubated at 8 µg/ml) for the indicated times.
Flow cytometric analysis
Cells were incubated in staining buffer (1x BSS/5% FBS/0.1% sodium azide) with the appropriate biotin, FITC, PE, or Cyanine 5-18-conjugated Abs for 30 min at 4°C in the presence of normal rat serum (Pel-Freez, Rogers, AR) and rat anti-CD16/32 (2.4G2) to eliminate FcR-mediated background staining. Following two washes with staining buffer, samples were incubated with the appropriate avidin conjugate (Texas Red, FITC, or Cyanine 5-18) for 30 min at 4°C. After two more washes with staining buffer, cells were fixed and permeabilized using Permeafix (Ortho Diagnostic Systems, Raritan, NJ). Cytoplasmic staining was then carried out using either PE-conjugated goat IgG or PE-conjugated murine SLP-76 in wash buffer (PBS/5% FBS/1.5% BSA/0.005% EDTA) at room temperature. Normal goat serum (Jackson ImmunoResearch, West Grove, PA) was included at a 20% final concentration to reduce nonspecific binding. Cells were washed once in wash buffer and analyzed using a Coulter EPICS 753 instrument at the University of Iowa Flow Cytometry Facility. Approximately 10,000 events were analyzed for each sample unless indicated otherwise and spectral overlap was corrected by electronic compensation when necessary. Data analysis and display were performed using Flowjo version 2.3.3 software (Treestar, San Carlos, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To prepare a fluorochrome-conjugated SLP-76-specific Ab for use in
flow cytometric analysis of murine hemopoietic cell subsets, we
affinity purified an existing polyclonal Ab raised in sheep against a
GST-SLP-76 fusion protein. This serum was precleared against GST alone
to remove any Abs specific for the GST component of the immunogen
followed by affinity column purification using immobilized GST-SLP-76
fusion protein. Purified SLP-76-specific Abs were then eluted from the
column, concentrated, and directly conjugated to PE. SLP-76 expression
has been detected by Northern blot analysis in virtually all T cell
lines tested and in several B cell lines (7). As shown in
Figure 1
, the murine T cell line 2B4
displays specific intracellular staining with the affinity-purified
PE-conjugated SLP-76-specific Ab (SLP-76-PE). The murine B cell line
CH12 exhibits heterogeneous SLP-76 staining, while the murine B cell
line WEHI-231 does not express detectable levels of SLP-76 (Fig. 1A). These data agree with our observation that RT-PCR
analysis reveals expression of SLP-76 mRNA in the 2B4 and CH12 cell
lines but not in our WEHI-231 cell line (data not shown). We confirmed
the specificity of the affinity-purified SLP-76 antiserum by using this
reagent for immunoblot analysis of lysates obtained from the above cell
lines. The affinity-purified SLP-76-specific antisera clearly reacts
with a 76-kDa protein in lysates obtained from the 2B4 and CH12 cell
lines, but not from the WEHI cell line (Fig. 1B). In
addition, our murine SLP-76-specific antiserum demonstrates limited
cross-reactivity with human SLP-76 by immunoblot analysis (Fig. 1B) and, as such, does not significantly stain the human T
cell line Jurkat (Fig. 1A).
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A number of Abs that recognize surface markers expressed during
hemopoiesis are currently available which facilitate the identification
of various cell lineages in the adult bone marrow. By using a
combination of fluorochrome-labeled Ly-6C and B220-specific Abs with
WGA, it is possible to identify at least six distinct lineage subsets
within the bone marrow (19, 20). A number of additional
surface markers have been used to confirm the identity of these
populations (S. Bhatia, P. de Vries, and T. Waldschmidt, manuscript in
preparation). When SLP-76-PE is used in conjunction with Ly-6C, B220,
and WGA to stain adult murine bone marrow cells, it is apparent that
the predominant population that expresses SLP-76 is of myeloid
derivation (population 5, Fig. 2A). This observation agrees
with the reported expression of SLP-76 in several monocytic and in a
rat basophilic cell line (7, 18). Monocytic precursors
(population 6) also display appreciable SLP-76 expression, although the
level of background staining in this subset is substantial compared
with the other major developmental subsets in the bone marrow. A
population of cells with a phenotype consistent with stem cell
precursors (population 4) express low to intermediate levels of SLP-76.
