Abstract
Intracellular signaling from the T cell receptor (TCR) is important in the development
of immature T cells in the thymus before they migrate to the peripheral tissues as mature T
cells. TCR-mediated signaling events continue to be crucial in the activation of mature T
cells and in immune responses against foreign pathogens. In T cells, a cytosolic adapter
protein, SLP-76 (SH2 domain-containing leukocyte phosphoprotein of 76 kDa), is critical in
the assembly of the TCR signaling complex following T cell activation. Adaptors are
signaling proteins that have no enzymatic or transcriptional activities. These proteins serve as
scaffolds to nucleate multiple proteins in close proximity to form signaling complexes. SLP76 contains four structural domains – three of the domains have been well-studied, but the
function of the N-terminal sterile α-motif (SAM) domain at the N-terminus has yet to be
characterized.
Unlike other frequently encountered protein domains, the SAM domain has not been
associated with any unifying function. SAM domain-containing proteins bind to a wide
variety of proteins and are involved in numerous biological processes. The SAM domain was
deleted from SLP-76 in mice to probe its role in SLP-76. Thymocyte development in the
mutant mice was partially blocked, leading to reduced output of mature T cells. TCRmediated calcium mobilization was reduced in the thymocytes. Calcium mobilization, MAP
kinase activation and IL-2 production were reduced in mature mutant T cells. These results
suggest that the SAM domain in SLP-76 plays an important role in the TCR-mediated
signaling that is necessary for thymocyte development and activation of mature T cells.
Introduction
T cells are a type of white blood cells that play an important role in cell-mediated
immunity. T cells express membrane-bound T cell receptors (TCRs) on their cell surface to
recognize antigens in the form of short peptides presented by major histocompatibility
complex (MHC) molecules on antigen-presenting cells, such as dendritic cells and
macrophages (figure 1) (1). Antigen stimulation through the TCR will trigger downstream
mechanisms such as cell proliferation and differentiation, cytokine production, and direct
killing of the pathogen-infected cells (2).
Figure 1. Engagement of the TCR with peptide/MHC complex on antigen-presenting cells.
Modified from http://library.med.utah.edu/kw/ms/mml/ms_etiology01.html
T cells originate in the bone marrow as hematopoietic stem cells that migrate to the
thymus where they undergo differentiation (3, 4). These precursor cells undergo a series of
arrangements of the TCR α and β gene fragments to generate a large diversity of antigenspecific TCRs that recognize a unique antigen (5, 6). However, amongst the large pool of
immature thymocytes produced, only about 2% of them survive the stringent selection
processes described below to become mature T cells that are released into peripheral tissues,
such as the spleen and lymph nodes (7, 8).
The T cell population and the specific stages of their development can be identified
by the presence of specific cell surface marker proteins. In the early stages, pre-T cells
2
express neither CD4 nor CD8 markers (9, 10). This population of cells is termed the doublenegative or ‘DN’ cells. The DN thymocytes can be further subdivided into four
subpopulations by surface expression of cell markers CD44 and CD25 (11, 12). Maturation
within the DN population follows the sequence CD25–CD44+ → CD25+CD44+ →
CD25+CD44– → CD25–CD44– (figure 2) (11, 12). At the CD25+CD44– DN3 stage, the
TCRβ chain is first expressed in the thymocytes. TCRβ chain associates with the surrogate α
chain and its accessory chains to form the pre-TCR complex (13). Signaling through the preTCR drives the development of these cells into CD4+CD8+ immature double positive (DP) T
cells, during which the TCRα chain is expressed and replaces the surrogate α chain (14, 15).
TCRα chain
rearrangement and
expression
TCRβ chain
rearrangement and
expression
Precursor
T cell
–
–
CD4 CD8
DN1
–
CD25
CD44+
DN2
CD25+
CD44+
DN3
CD25+
–
CD44
DN4
–
CD25
–
CD44
DP immature
T cell
CD4+CD8+
Figure 2. Maturation of the subsets of DN population. The precursor T cells (yellow) develop
following the sequence DN1 to DN4 (green), upregulating or downregulating the cell markers, CD44
and CD25. TCRβ chain is first expressed at the DN3 stage and the α chain is then expressed in the
immature T cells (blue).
The DP immature T cells expressing functional TCRs will undergo selection
processes (figure 3), after which they downregulate the expression of one of the cell surface
markers, CD4 or CD8 (4, 16). The cells then differentiate to either CD4+ or CD8+ cells.
These mature T cells then leave the thymus to the peripheral lymphoid organs such as the
spleen and lymph nodes (figure 3) (16).
3
Figure 3. Stages of T cell maturation. Modified from Kuby, Immunology (W.H. Freeman), 2006.
Pre-T cells from the bone marrow are DN cells (yellow). After rearrangement of TCR genes,
immature DP cells (pink) express both CD4 and CD8 markers alongside with the functional TCR and
associated CD3 chains (figure 5). CD4 and CD8 are also the co-receptors that are part of the TCR
complex. After selection (figure 4), the cells mature into CD4+ or CD8+ T cell populations (red).
T cells are an integral part of the immune system to protect the host and therefore it is
crucial that T cells are able to distinguish self from foreign antigens (16, 17). The selection
processes in the thymus ensures that only T cells with TCRs that are able to recognize
foreign peptides to survive and mature, while eliminating those which are reactive to selfantigens (4, 16, 17).
There are two selection processes – positive and negative selection – in thymocyte
development (figure 4) (16). In positive selection, T cells which bind with low or
intermediate affinity to peptides presented by an MHC molecule are protected from cell
death (18, 19). This also ensures that the thymocyte receptors are capable of binding selfMHC molecules. Cells which fail to recognize self-MHC are eliminated by apoptosis (19,
20).
Following positive selection, the population of MHC-restricted thymocytes is
subjected to negative selection (21). The cells express TCRs recognizing self antigens
presented by self-MHC molecules with varying affinities from low to high (8, 16).
Thymocytes with high-affinity receptors will be directed towards apoptosis, resulting in self-
4
tolerance, while the rest of the thymocytes are selected for expansion and differentiation
towards mature T cells (16, 21).
Figure 4. Positive and negative selection of DP T cells. Modified from Kuby, Immunology (W.H.
Freeman), 2006. The color schemes of the cells are as previous. The epithelial cells, macrophages and
dendritic cells (colored blue) are antigen-presenting cells displaying the antigens on MHC molecules.
Active signaling from mature TCR initiated by engagement with an intrathymic
ligand has been found to be essential in the selection processes of T cell development and
mature T cell activation and proliferation (22, 23). As shown in figure 5, the αβ TCR
contains the antigen binding site to recognize the peptide/MHC complex (8). However, these
chains have very short cytoplasmic tails and are not able to associate with intracellular
molecules alone to mediate downstream signal transduction (23). The αβTCR associates
with CD3 chains to form the TCR complex. CD3 is a complex of 5 polypeptide chains that
5
form 3 dimers, as shown in figure 5 (23). The CD3 chains have short extracellular regions
and long cytoplasmic chains containing immunoreceptor tyrosine-based activation motifs
(ITAMs) that are initiators of the downstream signal transduction (24).
Figure 5. T cell receptor with its associated CD3 chains. From Janeway, Immunobiology Fig. 6.8,
Garland Pub, 2001. The ITAMs on the associated CD3 chains (εδ and γε heterodimers and ζζ
homodimer) are represented by the yellow bars. The horizontal black linkers between α and β or ζ
and ζ subunits indicate the disulphide bonds between the subunits. The cell membrane is represented
as a gray layer.
Figure 6 summarizes the TCR-initiated signal transduction cascades. Following TCRpeptide/MHC engagement, Src kinase Lck is activated and phosphorylates tyrosine residues
within the ITAMs of CD3 chains (25, 26). Protein tyrosine kinase ZAP-70 is recruited to the
TCR via its interaction with ITAMs (25, 27). ZAP-70 is phosphorylated by Lck and in turn
phosphorylates LAT, a membrane adaptor protein (27-30). LAT interaction with Grb2/Sos
(an adaptor protein-enzyme complex) activates G-protein Ras which induces the Ras-RafMEK-ERK MAP kinase cascade (31, 32), while LAT interaction with Gads and SLP-76, two
other adaptor proteins (33-38), activates Vav (a guanine nucleotide exchange factor) which
further activates the Rac/JNK and the p38 MAP kinase cascades (39-43). SLP-76, the focus
of my work (see below), also recruits tyrosine kinase Itk to the LAT complex, and the
complex binds to intracellular enzyme PLCγ1 (44, 45). Upon phosphorylation, PLCγ1
6
enzyme is activated and cleaves phosphatidylinositol biphosphosphate (PIP2) into
diacylglycerol (DAG) and second messenger inositol triphosphate (IP3). IP3 induces
downstream calcium mobilization, while DAG activates enzyme protein kinase C (PKC)
which initiates IκB kinase activation of transcription factor NFκB (46-48). Activation of
three MAP kinase cascades, the calcium mobilization pathway and the IκB/ NFκB pathway
leads to production of cytokine IL-2 which stimulates the survival, proliferation and
differentiation of T cells (25, 49, 50).
Figure 6. Downstream TCR signaling pathways of SLP-76 and LAT. Modified from
http://www.med.uni-magdeburg.de/fme/institute/iim/imd30302.html. TCR with its associated CD3
chains are shown at the top of the diagram. The vertical blue bars represent the ITAMs. Certain
phosphorylation events are indicated by black circles with “P”. As shown, phosphorylated SLP-76
and LAT serve as major docking scaffolds for several downstream signaling molecules to form a
large complex at the membrane. Dotted arrows represent signaling events. Perpendicular cross arrows
from PIP2 indicate cleavage of the molecule. Solid arrows at the bottom of the diagram represent the
activation of IL-2 promotor to initiate IL-2 gene transcription. Details in text.
7
The molecule of interest in my thesis is SH2 domain-containing leukocyte-specific
phosphoprotein of 76 kDa (SLP-76). SLP-76 is a cytosolic adaptor protein that is critical in
the nucleation of the TCR signaling complex (29). Adaptor proteins are signaling proteins
that have no enzymatic or transcriptional activities (25). These proteins serve as scaffolds to
nucleate multiple proteins in close proximity to form signaling complexes (25).
Previous studies have shown that SLP-76 is essential for thymocyte development and
mature T cell activation (51). SLP-76 deficient mice have a profound block in thymic
development, leading to a lack of DP cells and mature SP T cells (30, 52-54). Jurkat T cell
lines deficient in SLP-76 also exhibit defects in TCR-induced ERK activation, PLCγ1
phosphorylation, and calcium influx, resulting in a defect in IL-2 production (55). Other
experiments have also shown that overexpression of SLP-76 results in increased TCRmediated production of IL-2 (56, 57). SLP-76 is therefore speculated to act as a common
downstream target for upstream TCR signals that are important to drive the development and
expansion of DP thymocytes, as well as the activation of mature T cells to lead to their
expansion and proliferation (3).
Four structural domains of SLP-76 have been identified (figure 7): (1) a sterile αmotif (SAM) domain at the N-terminus; (2) an acidic region with three tyrosine residues
within consensus motifs for SH2 (Src homology 2) binding with phosphorylated tyrosine
residues in proteins (29, 45, 58). Phosphorylated SLP-76 binds to Vav and Itk in this region
to activate the JNK and PLCγ1 downstream pathways (figure 6) (58-60); (3) a central
proline-rich region that binds the SH3 (Src homology 3) domain of Gads (35, 55, 61, 62);
and (4) a carboxy-terminal SH2 domain that interacts with adaptor protein SLAP, a negative
regulator of TCR signaling and kinase HPK1 (63).
8
In B cells, the homologous cytosolic adaptor protein of SLP-76, named BLNK (or
SLP-65), has been identified as a signaling element important in the nucleation of the B-cell
receptor (BCR) signaling complex (64-66). BLNK has a strikingly similar domain structure
as SLP-76 despite only 33% protein sequence homology (64, 66). Like SLP-76, BLNK
contains an amino-terminal acidic region with five (instead of three) tyrosine
phosphorylation motifs for SH2 binding, a central proline-rich domain and a carboxyterminal SH2 domain (64, 66) (figure 7). The functions of the acidic SH2 domain, prolinerich region and the C-terminal SH2 domain have been studied in both SLP-76 and BLNK.
However, the additional SAM domain at the N-terminus of SLP-76 (absent in BLNK) has yet
to be characterized.
SLP-76
BLNK
Figure 7. Comparison of structural domains of SLP-76 and BLNK. Modified from SLP76 and
SLP65: complex regulation of signaling in lymphocytes and beyond, Gary A. Koretzky, Farhad
Abtahian and Michael A. Silverman, Nature reviews, 2006. As shown, SLP-76 and BLNK share very
similar domain structure, represented by the different colored regions (cyan, blue and green). SLP-76
has an additional SAM domain at the N-terminus (colored orange) that is absent in BLNK. See text
for details.
The SAM domain is known to be a protein-protein interaction motif and it has been
reported that there are over 1300 SAM-containing proteins (67). This number is comparable
to that of the common SH2 domain (68, 69). However, unlike SH2 and SH3 (70, 71), no
common function is known for SAM domains, as they have several binding properties (72).
SAM domains have been found to interact with each other to form homodimers or polymers.
They can also bind to other related SAM domains or non-SAM domain-containing proteins,
9
and have been also reported to bind to RNA (73-75). This versatility accounts for the wide
range of roles the SAM domain plays in biological processes, from signal transduction to
transcriptional and translational regulation (67, 72). For example, SAM domains can serve as
kinase docking sites in the ETS family of transcription activators that are involved in cell
growth control, differentiation and embryonic development, leading to the activation of the
Ras/MAP kinase pathway (76, 77). The SAM domain is also found in several Drosophila
Polycomb group proteins and their mammalian relatives (78, 79). These proteins are
transcriptional repressors that are essential for restricting Hox gene expression along the
anterior-posterior axis. An example is the Polyhomeotic (Ph) and Sex comb on midleg (Scm)
gene, both containing C-terminal SAM domains (79, 80). The SAM domains of these two
proteins interact with themselves, as well as with each other, each forming a polymer that has
been shown to be crucial in transcriptional repression (80).
Given the diverse functions of the SAM domain, I was curious to investigate the
function of the SAM domain in SLP-76 in mediating T cell development and mature T cell
activation. To elucidate the role of the SAM domain of SLP-76, we generated knock-in mice
homozygous for SLP-76 with the SAM domain deleted (SLP76m/m).