Neither the early (population 2) or recirculating mature (population 3)
B cell subsets express detectable levels of SLP-76. In addition,
erythroid precursors (population 1) also exhibit minimal SLP-76
staining.
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C). Note that a subset of this
heterogeneous population displays very low levels of SLP-76, although
this level of expression is not significantly above background
staining. SLP-76 is not expressed in any of the
HSAhighB220int susbets identified based on CD43
or BP-1 expression. If SLP-76 is expressed in a multipotent lymphocyte
precursor, expression is down-regulated prior to the
HSAlowB220int stage of B lymphocyte development
and is not detectable at any subsequent stage of B cell maturation.
To confirm the myeloid-restricted expression of SLP-76 in the bone
marrow, the SLP-76-specific reagent was used in combination with
various surface markers specific for cells of the monocyte (Mac-1),
granulocyte (Gr-1), or erythroid (Ter-119) lineage. When bone marrow
cells are analyzed for SLP-76 expression based solely on side-scatter
phenotype, it is apparent that the more granular subset contains
SLP-76-positive cells, consistent with the predominance of myeloid
lineage cells in this subset (data not shown). Indeed, SLP-76
expression is restricted to the granulocyte (Gr-1+) and
monocyte (Mac-1+) subsets (Fig. 2B). The
erythroid compartment (Ter-119+) does not express SLP-76.
Consistent with the data obtained from bone marrow, SLP-76 expression
is also detected in Gr-1+ and Mac-1+ cells in
the peripheral blood but not in the Ter-119+ population
(data not shown).
SLP-76 expression is regulated during thymic development
The process of T cell maturation in the thymus can be followed
closely by monitoring the surface expression levels of several markers,
including the CD4 and CD8 coreceptors as well as CD3. Using these
markers in conjunction with SLP-76, we assayed distinct thymocyte
maturational subsets for SLP-76 expression. As shown in Figure 3A, the
CD4-CD8- thymocyte compartment displays a
heterogenous level of SLP-76 expression. The
CD4+CD8+ thymocytes express a uniform albeit
intermediate level of SLP-76. Within this population, the small subset
of CD3high cells (approximately 5%) express higher levels
of SLP-76 than the predominant CD3-/low subset (see arrow,
Fig. 3B). This CD3high population may represent
those cells that have been recently selected for further maturation.
This notion is supported by the observation that the level of SLP-76
expression in the mature thymocytes (CD4+ or
CD8+) is approximately twofold higher than that observed
for the majority of CD4+CD8+ thymocytes and
more comparable with the level of SLP-76 expression seen in the
CD3+CD4+CD8+ subset.