We showed that SLP76m/m mice exhibited a partial block in T-cell development. In
the thymocytes, there was a decreased ratio of CD4+:CD8+ thymocytes and significant
reduction in the absolute thymocyte cell count compared to SLP76m/+ mice. In the mature
peripheral T cells, there was a corresponding decrease in the proportion of mature SP T cells
in the spleen of SLP76m/m mice. TCR-mediated MAP kinase activation, calcium flux, and IL2 production were decreased in mature T cells from SLP76m/m mice, suggesting that the SAM
10
domain in SLP-76 plays an important role in the TCR-mediated signaling that is crucial in T
cell development and activation of mature T cells.
Results
Generation of SLP∆SAM mutant mice
To generate knock-in mice expressing SLP-76 lacking the SAM domain, the genomic
fragment that contains exons 1 to 3 of the SLP-76 gene (encoding SAM amino acids 1-80
region of murine SLP-76) was replaced by a PGK-neo cassette flanked by two loxP sites in
embryonic stem (ES) cells (figure 8). The Pgk-1 promoter was used to drive strong
ubiquitous expression of neo (neomycin resistance gene) (81). For negative selection of
random recombinants, the diphtheria toxin-A fragment (DTA) gene was added to the
upstream of the homologous recombination sites. The DTA gene will be lost upon
homologous recombination with the genomic fragment but may be maintained in the nonhomologous recombinants (82, 83). When the neomycin analog G418 was added, cells which
underwent random integration and contained the DTA gene would not survive the negative
selection process.
The short arm and long arm areas indicate the homologous recombination sites to
delete exons 1, 2 and 3, which encode the SAM domain. Successful homologous
recombinant ES cells were injected into blastocysts and implanted into pseudopregnant
female recipient mice to establish chimeric mice. The neo cassette, flanked by loxP sites, was
subsequently removed by crossing the chimeric mice with β-actin-Cre transgenic mice,
which expresses the Cre recombinase enzyme in all cells following the ubiquitous expression
of the housekeeping gene β-actin. Heterozygous mice with deletion of exons 1, 2 and 3
(SLP76m/+) were inbred to obtain mice homozygous for the SAM deletion in SLP-76
11
(SLP76m/m). SLP76m/m mice were generated at the expected Mendelian ratio and appeared
healthy under specific pathogen-free conditions. The deletion of exons 1, 2 and 3 was
confirmed by PCR and sequencing (data not shown).
1
6
2
3
45
*
genome
Short
arm
5.0 kb
1.7 kb
DT-A
*
Neo
PGK
*
Neo
PGK
Long
arm
targeting construct
targeted allele
cross with Cre mice
*
LoxP site
exon
non-coding
*
start codon
Figure 8. Cloning strategy for generating SLP76m/m knock-in mice. Top panel shows the targeting
construct with the mouse genomic fragment containing the SLP-76 gene. The symbols are as
indicated in the legend. The two crosses ‘X’ indicate the homologous recombination sites at the short
arm and long arm areas. Middle panel shows the successful homologous recombination event with
exons 1, 2, and 3 replaced by the PGK-Neo cassette. Bottom panel shows the resulting genome
fragment of the SLP76m/m mutant mice after crossing with the Cre transgenic mice to delete the
cassette.
To confirm the deletion of the SAM domain in the SLP-76 protein in SLP76m/m mice,
I analyzed the lysates of T cells by immunoblotting with an anti-SLP-76 antibody. As shown
in figure 9, mutant SLP-76 indeed had a smaller molecular weight than wild-type SLP-76 on
SDS-PAGE/Western analysis. Lysates of heterozygotes gave a double band showing both the
wild-type and mutant copy of SLP-76.
12
WB: anti-SLP-76 (mAb)
SLP76+/+ SLP76m/+ SLP76m/m
Figure 9. Expression of SLP-76 in T cells from SLP76+/+, SLP76m/+ and SLP76m/m mice. Cells
were solubilized with RIPA lysis buffer and the extracted proteins were subjected to Western blot
analysis with anti-SLP-76 antibodies. Calculated molecular weight of mutant SLP-76 should be 8.8
kiloDaltons (kD) smaller than wild-type SLP-76. The difference in protein size is indicated by the
horizontal line markers at the sides.
Analysis of Thymocytes in SLP76m/m mice
SLP76m/+ mice displayed a phenotype identical to wild-type mice. The thymi from
SLP76m/+ mice did not show any significant differences in size and cellularity compared with
SLP76+/+ mice. However, the thymi from SLP76m/m mice showed a significant 30% decrease
in thymocyte numbers from 1.07x108 to 7.6x107 cells.
Partial Defect in Thymocyte Development
To examine if the decrease in thymocyte numbers was a result of their defective
development, thymocytes were isolated from SLP76m/+ and SLP76m/m mice for flow
cytometry analysis. Cells were stained with CD8 and CD4 fluorescent dyes to examine their
expression levels and identify the various stages of the thymocyte development from DN →
DP → SP stages. The analysis revealed that there was a partial defect in thymocyte
development in SLP76m/m mice, as the percentage of CD4+ cells decreased (7.39 to 4.96%)
(figure 10, top panel). The thymocyte counts confirmed the decrease in CD4+ cell numbers,
as shown below in figure 11. There was no significant difference in the CD8+ population.
To further determine if the defect in thymocyte development occurred at the early
maturation stages from DN1 → DN4, we conducted further analysis on the DN
subpopulations by examining the expression of cell surface markers CD44 and CD25. The
results showed that the development of thymocytes in the DN stages was normal, as the
13
percentages of cells at each of the DN subsets were similar in the SLP76m/m and SLP76m/+
thymocytes (figure 10, bottom panel). This suggests that the defect in T cell development in
the SLP76m/m mice occurred mainly at the DP to SP transition, where positive and negative
selection takes place.
Figure 10. Flow cytometric analysis of thymocytes in SLP76m/m mice. Direction of axes indicates
increasing levels of cell marker expression. Numbers in quadrants represent percentages of cell
populations. Top panel: Four quadrants boxed up in pink represent each of the four cell populations –
clockwise from upper left: CD8+ SP cells, DP cells, CD4+ SP cells, DN immature T cells. There was a
significant decrease in the CD4+ population in the SLP76m/m thymocytes compared to the SLP76m/+
thymocytes. Bottom panel: Red arrows represent analysis on the DN population in the bottom left
quadrant of the plots in top panel. Four quadrants represent each of the four DN subpopulations in
developmental order – clockwise from upper left: DN1, DN2, DN3, DN4.
14
Figure 11. Thymocyte cell counts of CD4+ and CD8+ populations. The CD8+ cell numbers (left)
were comparable in the SLP76m/+ mice (1.07 million cells) and the SLP76m/m mice (0.829 million
cells). The CD4+ cell numbers (right) were significantly lower in the SLP76m/m mice (1.44 million
cells) compared to the SLP76m/+ mice (3.88 million cells).
Normal αβTCR and CD3 cell surface expression
A defect of thymocyte development in the selection processes might be due to
reduced cell surface expression of TCR and its associated CD3 chains, resulting in
insufficient TCR-mediated signaling. To test this possibility, we examined the αβTCR and
CD3 surface expression by staining thymocytes with antibodies specific for TCRβ and CD3
chains. Analysis of the DP, CD4+ and CD8+ SP cells showed that SLP76m/m thymocytes
expressed similar levels of TCRβ and CD3 as SLP76m/+ thymocytes (figure 12). This
suggests that SLP76m/m thymocytes had similar numbers of TCRs and the associated CD3
chains to receive activation signals, compared to SLP76m/+ thymocytes.
15
Figure 12. Expression of TCRβ and CD3 in SLP76m/m thymocytes by flow cytometry analysis.
Direction of horizontal axes indicates increasing levels of TCRβ or CD3 respectively. Direction of
vertical axes indicates number of cells. In all three populations analyzed, TCRβ (top panel) and CD3
(bottom panel) expression levels have overlapping graphs (shaded area and red line), indicating that
expression levels are similar in SLP76m/m and SLP76m/+ thymocytes.
Defective Calcium Mobilization
The SAM domain could be involved in mediating the downstream signaling events
from SLP-76; therefore, a deletion of the SAM domain might result in the defect in
thymocyte development in the selection processes. To probe this possibility, TCR-mediated
calcium flux that is at the downstream of SLP-76 was analyzed in the thymocytes. The
thymocytes were loaded with calcium indicator Indo-1 AM fluorescence dye and were then
stimulated by adding anti-CD3 antibody. An anti-CD3 antibody is often used to cross-link
TCR-CD3 chains to mimic engagement of MHC-antigen complexes (84). The cells were
analyzed by flow cytometry, where the ratio of calcium-bound Indo-1 AM dye to calciumfree dye is a measure of the calcium concentration, as the calcium-bound dye corresponds to
16
a higher energy violet fluorescence (405nm wavelength) which is normalized to the calciumfree dye at a lower energy blue fluorescence (510nm wavelength). As shown in figure 13,
anti-CD3-induced calcium flux was reduced in the DP, CD4+ and CD8+ SP thymocyte
populations of the SLP76m/m mutant mice, suggesting that the SAM domain indeed plays an
important role in mediating TCR downstream signaling. Defective signaling in the
Ratio 405nm/510nm
thymocytes may therefore result in a defect in the selection processes during their maturation.
DP thymocyte
anti-CD3
CD4 SP thymocyte
SLP76m/+
anti-CD3
SLP76m/m
CD8 SP thymocyte
anti-CD3
Time
Figure 13. Flow cytometric analysis of calcium flux of DP, CD4+ and CD8+ thymocytes
following stimulation with anti-CD3 antibody. Directions of axes indicate increasing calcium
concentration (measured by ratio of fluorescence intensity at 405nm to 510nm) and progression of
time. Vertical arrows mark the time point when anti-CD3 was added to initiate TCR stimulation. The
peak (shown in red) represented the wave of calcium release after signaling pathway activation.
SLP76m/m thymocytes showed a significant decrease in calcium flux.
17
Analysis of Peripheral Mature T cells in SLP76m/m mice
Corresponding decreased output of mature T cells from maturing thymocytes
The flow cytometry analysis of thymocytes revealed a partial defect in development,
resulting in a decreased CD4+ population. As mentioned earlier, mature T cells migrate to the
peripheral tissues after differentiation. Thus, we analyzed the mature T cell populations in the
spleen and lymph nodes of SLP76m/m mutant mice to examine if the defect in thymocyte
development is reflected in these mature T cells. We isolated the peripheral T cells and
stained them as above. The analysis revealed that the mature T cell populations in both the
lymph node and the spleen in the SLP76m/m mutant mice had a significantly reduced CD4+
population compared to that of the SLP76m/+ mice (figure 14). These results are consistent
with the results obtained from thymocytes.
Normal αβTCR and CD3 cell surface expression
We examined the αβTCR and CD3 surface expression by staining mature T cells,
pooled together from spleen and lymph nodes, with antibodies specific for TCRβ and CD3
chains. Analysis of the peripheral mature T cells showed that SLP76m/m T cells expressed
similar levels of TCRβ and CD3 as those of SLP76m/+ (data not shown). This data is
consistent with previous data from the thymocytes.
18
Figure 14. Flow cytometric analysis of peripheral mature T cells from spleen and lymph nodes
in SLP76m/m mice. Direction of axes indicates increasing levels of cell marker expression. Numbers
in quadrants represent percentages of cell populations. Horizontal rectangles enclose the CD8+ SP
cells and vertical rectangles enclose the CD4+ SP cells. Cell population in bottom left corner of
figures represent non-T-cells such as B cells and dendritic cells and are irrelevant in this context. Note
that the CD4+ SP cell populations are significantly smaller in the SLP76m/m samples compared to
SLP76m/+.
Normal upregulation of CD25 and CD69 early activation markers
Upon T cell activation via TCR stimulation, a characteristic downstream event is the
upregulation of expression of T cell early activation markers on the cell surface. Two of these
cell surface antigen proteins expressed are CD69 and CD25 (85-87). To investigate if the
deletion of the SAM domain in SLP-76 affects the upregulation of these activation markers,
CD4+ T cells were isolated and purified from the lymph nodes of SLP76m/m and SLP76m/+
mice and analyzed for the expression of these proteins following stimulation with anti-CD3.
As shown in figure 15, T cells from both mice demonstrated normal upregulation of these
19
two markers with anti-CD3 stimulation at 6 hours, suggesting that this process is not affected
by the deletion of the SAM domain of SLP-76.
Figure 15. Flow cytometric analysis of upregulation of early activation markers in CD4+ lymph
node T cells at 6 hr after stimulation. Direction of horizontal axes indicates increasing levels of
CD25 or CD69 respectively. Direction of vertical axes indicates number of cells. In unstimulated
cells, there was no upregulation of either marker as shown by the shaded area. Stimulation using
PMA and ionomycin (P+I) acts as a positive control, as indicated by the dotted blue lines. PMA and
ionomycin are IP3 and DAG analogs and mimic their function to result in T cell stimulation which
bypasses the TCR. Stimulation via the TCR using anti-CD3 antibody results in similar levels of CD25
and CD69 expression, as indicated by the red solid lines.
Defective TCR-mediated Calcium Mobilization in mutant T cells
As shown in figure 13, the deletion of the SAM domain of SLP-76 affected TCRmediated calcium mobilization pathway that is downstream of SLP-76 in thymocytes,
resulting in a defect in thymocyte development. TCR-mediated signaling is also important in
the activation of mature T cells to drive their expansion and proliferation upon ligand
engagement. Thus, we wanted to examine if TCR-mediated downstream signaling events
were also affected by the deletion of the SAM domain in SLP-76. We analyzed the ability of
SLP76m/m lymph node T cells to mobilize calcium upon anti-CD3 stimulation, following the
20
same procedure as before. As shown in figure 16, anti-CD3-induced calcium flux was
reduced in both the CD4+ and CD8+ SP mature T cell populations of the SLP76m/m mutant
mice, suggesting that the SAM domain also plays an important role in activating downstream
calcium mobilization in the mature T cells.
Ratio 405nm/510nm
CD4+ LN T cells
anti-CD3
SLP76m/+
CD8+ LN T cells
SLP76m/m
anti-CD3
Time
Figure 16. Calcium flux of CD4+ and CD8+ lymph node mature T cells following TCR
stimulation with anti-CD3 treatment. Graphics are as in figure 13.
Defective Phosphorylation of Downstream Signaling Molecules
The above results suggest that the deletion of the SAM domain of SLP-76 affects
TCR-mediated signaling in mature peripheral T cells. To explore this further, we examined
the phosphorylation of various TCR downstream signaling molecules (see outline in figure 6).