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B). In addition, SLP-76 expression remains high in the
CD69+CD4+ and CD69+CD8+
thymocytes. Interestingly, SLP-76 expression is slightly down-regulated
in both the CD4+ and CD8+ compartments, with a
more pronounced effect in the CD8+ T cells, as CD69
expression is lost and these cells are prepared for export from the
thymus to the peripheral lymphocyte compartments (Fig. 3B). SLP-76 is expressed most abundantly at the proT2 and proT3 stages of development within the CD3-CD4-CD8- thymocyte compartment
The heterogeneous nature of SLP-76 expression within the
CD4-CD8- thymocyte subset prompted us to
explore in more detail the expression pattern of SLP-76 in this
compartment. Within the
CD3-CD4-CD8- (triple-negative)
thymocyte subset, the maturation process can be delineated further
based on surface expression of CD44 and CD25 (23). The
earliest thymic immigrants are CD44+CD25-
(proT1). These cells then acquire CD25
(CD44+CD25+ or proT2) and mature further to a
CD44-CD25+ stage (proT3). The generation and
expression of a productive TCRß-chain in a complex with the surrogate
pT-chain triggers cell division, expansion, and further maturation
to a CD44-CD25- phenotype (ProT4) (24, 25). In the adult thymus, the proT3 and T4 populations are
readily identified based on CD44 and CD25 expression when those cells
that express CD3, CD4, or CD8 are excluded from analysis (Fig. 4A). The proT2 and proT3
subsets can also be visualized, but represent a small fraction of the
total triple-negative compartment (Fig. 4A). SLP-76
expression is detectable in all four early thymocyte developmental
subsets, but subtle differences in expression are worth noting. SLP-76
expression is heterogenous in the proT1 subset, but uniformly high in
the proT2 and proT3 cells (Fig. 4). Interestingly, the level of SLP-76
expression in the proT4 subset is less than that observed in the proT2
and proT3 population and more comparable with that found in the
CD4+CD8+ compartment.
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B). A significantly larger fraction
of the CD25- cells, which contain mostly proT4 thymocytes,
exhibit large forward scatter characteristics (Fig. 4B).
Within the CD25+ population, the level of SLP-76 expression
is comparable in both the small resting cells and large cycling cells,
suggesting that SLP-76 expression remains elevated as cells transit
from the proT2 to proT3 stage of development and does not appreciably
change as these cells enter the cell cycle (Fig. 4B). In
contrast, SLP-76 expression is higher in the large CD25-
triple-negative thymocytes when compared with CD25- cells
with a smaller phenotype. In this case, SLP-76 expression may decrease
concomitant with loss of surface CD25 expression and increase again as
proT4 thymocytes enter the cell cycle. Alternatively, SLP-76 expression
may remain elevated as thymocytes transit from a proT3 phenotype to the
proT4 stage of development and gradually falls as thymocytes lose CD25
and attain a quiescent state prior to acquisition of CD4 and
CD8. SLP-76 expression is restricted to T cells in the peripheral lymphoid organs and is highest in T cells bearing a memory cell phenotype
In the bone marrow, no SLP-76 expression is detected in either the
immature or mature recirculating B cell subsets (Fig. 2). In agreement
with this observation, SLP-76 expression in lymph node and spleen is
restricted to T lymphocytes (CD3+) with no appreciable
levels observed in the B cell (B220+) compartment (Fig. 5A). The T cell-restricted
expression of SLP-76 is supported by the observation that no SLP-76
mRNA was detected via RT-PCR in sorted
B220+CD23+ and
B220+CD23- splenic populations (M. Musci and
G. Koretzky, unpublished observations). When the T cell compartment is
divided into CD4+ and CD8+ subsets, no obvious
difference in SLP-76 expression is seen (Fig. 5B). However,
it is evident that the memory cell subset within the CD4+
compartment as defined by CD62L (Mel-14) and CD44 expression contains
more SLP-76 than the naive population, with the Mel-14low
subset displaying approximately twofold higher SLP-76 expression than
the Mel-14high subset (see 
MFI for each population, Fig. 5C). The elevated expression of SLP-76 might therefore
relate to the lower threshold of TCR-mediated activation observed in
CD4+ memory T cells (26). In our experiments
with BALB/c mice, it was difficult to identify a memory population in
the CD8+ subset based on Mel-14 and CD44 expression,
preventing the stringent analysis of SLP-76 expression in this
population.