We isolated lymph node T cells from SLP76m/m and SLP76m/+ mice and expanded the T cell
population in vitro to obtain sufficient numbers of T cells for biochemical studies. After
activation with anti-CD3 for two days and expansion in IL-2 medium, the cells were then
analyzed for their ability to activate various signaling pathways following TCR stimulation.
21
We analyzed TCR-mediated tyrosine phosphorylation by western blotting with
phosphotyrosine antibodies. Figure 17a shows that there was some reduction in the tyrosine
phosphorylation of cellular proteins of SLP76m/m T cells upon TCR stimulation. Some of the
protein bands were similar in intensity for both samples after stimulation (dashed arrows),
while some bands in the SLP76m/m samples appear to be weaker than the SLP76m/+ samples
(asterisks). It is likely that the former represents the proteins upstream of SLP-76 and the
phosphorylation of these proteins is not affected, while the latter are the proteins downstream
of SLP-76 whose phosphorylation was affected by the deletion of SAM.
Immunoblotting of pPLCγ1 (figure 17b), when adjusted for uneven loading, showed
only a slight decrease in phosphorylated PLCγ1 protein levels in SLP76m/m T cells following
TCR stimulation as compared to SLP76m/m T cells. This result is consistent with the decrease
in calcium flux as shown above, as pPLCγ1 is directly upstream of the calcium mobilization
pathway.
Immunoblotting of pERK and pp38 also showed significant decrease in ERK and p38
phosphorylation in SLP76m/m mice (figure 17c, d), indicating that SAM is involved in the
activation of these two MAP kinase signaling cascades downstream of SLP-76.
22
*
*
*
Figure 17. Western blot analysis of phosphorylation of downstream signaling molecules of SLP76 in response to TCR engagement. (a) Analysis of protein tyrosine kinase phosphorylation activity
using phosphotyrosine 4G10 antibody. Asterisks on the right of the figure indicate protein bands
whose phosphorylation is reduced in SLP76m/m samples 1.5 minutes after stimulation (lanes 2 and 4).
(b) Analysis of PLCγ1 phosphorylation level. Upper panel: In SLP76m/m cells, phosphorylation of
PLCγ1 is slightly reduced (lane 2). Lower panel: Blotting with anti-PLCγ1 shows that more SLP76m/m
lysates were loaded (lanes 3 and 4) than SLP76m/+ cells (lanes 1 and 2). Bottom graph show
normalized intensity of PLCγ1 phosphorylation band after dividing the intensities by the intensities of
the corresponding loading control. (c), (d) Analysis of Erk and p38 phosphorylation levels. Upper
panels: SLP76m/m T cells (lane 4) show weaker phosphorylation compared to SLP76m/m T cells. Lower
panels: Protein loading controls show that equal amounts of lysates were loaded.
Impaired IL-2 Production in SLP76m/m T cells
Activation of TCR-mediated signaling pathways leads to the production of cytokines,
such as IL-2 (figure 6). As shown above, the calcium mobilization and MAP kinase pathways
are affected by the deletion of the SAM domain. Therefore, we continued to investigate if the
production of IL-2 is affected as well. To examine this, CD4+ lymph node T cells were
purified from SLP76m/+ and SLP76m/m mice and stimulated with anti-CD3 and anti-CD28. IL2 production was assayed by ELISA (enzyme-linked immunosorbent assay). As shown in
23
figure 18, SLP76m/m T cells showed significantly reduced IL-2 production as compared to
that of SLP76m/+. In contrast, SLP76m/m CD4+ T cells secreted IL-2 at levels comparable to
that of SLP76m/+ in response to PMA and ionomycin, which bypass TCR-mediated signaling.
This result strongly suggests that the defective TCR-mediated signaling pathways due to the
deletion of the SAM domain of SLP-76 also continue to affect IL-2 production.
Figure 18. Impaired IL-2 production in SLP76m/m CD4+ lymph node T cells. As indicated, cells
were left unstimulated (negative control), stimulated with anti-CD3 and anti-CD28 or stimulated with
PMA and ionomycin (P+I) (positive control). All cultures were tested in triplicates (error bars).
Cytokine production was assayed by ELISA at 4h, 6h and 8h of culture. The brackets with the
asterisks indicate that the difference between the amount of IL-2 produced by SLP76m/m and SLP76m/+
cells is statistically significant. Unstimulated cells (left) show almost no IL-2 production. Stimulation
with P+I (right) yields comparable levels of IL-2 production between SLP76m/m and SLP76m/+ cells.
24
Discussion
Deletion of the SAM domain in SLP-76 resulted in a partial block in thymocyte
development. In the thymocytes of SLP76m/m mice, there was a significant decrease in the
CD4+ SP population, decreased thymocyte cell counts and decreased TCR-mediated calcium
mobilization compared to that of SLP76m/+ mice. In the peripheral mature T cells of
SLP76m/m mice, there was a corresponding decrease in the output of CD4+ population and
decreased T cell counts. Downstream TCR-mediated signaling pathways were also affected;
there was decreased calcium mobilization, decreased phosphorylation of ERK and p38 in the
MAP kinase pathways, and decreased IL-2 production. Altogether, these results suggest that
the SAM domain in SLP-76 plays a crucial role in the selection processes of T cell
development and TCR-mediated signaling in peripheral mature T cells.
The decreased output of mature CD4+ SP T cells suggests that the deletion of SAM
from SLP-76 results in defective positive selection processes during T cell development. The
decision to downregulate either co-receptor to mature CD4+ or CD8+ SP T cells has been
reported to depend on the strength of the activation signals that the thymocytes receive (88).
CD4 co-receptors transduce stronger signals than the CD8 co-receptors. This accounts for the
high CD4+: CD8+ T cell ratio, as the development of CD8+ T cells require a lower signal
threshold of activation signals compared to the CD4+ T cells to direct their commitment
towards the respective lineages. The decreased CD4+ cell population thus suggests that the
TCR-mediated signaling events in SLP76m/m mice decrease in intensity.
The partial defect in ERK, p38 and PLCγ1/calcium mobilization pathways in
SLP76m/m T cells suggests that there is a decrease in signal intensity downstream of SLP-76.
How the SAM domain contributes to SLP-76 function is not clear and remains to be
25
determined. One possibility is that the SAM domain could be involved in the recruitment of
SLP-76 to LAT or the membrane. SLP-76 associates with LAT in the signaling complex to
initiate the downstream events. It is possible that SLP-76 interacts with the LAT adaptor
protein via two ways: (i) via tethering of SLP-76 to LAT or the membrane via the SAM
domain and (ii) via the adaptor protein Gads which is first recruited to LAT. Without the
SAM domain, SLP-76 can still bind LAT although less efficiently. This hypothesis can be
tested by conducting further experiments. Double-mutant mice of Gads and SAM of SLP-76
can be generated by crossing the current SLP76m/m mice with Gads knockout mice. Previous
studies have shown that Gads-deficient mice also exhibit an incomplete block in thymocyte
development (89, 90). If SAM is indeed responsible for the tethering of SLP-76 to the
membrane or LAT, there will be a complete disruption of signaling events downstream of
SLP-76. As mentioned above, intracellular signaling from the TCR is crucial in thymocyte
development in positive selection processes. Thus, it is expected that the mice will exhibit a
complete block in thymocyte development and that the DP population will be absent.