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To address SLP-76 regulation following TCR stimulation, we used a
plate-bound CD3-specific Ab (145-2C11) as a means to stimulate
freshly isolated murine T lymphocytes. The early activation marker CD69
was used to identify activated T cells following 14 h of culture
in the absence or presence of anti-CD3-specific Ab. As
demonstrated in Figure 6, SLP-76
expression is augmented approximately three- to fourfold within 14
h after stimulation via the TCR. This effect correlates with an
activated phenotype as increased SLP-76 levels are found in cells that
express CD69 (lower panels) and have internalized
surface TCR (upper panels). This increased level of SLP-76
expression is characterized by a loss of the diffuse staining pattern
of SLP-76 observed in freshly isolated T cells. TCR induced
augmentation of SLP-76 expression has also been observed within 4
h following exposure to plate-bound 2C11, suggesting that the
activation-dependent up-regulation of SLP-76 is rapid (data not shown).
It should be noted that these experiments were performed in the
presence of reduced serum levels (1% FBS). Culture in higher levels of
serum alone results in significant augmentation of SLP-76 expression,
suggesting that factors present in serum are also capable of
activation-dependent augmentation of SLP-76 expression (data not
shown).
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The observations that SLP-76 is expressed more abundantly in
CD4+ T cells bearing a memory phenotype and that TCR
ligation augments SLP-76 expression led us to hypothesize that SLP-76
might be up-regulated following antigenic exposure. To address further
the activation-dependent expression of SLP-76, mice were treated with
the superantigen SEB. Exposure to SEB in the appropriate genetic
background leads to the specific stimulation of T cells bearing a TCR
with a Vß8 component (27). These SEB-reactive cells
expand to roughly twice the original number followed by a rapid
deletion phase that is characterized by apoptosis of the SEB-reactive
cells (28). In our experiments, we also observed a
dramatic increase in the Vß8+ T cell compartment with
maximum expansion at approximately 2 days post-SEB treatment (Table I). This expansion phase was followed by
a gradual decrease in Vß8+ T cell frequency, consistent
with apoptosis of the SEB-reactive subset. Interestingly, SLP-76
expression was substantially higher in the Vß8+ T cell
subset compared with the non-SEB-reactive Vß6+ control
population at day 2 posttreatment (Table I), suggesting that SLP-76
expression is augmented following ligation of the TCR. However, SLP-76
expression decreased to a level comparable with naive T cells as the
SEB-reactive subset was deleted.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our observation that SLP-76 is expressed in cells of myeloid lineage
suggests that SLP-76 may also function to couple receptor-mediated
signaling in granulocytes and monocytes. In support of this notion,
SLP-76 is tyrosine phosphorylated following ligation of the high
affinity IgE receptor in a RBL cell line and associates with
phosphoproteins that display m.w. consistent with those of SLAP-130 and
pp62 (18). A role for the adapter proteins Shc and Grb2 in
the regulation of respiratory burst following Fc
RI ligation has also
been demonstrated (30). Thus, adapter proteins such as
SLP-76 may dictate the activation-dependent formation of one or more
distinct signaling complexes within several hemopoietic cell lineages.
These signaling complexes may vary depending on the cell type, and may
promote positive or negative signals depending on the molecular
composition of the complex.
Our data document that SLP-76 expression is first detected in the T cell lineage at the proT1 stage of thymic development. The heterogeneous nature of SLP-76 expression within the proT1 subset may reflect the continuum of maturation within this population. The population of proT1 cells with the lowest SLP-76 expression may represent those cells that are the most immature and have recently immigrated to the thymus from the bone marrow. As cells mature within the proT1 subset, SLP-76 expression may gradually increase until it reaches a level comparable with that observed in the proT2 susbset. Those cells with the highest SLP-76 expression within the proT1 subset would presumably represent the most mature cells in this subset at a stage of development just prior to aquisition of CD25. Alternatively, the SLP-76-negative cells within the proT1 subset may represent a previously described population of cells that are not true T lymphocyte precursors and exhibit the potential to give rise to B cells or thymic dendritic cells (31, 32). Thus, it is possible that SLP-76 expression may serve as a useful marker for the identification of cells that have recently homed to the thymus and are committed to the T cell lineage.
Given the ability of SLP-76 to promote TCR-mediated signaling events, it is intriguing that SLP-76 expression is highest in those thymocytes that express significant levels of pre-TCR or mature TCR-signaling complexes. During thymocyte development, there are several developmental checkpoints that require a pre-TCR or mature TCR-dependent signal. Within the triple-negative population, SLP-76 expression is highest within the CD25+ subset, which contains thymocytes at the proT2 and proT3 stages of maturation. Expression of a competent pre-TCR complex at the proT3 developmental stage is required for signaling cell cycle initiation and further maturation (24, 25). Within the CD25+ triple-negative thymocyte compartment, SLP-76 expression is elevated in both the small resting and large cycling cells, suggesting that SLP-76 levels rise prior to initiation of cycling. However, SLP-76 expression is higher in the large CD25- thymocyte subset compared with the small CD25- cells. Given the low level of SLP-76 expression observed at the next stage of maturation (CD4+CD8+), we favor the hypothesis that SLP-76 expression is gradually down-regulated as cells transit from the proT3 to proT4 stage of development and acquire a resting phenotype. Taken together, these data suggest that SLP-76 expression increases as cells approach a stage of thymocyte development (proT3) in which pre-TCR-mediated signaling events are required for further maturation and down-regulated once cells have passed through this pre-TCR-dependent checkpoint.
Engagement of the pre-TCR is known to activate several signaling cascades similar to those elicited by ligation of the mature TCR complex, including activation of tyrosine kinases (15). To date, the best candidate for the tyrosine kinase responsible for the TCR-dependent phosphorylation of SLP-76 is the Syk kinase family member ZAP-70 (33). The requirement for Syk family tyrosine kinases in pre-TCR-driven thymocyte development has beeen demonstrated by the generation of mutant strains of mice that lack both ZAP-70 and Syk (16). These mice manifest a severe block in the transition from the proT3 to proT4 stage of thymic development. If SLP-76 is required to translate ZAP-70 and/or Syk activation into downstream signaling events following ligation of the pre-TCR, one might predict that a SLP-76 deficiency would result in a phenotype similar to ZAP-70/Syk-deficient mice. It is also possible that additional adapter-like proteins are present, which serve a role similar to that of SLP-76 during thymocyte development. The generation of SLP-76-deficient and transgenic strains will facilitate the determination of a more definitive role for SLP-76 in these processes.
The second round of selection in the thymus is thought to be dictated by the mature TCR complex and is required to eliminate potentially autoreactive T cell clones and perpetuate those thymocytes that express a TCR complex capable of conferring a protective role. Again, the involvement of Syk family tyrosine kinases in this process has been demonstrated by the observation that ZAP-70 deficiency in mice prevents maturation from a CD4+CD8+ phenotype to a mature CD4+ or CD8+ phenotype (34). The processes of negative and positive selection are thought to be mediated by the affinity of the TCR complex for self-MHC expressed on accessory cells in the thymus and the subsequent "strength" of the TCR signal initiated by this interaction (reviewed in Refs. 17 and 35). Those TCRs with a high affinity for self MHC mediate signals that lead to apoptosis (negative selection) while those with an intermediate affinity promote survival (positive selection). Thymocytes that contain a TCR with low or no affinity for self-MHC are proposed to die by "neglect." As a result, relatively few thymocytes mature to a functional, immunocompetent T cell. Several markers, including CD3 and CD69, have been identified that correlate with the onset of positive selection (22). Interestingly, CD3+ or CD69+ CD4+CD8+ thymocytes express a higher level of SLP-76 than their CD3- or CD69- counterparts. Given the effect of TCR ligation on SLP-76 expression, it is possible that TCR ligation during selection augments SLP-76 expression. Alternatively, it may be necessary to maintain a low level of SLP-76 expression at the CD3low CD4+CD8+ stage of development to prevent the conversion of low affinity TCR interactions into high affinity signals. The low expression of SLP-76 in the majority of CD4+CD8+ thymocytes correlates well with the reduced signaling potential of additional TCR-coupled signaling molecules, including the transcription factors NF-AT and AP-1. Both NF-AT and AP-1 are inducible in CD4-CD8- and mature CD4+ or CD8+ thymocytes, but inducible DNA-binding activity is lost in the CD4+CD8+ subset (36). While SLP-76 demonstrates the capacity to augment both NF-AT and AP-1 when overexpressed in Jurkat T cells, a causal relationship between low SLP-76 expression and reduced NF-AT and AP-1-binding activity in the CD4+CD8+ population remains to be determined.