The second possibility is that the SAM domain in SLP-76 mediates the binding of
SLP-76 with other proteins in the signaling complex. The SAM domain has been shown to be
involved in mediating protein-protein interactions (see introduction). I have previously tried
GST pull-down assays in T cell lines using purified SAM domain fused with glutathione Stransferase (GST) to detect any potential binding partners of the SAM domain. However, the
results did not reveal any proteins that bound to the SAM domain. One possibility of failing
to detect any binding partners is that I had probed for phosphorylated proteins by
immunoblotting with phosphotyrosine antibodies. The proteins interacting with the SAM
domain in SLP-76 could have been proteins that are not phosphorylated and thus were not
26
detected. To investigate this possibility, metabolic labeling experiments can be conducted.
J14 T cells (T cells with SLP-76 deleted) can be reconstituted with either wild-type SLP-76
protein or mutant SLP-76 with the SAM domain deleted. These cells can then be grown in
radioactive
35
S methionine or cysteine so that these amino acids will be incorporated into
newly synthesized proteins. The proteins that are bound to SLP-76 in the signaling complex
can then be co-immunoprecipitated and analyzed by SDS-PAGE. Autoradiography can be
used to reveal all radioactive proteins. These proteins can be identified by mass spectrometry.
In the phosphotyrosine western blot shown in figure 17a, the lowest protein band with
the asterisk has a size that corresponds to the LAT protein. This result is puzzling as
phosphorylation of LAT is upstream of SLP-76 and should therefore be unaffected. However,
the SLP76m/m T cells shows a decrease in protein phosphorylation compared to SLP76m/+ T
cells. One possibility of this decreased LAT phosphorylation could be due to a positive
feedback loop downstream of SLP-76 that maintains the phosphorylation of LAT. With the
deletion in the SAM domain, the partial disruption of downstream signaling events may have
reduced the strength of the feedback loop, therefore leading to decreased LAT
phosphorylation.
There is an apparent discrepancy between the decrease in ERK phosphorylation
(figure 17c) and the upregulation of the early activation markers CD69 and CD25 (figure 15).
The upregulation of these markers CD69 and CD25 has been reported to be dependent on the
Ras/MEK/ERK pathway activation (91, 92). However, we observe that the upregulation of
the activation markers seem to be normal in the SLP76m/m mice, although there is an obvious
decrease in ERK phosphorylation. One reasonable explanation would be that the level of
phosphorylated ERK required for the activation of the marker expressions is rather low. Thus,
27
the decrease in ERK phosphorylation does not lead to a defective regulation of these cell
surface markers.
To fully characterize the role of the SAM domain of SLP-76, more analysis of the
downstream signaling pathways from SLP-76 is required. In the ERK MAP kinase pathway
(figure 6), I have only examined the phosphorylation of ERK. Phosphorylation of the other
members of the ERK MAP kinase cascade should be analyzed. It is expected that expression
of Grb2/Sos, Ras, Raf and MEK1/2 will be lower in SLP76m/m T cells compared to that of
SLP76m/+, as these proteins are all downstream of SLP-76 activation. In the same way, only
phosphorylation of p38 has been examined and other members of the cascade should be
examined to confirm the result. The third MAP kinase pathway – Rac/JNK cascade – has not
been examined. It is likely that there will be defective signaling in the JNK cascade as well.
The decrease in phosphorylation of PLCγ1 expression in SLP76m/m T cell lysates
compared to that of SLP76m/+ was only evident after adjusting for the differential loading of
protein between the SLP76m/m and SLP76m/+ samples. The experiment should to be repeated
to assess whether decreased PLCγ1 phosphorylation is real. PLCγ1 phosphorylation activates
calcium mobilization which in turn activates NFAT. NFAT is a transcription factor which
regulates IL-2 transcription, along with MAP kinase cascades. The activity of NFAT could
be measured by performing an NFAT-luciferase reporter assay. The activity of NFAT in
SLP76m/m T cells is expected to be lower compared to that of SLP76m/+ T cells.
Another downstream pathway not previously mentioned is the Akt pathway. In
addition to activating Rac to lead to the downstream JNK pathway (figure 6), activated Vav
also activates PI3 kinase. A major target of PI3 kinase is another kinase Akt, which activates
the IκB kinase/ NFκB transcription factor downstream pathway (figure 6) (93-95). This is a
28
common downstream pathway that is also activated by the phosphorylation of PLCγ1.
Further experiments to assess the role of the SAM domain in SLP-76 in the downstream
signaling pathways include investigating the phosphorylation of Akt and members of the IκB
kinase pathway by western blotting, and the activity of NFκB.
Examination of events upstream of SLP-76 is also important to confirm that these
signaling events are not affected in the SLP76m/m mice. Figure 17a showed that the
phosphorylation of some proteins are unaffected by the SAM deletion and it is likely that
these proteins are those upstream of SLP-76. Examples of such molecules include ZAP-70,
LAT and Gads. However, immunoblotting with specific antibodies to these molecules should
be done to confirm the results of figure 17a. In addition, the phosphorylation of SLP-76 itself
upon stimulation should be similar as well. However, experiments conducted on ZAP-70 and
SLP-76 phosphorylation thus far have been unsuccessful as the antibodies available for
immunoblotting are weak and failed to detect these proteins.
Detection of IL-2 production by ELISA was conducted using CD4+ primary lymph
node T cells, which are T cells obtained directly from the mice without expansion of the cell
population. On the other hand, the immunoblotting data, which required more cells to obtain
detectable levels of proteins, were carried out using activated T cells. Although the behavior
of primary T cells is known to be similar to activated T cells at large, some differences may
be present. The IL-2 production assay should be repeated using activated T cells and using
total CD4+ and CD8+ populations instead of the isolated populations. Likewise, the calcium
mobilization assays were conducted using primary T cells. The experiments need to be
repeated using activated T cells.
29
The biochemistry results will also have to be repeated to obtain data using the
primary T cells. However, as the number of primary T cells numbers from each mouse is low,
these experiments can only be performed when the mouse colonies are larger. Cells from
several mice of each genotype can be pooled together to obtain detectable levels of proteins.
Furthermore, I can examine the proliferation of T cells using the thymidine
incorporation assay (96) or CFSE (carboxyfluoroscein succinimidyl ester) fluorochrome
intracellular staining (97, 98). As my results suggest that there is a partial block in thymocyte
development, especially in the DP to SP transition, I expect that there would be less
proliferation in the SLP76m/m T cells compared to the SLP76m/+ T cells.
These preliminary results strongly suggest that the SAM domain in SLP-76 plays an
important role in TCR-mediated signaling. Signaling events start to play an important role in
the development of the thymocytes which, when impaired, led to the partial block in T cell
development and in a reduced output of mature T cells. TCR-mediated signaling continues to
be important in activating the mature T cells to stimulate their proliferation and effector
functions, initiating an immune response. In the SLP76m/m mice, several downstream
signaling pathways were affected. T cells are an integral part of our cell-mediated immunity
that serves to defend our bodies from the wide range of pathogens. Therefore, characterizing
the role of SAM domain in SLP-76 is important to enable us to gain a more complete
understanding of T cell development and mature T cell activation. Moreover, understanding
the function of the SAM domain in SLP-76 will add to our knowledge of the binding
properties of this motif. This will lead us closer to identifying a unifying function of this
commonly found domain.