While it is clear that TCR ligation augments SLP-76 expression,
the actual mechanism by which SLP-76 protein expression is regulated
during thymic development and following TCR-dependent activation is not
known. We have made the observation that SLP-76 contains a single,
amino-terminal proline-glutamic acid-serine-threonine rich (PEST)
domain (amino acid residues 99–163), a motif that has been implicated
in the metabolic stability of many proteins, including members of the
I-
B family and specific components of the cell cycle control
machinery (37, 38, 39, 40). Interestingly, the SLP-76 PEST domain
contains the tyrosine residues implicated in Vav recruitment. It is
possible that the rapid recruitment of Vav or additional molecules to
SLP-76 following TCR ligation masks the PEST domain and stabilizes
SLP-76 expression. The diffuse nature of SLP-76 expression in freshly
isolated T cells and the rapid increase in SLP-76 levels following TCR
ligation support the hypothesis that existing SLP-76 protein is
stabilized following activation. Alternatively, increased SLP-76
expression following TCR ligation may be a function of increased mRNA
production or transcript stability. Detailed kinetic studies of SLP-76
mRNA and protein expression following TCR ligation will be required to
determine the mechanism by which SLP-76 expression is regulated.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Gary A. Koretzky, Department of Internal Medicine, University of Iowa College of Medicine, 540 EMRB, Iowa City, IA 52242. E-mail address:
4 Abbreviations used in this paper: NF-AT, nuclear factor of activated T cells; RBL, rat basophilic leukemia; PE, phycoerythrin; WGA, wheat germ agglutinin; SEB, staphylococcal enterotoxin B; MFI, mean fluorescence intensity; GST, glutathione-S-transferase; BSS, balanced salt solution.
Received for publication February 11, 1998. Accepted for publication June 9, 1998.
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 N. A. Luckashenak, R. L. Ryszkiewicz, K. D. Ramsey, and J. L. Clements The Src Homology 2 Domain-Containing Leukocyte Protein of 76-kDa Adaptor Links Integrin Ligation with p44/42 MAPK Phosphorylation and Podosome Distribution in Murine Dendritic Cells J. Immunol., October 15, 2006; 177(8): 5177 - 5185. [Abstract] [Full Text] [PDF]
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 F. Abtahian, N. Bezman, R. Clemens, E. Sebzda, L. Cheng, S. J. Shattil, M. L. Kahn, and G. A. Koretzky Evidence for the Requirement of ITAM Domains but Not SLP-76/Gads Interaction for Integrin Signaling in Hematopoietic Cells. Mol. Cell. Biol., September 1, 2006; 26(18): 6936 - 6949. [Abstract] [Full Text] [PDF]
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M. S. Jordan, J. Sadler, J. E. Austin, L. D. Finkelstein, A. L. Singer, P. L. Schwartzberg, and G. A. Koretzky Functional Hierarchy of the N-Terminal Tyrosines of SLP-76 J. Immunol., February 15, 2006; 176(4): 2430 - 2438. [Abstract] [Full Text] [PDF]
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 J. S. Maltzman, L. Kovoor, J. L. Clements, and G. A. Koretzky Conditional deletion reveals a cell-autonomous requirement of SLP-76 for thymocyte selection J. Exp. Med., October 3, 2005; 202(7): 893 - 900. [Abstract] [Full Text] [PDF]
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S.-H. Lee, Y.-H. Chung, N.-H. Cho, Y. Gwack, P. Feng, and J. U. Jung Modulation of T-Cell Receptor Signal Transduction by Herpesvirus Signaling Adaptor Protein Mol. Cell. Biol., June 15, 2004; 24(12): 5369 - 5382. [Abstract] [Full Text] [PDF]