30
Experimental Procedures
Mice
All mice were used in accordance with the National Institutes of Health guidelines. The
experiments described above were reviewed and approved by the Duke University
Institutional Animal Care and Use Committee. Mice were housed in specific pathogen-free
conditions at the Duke University Animal Care facility.
Expanding T cell population
To prepare activated T cells, lymph nodes were excised from the mice and the cells were
cultured in CD3 antibody (2C11)-coated 6-well plates (3µg/ml) in the presence of murine IL2 (10ng/ml) for 2 days. The cells were then transferred to flasks with complete RPMI 1640x
growing media in the presence of IL-2 and expanded for another 4 days. Before cells were
used, the activated T cells were rested in medium with IL-2 for 6 hours to return them to their
inactivated states (99).
Western Blotting
Lymph node activated T cells from SLP76m/+ and SLP76m/m mice were stained with antiCD3-biotin (5µg/ml) and anti-CD28-biotin (2.5µg/ml) antibodies and then stimulated by
crosslinking with streptavidin (40µg/ml) that binds to biotin molecule for 90 seconds. The
cells were then lysed in 250µl RIPA lysis buffer on ice for 10 minutes. After centrifugation,
the supernatant was then mixed with 2x SDS sample buffer for protein denaturation and as a
loading buffer. Investigation of SLP-76 protein size was conducted using activated spleen
cells from SLP76+/+, SLP76m/+ and SLP76m/m mice. The cells were lysed directly in RIPA
lysis buffer on ice without stimulation. The supernatant was mixed with 5x SDS sample
buffer to obtain a more concentrated protein level. The samples were resolved by SDS-PAGE
31
and transferred onto nitrocellulose membranes. The membranes were incubated with primary
antibodies 4G10 PY, anti-pPLCγ1, anti-PLCγ1, anti-pErk, anti-Erk2, anti-pp38 and anti-p38
(Cell signaling) (as indicated in figure 17) at 1:1000 dilution. After washing with PBST,
membranes were then probed with secondary antibodies (1:5000 dilution), using either goat
anti-mouse or anti-rabbit Ig conjugated with Alexa Fluor 680 (Molecular Probes) or IRDye
800 (Rockland). Membranes were then visualized with an infrared fluorescence imaging
system (LI-COR Bioscience Odyssey system) (99, 100).
FACS analysis
Thymocytes, lymph node and spleen T cells were excised from mice and single cell
suspensions were prepared according to Zhang et al. (1999). The cells were stained with the
following antibodies: Texas-red - conjugated anti-CD8α, FITC-conjugated anti-CD4,
phycoerythrin (PE)-conjugated anti-CD69, CD3 and CD25, APC-conjugated anti-CD44 and
TCRβ and 7AAD (7-amino-actinomycin D). Standard flow cytometry was performed using a
Becton Dickinson FACScan and FlowJo software. Investigation of the upregulation of CD25
and CD69 was conducted by incubating T cells either with medium alone, with anti-CD3
antibody (3µg/ml) or with PMA (40ng/ml) plus ionomycin (500ng/ml).
Calcium Mobilization
Thymocytes and lymph node T cells were first loaded with indo-1 in Ca2+ loading buffer (1x
Hanks’ Balanced Salt Solution with 10mM HEPES and 1% FBS) for 30 min and then stained
with FITC-conjugated anti-CD4, PE conjugated anti-CD8 and 7AAD (7-amino-actinomycin
D). Ca2+ flux was assayed by monitoring the fluorescence emission ratio at 405/510nm with
FACS. Calcium flux was initiated by addition of anti-CD3-biotin and anti-CD4-biotin or
anti-CD8-biotin (1µg/ml) followed by crosslinking with strepavidin (25µg/ml) (99).
32
Enzyme-Linked Immunosorbent Assay (ELISA)
Cells were either incubated with RPMI 1640x medium alone (unstimulated), with anti-CD3
and anti-CD28 antibodies, or with PMA (40ng/ml) plus ionomycin (500ng/ml). All cultures
were stimulated in triplicates. Supernatents were collected at 4, 6, and 8 hours of culture and
IL-2 cytokine production was assayed. Samples and IL-2 standards were incubated in a 96well flat-bottom polyvinylchloride microtiter plate pre-coated with anti-IL-2 capture
antibody (diluted 1:500) for two hours. Wells were washed with PBS and incubated with
biotinylated anti-IL-2 detection antibody (diluted 1:2000) with peroxidase labeled
streptavidin (diluted 1:1000) for one hour. The substrate buffer (TMB peroxidase EIA) was
added and incubated until color development was sufficient before adding the stop solution
(10% NaN3) to halt the reaction. The color intensity was read by EIA reader (BioRad) (101).
Acknowledgements
I would like to thank my supervisor, Dr. Weiguo Zhang, Dr. Minghua Zhu, my thesis reader,
Dr. Daniele Armaleo, Shudan Shen, and all other members of the Zhang Lab for their support,
advice and encouragement. I would also like to thank the Duke University Cancer Flow
Cytometry and DNA sequencing facilities, as well as the Biology Department for their
services.
33
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Zinkernagel, R. M., and P. C. Doherty. 1974. Restriction of in vitro T cell-mediated
cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic
system. Nature 248:701-702.
Cantrell, D. A. 2002. T-cell antigen receptor signal transduction. Immunology
105:369-374.
Pivniouk, V. I., and R. S. Geha. 2000. The role of SLP-76 and LAT in lymphocyte
development. Curr Opin Immunol 12:173-178.
Moroy, T., and H. Karsunky. 2000. Regulation of pre-T-cell development. Cell Mol
Life Sci 57:957-975.
Fehling, H. J., and H. von Boehmer. 1997. Early alpha beta T cell development in the
thymus of normal and genetically altered mice. Curr Opin Immunol 9:263-275.
Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302:575-581.
Shortman, K., and L. Wu. 1996. Early T lymphocyte progenitors. Annu Rev Immunol
14:29-47.
Werlen, G., B. Hausmann, D. Naeher, and E. Palmer. 2003. Signaling life and death
in the thymus: timing is everything. Science 299:1859-1863.
Boyd, R. L., and P. Hugo. 1991. Towards an integrated view of thymopoiesis.
Immunology today 12:71-79.
Anderson, G., N. C. Moore, J. J. Owen, and E. J. Jenkinson. 1996. Cellular
interactions in thymocyte development. Annu Rev Immunol 14:73-99.
Godfrey, D. I., and A. Zlotnik. 1993. Control points in early T-cell development.
Immunology today 14:547-553.
Godfrey, D. I., J. Kennedy, T. Suda, and A. Zlotnik. 1993. A developmental pathway
involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J
Immunol 150:4244-4252.
von Boehmer, H., and H. J. Fehling. 1997. Structure and function of the pre-T cell
receptor. Annu Rev Immunol 15:433-452.
Zuniga-Pflucker, J. C., and M. J. Lenardo. 1996. Regulation of thymocyte
development from immature progenitors. Curr Opin Immunol 8:215-224.
Rodewald, H. R., and H. J. Fehling. 1998. Molecular and cellular events in early
thymocyte development. Advances in immunology 69:1-112.
Sebzda, E., S. Mariathasan, T. Ohteki, R. Jones, M. F. Bachmann, and P. S. Ohashi.