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J. N. Wu, M. S. Jordan, M. A. Silverman, E. J. Peterson, and G. A. Koretzky Differential Requirement for Adapter Proteins Src Homology 2 Domain-Containing Leukocyte Phosphoprotein of 76 kDa and Adhesion- and Degranulation-Promoting Adapter Protein in Fc{epsilon}RI Signaling and Mast Cell Function J. Immunol., June 1, 2004; 172(11): 6768 - 6774. [Abstract] [Full Text] [PDF]
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 A. L. Singer, S. C. Bunnell, A. E. Obstfeld, M. S. Jordan, J. N. Wu, P. S. Myung, L. E. Samelson, and G. A. Koretzky Roles of the Proline-rich Domain in SLP-76 Subcellular Localization and T Cell Function J. Biol. Chem., April 9, 2004; 279(15): 15481 - 15490. [Abstract] [Full Text] [PDF]
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K. E. Nichols, K. Haines, P. S. Myung, S. Newbrough, E. Myers, H. Jumaa, D. J. Shedlock, H. Shen, and G. A. Koretzky Macrophage activation and Fc{gamma} receptor-mediated signaling do not require expression of the SLP-76 and SLP-65 adaptors J. Leukoc. Biol., March 1, 2004; 75(3): 541 - 552. [Abstract] [Full Text] [PDF]
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X.-P. Zhong, J. S. Maltzman, E. A. Hainey, and G. A. Koretzky Transcriptional Regulation of Src Homology 2 Domain-Containing Leukocyte Phosphoprotein of 76 kDa: Dissection of Key Promoter Elements J. Immunol., December 15, 2003; 171(12): 6621 - 6629. [Abstract] [Full Text] [PDF]
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K. N. Kremer, T. D. Humphreys, A. Kumar, N.-X. Qian, and K. E. Hedin Distinct Role of ZAP-70 and Src Homology 2 Domain-Containing Leukocyte Protein of 76 kDa in the Prolonged Activation of Extracellular Signal-Regulated Protein Kinase by the Stromal Cell-Derived Factor-1{alpha}/CXCL12 Chemokine J. Immunol., July 1, 2003; 171(1): 360 - 367. [Abstract] [Full Text] [PDF]
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A. Kettner, V. Pivniouk, L. Kumar, H. Falet, J.-S. Lee, R. Mulligan, and R. S. Geha Structural Requirements of SLP-76 in Signaling via the High-Affinity Immunoglobulin E Receptor (Fc{varepsilon}RI) in Mast Cells Mol. Cell. Biol., April 1, 2003; 23(7): 2395 - 2406. [Abstract] [Full Text] [PDF]
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B. Guyot and G. Mouchiroud Characterization of promoter elements directing Mona/Gads molecular adapter expression in T and myelomonocytic cells: involvement of the AML-1 transcription factor J. Leukoc. Biol., February 1, 2003; 73(2): 263 - 272. [Abstract] [Full Text] [PDF]
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S. Kabak, B. J. Skaggs, M. R. Gold, M. Affolter, K. L. West, M. S. Foster, K. Siemasko, A. C. Chan, R. Aebersold, and M. R. Clark The Direct Recruitment of BLNK to Immunoglobulin {alpha} Couples the B-Cell Antigen Receptor to Distal Signaling Pathways Mol. Cell. Biol., April 15, 2002; 22(8): 2524 - 2535. [Abstract] [Full Text] [PDF]
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B. A. Judd, P. S. Myung, A. Obergfell, E. E. Myers, A. M. Cheng, S. P. Watson, W. S. Pear, D. Allman, S. J. Shattil, and G. A. Koretzky Differential Requirement for LAT and SLP-76 in GPVI versus T Cell Receptor Signaling J. Exp. Med., March 18, 2002; 195(6): 705 - 717. [Abstract] [Full Text] [PDF]
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S. F. Hussain, C. F. Anderson, and D. L. Farber Differential SLP-76 Expression and TCR-Mediated Signaling in Effector and Memory CD4 T Cells J. Immunol., February 15, 2002; 168(4): 1557 - 1565. [Abstract] [Full Text] [PDF]
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J. Yu, C. Riou, D. Davidson, R. Minhas, J. D. Robson, M. Julius, R. Arnold, F. Kiefer, and A. Veillette Synergistic Regulation of Immunoreceptor Signaling by SLP-76-Related Adaptor Clnk and Serine/Threonine Protein Kinase HPK-1 Mol. Cell. Biol., September 15, 2001; 21(18): 6102 - 6112. [Abstract] [Full Text] [PDF]
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D. Yablonski, T. Kadlecek, and A. Weiss Identification of a Phospholipase C-{gamma}1 (PLC-{gamma}1) SH3 Domain-Binding Site in SLP-76 Required for T-Cell Receptor-Mediated Activation of PLC-{gamma}1 and NFAT Mol. Cell. Biol., July 1, 2001; 21(13): 4208 - 4218. [Abstract] [Full Text] [PDF]
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 B. A. Judd, P. S. Myung, L. Leng, A. Obergfell, W. S. Pear, S. J. Shattil, and G. A. Koretzky Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through alpha IIbbeta 3 and collagen receptors PNAS, October 24, 2000; 97(22): 12056 - 12061. [Abstract] [Full Text] [PDF]
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N. J. Boerth, J. J. Sadler, D. E. Bauer, J. L. Clements, S. M. Gheith, and G. A. Koretzky Recruitment of Slp-76 to the Membrane and Glycolipid-Enriched Membrane Microdomains Replaces the Requirement for Linker for Activation of T Cells in T Cell Receptor Signaling J. Exp. Med., October 2, 2000; 192(7): 1047 - 1058. [Abstract] [Full Text] [PDF]
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 P. S. Myung, J. L. Clements, D. W. White, Z. A. Malik, J. S. Cowdery, L.-A. H. Allen, J. T. Harty, D. J. Kusner, and G. A. Koretzky In vitro and in vivo macrophage function can occur independently of SLP-76 Int. Immunol., June 1, 2000; 12(6): 887 - 897. [Abstract] [Full Text] [PDF]
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D. Peters, M. Tsuchida, E. R. Manthei, T. Alam, C. S. Cho, S. J. Knechtle, and M. M. Hamawy Potentiation of CD3-induced expression of the linker for activation of T cells (LAT) by the calcineurin inhibitors cyclosporin A and FK506 Blood, May 1, 2000; 95(9): 2733 - 2741. [Abstract] [Full Text] [PDF]
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M. Y. Cao, D. Davidson, J. Yu, S. Latour, and A. Veillette Clnk, a Novel Slp-76-Related Adaptor Molecule Expressed in Cytokine-Stimulated Hemopoietic Cells J. Exp. Med., November 15, 1999; 190(10): 1527 - 1534. [Abstract] [Full Text] [PDF]
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R. Sanzenbacher, D. Kabelitz, and O. Janssen SLP-76 Binding to p56lck: A Role for SLP-76 in CD4-Induced Desensitization of the TCR/CD3 Signaling Complex J. Immunol., September 15, 1999; 163(6): 3143 - 3152. [Abstract] [Full Text] [PDF]
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F. A. Bonilla, R. M. Fujita, V. I. Pivniouk, A. C. Chan, and R. S. Geha Adapter proteins SLP-76 and BLNK both are expressed by murine macrophages and are linked to signaling via Fcgamma receptors I and II/III PNAS, February 15, 2000; 97(4): 1725 - 1730. [Abstract] [Full Text] [PDF]
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