1999. Selection of the T cell repertoire. Annu Rev Immunol 17:829-874.
Grossman, Z., and A. Singer. 1996. Tuning of activation thresholds explains
flexibility in the selection and development of T cells in the thymus. Proc Natl Acad
Sci U S A 93:14747-14752.
von Boehmer, H. 1994. Positive selection of lymphocytes. Cell 76:219-228.
Santori, F. R., W. C. Kieper, S. M. Brown, Y. Lu, T. A. Neubert, K. L. Johnson, S.
Naylor, S. Vukmanovic, K. A. Hogquist, and S. C. Jameson. 2002. Rare, structurally
homologous self-peptides promote thymocyte positive selection. Immunity 17:131142.
Nikolic-Zugic, J., and M. J. Bevan. 1990. Role of self-peptides in positively selecting
the T-cell repertoire. Nature 344:65-67.
Nossal, G. J. 1994. Negative selection of lymphocytes. Cell 76:229-239.
34
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
Wange, R. L., and L. E. Samelson. 1996. Complex complexes: signaling at the TCR.
Immunity 5:197-205.
Weiss, A., and D. R. Littman. 1994. Signal transduction by lymphocyte antigen
receptors. Cell 76:263-274.
Marie-Cardine, A., and B. Schraven. 1999. Coupling the TCR to downstream
signalling pathways: the role of cytoplasmic and transmembrane adaptor proteins.
Cell Signal 11:705-712.
Zhang, W., and L. E. Samelson. 2000. The role of membrane-associated adaptors in T
cell receptor signalling. Semin Immunol 12:35-41.
Zamoyska, R., A. Basson, A. Filby, G. Legname, M. Lovatt, and B. Seddon. 2003.
The influence of the src-family kinases, Lck and Fyn, on T cell differentiation,
survival and activation. Immunol Rev 191:107-118.
Wardenburg, J. B., C. Fu, J. K. Jackman, H. Flotow, S. E. Wilkinson, D. H. Williams,
R. Johnson, G. Kong, A. C. Chan, and P. R. Findell. 1996. Phosphorylation of SLP76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function. The
Journal of biological chemistry 271:19641-19644.
Zhang, W., J. Sloan-Lancaster, J. Kitchen, R. P. Trible, and L. E. Samelson. 1998.
LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular
activation. Cell 92:83-92.
Jackman, J. K., D. G. Motto, Q. Sun, M. Tanemoto, C. W. Turck, G. A. Peltz, G. A.
Koretzky, and P. R. Findell. 1995. Molecular cloning of SLP-76, a 76-kDa tyrosine
phosphoprotein associated with Grb2 in T cells. The Journal of biological chemistry
270:7029-7032.
Zhang, W., C. L. Sommers, D. N. Burshtyn, C. C. Stebbins, J. B. DeJarnette, R. P.
Trible, A. Grinberg, H. C. Tsay, H. M. Jacobs, C. M. Kessler, E. O. Long, P. E. Love,
and L. E. Samelson. 1999. Essential role of LAT in T cell development. Immunity
10:323-332.
Gong, Q., A. M. Cheng, A. M. Akk, J. Alberola-Ila, G. Gong, T. Pawson, and A. C.
Chan. 2001. Disruption of T cell signaling networks and development by Grb2
haploid insufficiency. Nat Immunol 2:29-36.
Karin, M., and T. Hunter. 1995. Transcriptional control by protein phosphorylation:
signal transmission from the cell surface to the nucleus. Curr Biol 5:747-757.
Yoder, J., C. Pham, Y. M. Iizuka, O. Kanagawa, S. K. Liu, J. McGlade, and A. M.
Cheng. 2001. Requirement for the SLP-76 adaptor GADS in T cell development.
Science 291:1987-1991.
Asada, H., N. Ishii, Y. Sasaki, K. Endo, H. Kasai, N. Tanaka, T. Takeshita, S.
Tsuchiya, T. Konno, and K. Sugamura. 1999. Grf40, A novel Grb2 family member, is
involved in T cell signaling through interaction with SLP-76 and LAT. J Exp Med
189:1383-1390.
Liu, S. K., N. Fang, G. A. Koretzky, and C. J. McGlade. 1999. The hematopoieticspecific adaptor protein gads functions in T-cell signaling via interactions with the
SLP-76 and LAT adaptors. Curr Biol 9:67-75.
Peterson, E. J., J. L. Clements, N. Fang, and G. A. Koretzky. 1998. Adaptor proteins
in lymphocyte antigen-receptor signaling. Curr Opin Immunol 10:337-344.
Samelson, L. E. 1999. Adaptor proteins and T-cell antigen receptor signaling.
Progress in biophysics and molecular biology 71:393-403.
35
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
Rudd, C. E. 1999. Adaptors and molecular scaffolds in immune cell signaling. Cell
96:5-8.
Salojin, K. V., J. Zhang, and T. L. Delovitch. 1999. TCR and CD28 are coupled via
ZAP-70 to the activation of the Vav/Rac-1-/PAK-1/p38 MAPK signaling pathway. J
Immunol 163:844-853.
Cano, E., and L. C. Mahadevan. 1995. Parallel signal processing among mammalian
MAPKs. Trends Biochem Sci 20:117-122.
Coso, O. A., M. Chiariello, J. C. Yu, H. Teramoto, P. Crespo, N. Xu, T. Miki, and J.
S. Gutkind. 1995. The small GTP-binding proteins Rac1 and Cdc42 regulate the
activity of the JNK/SAPK signaling pathway. Cell 81:1137-1146.
Minden, A., A. Lin, F. X. Claret, A. Abo, and M. Karin. 1995. Selective activation of
the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases
Rac and Cdc42Hs. Cell 81:1147-1157.
Kogkopoulou, O., E. Tzakos, G. Mavrothalassitis, C. T. Baldari, F. Paliogianni, H. A.
Young, and G. Thyphronitis. 2006. Conditional up-regulation of IL-2 production by
p38 MAPK inactivation is mediated by increased Erk1/2 activity. J Leukoc Biol
79:1052-1060.
Stoica, B., K. E. DeBell, L. Graham, B. L. Rellahan, M. A. Alava, J. Laborda, and E.
Bonvini. 1998. The amino-terminal Src homology 2 domain of phospholipase C
gamma 1 is essential for TCR-induced tyrosine phosphorylation of phospholipase C
gamma 1. J Immunol 160:1059-1066.
Bunnell, S. C., M. Diehn, M. B. Yaffe, P. R. Findell, L. C. Cantley, and L. J. Berg.
2000. Biochemical interactions integrating Itk with the T cell receptor-initiated
signaling cascade. The Journal of biological chemistry 275:2219-2230.
Rhee, S. G., and Y. S. Bae. 1997. Regulation of phosphoinositide-specific
phospholipase C isozymes. The Journal of biological chemistry 272:15045-15048.
Dienz, O., A. Moller, A. Strecker, N. Stephan, P. H. Krammer, W. Droge, and M. L.
Schmitz. 2003. Src homology 2 domain-containing leukocyte phosphoprotein of 76
kDa and phospholipase C gamma 1 are required for NF-kappa B activation and lipid
raft recruitment of protein kinase C theta induced by T cell costimulation. J Immunol
170:365-372.
Dienz, O., S. P. Hehner, W. Droge, and M. L. Schmitz. 2000. Synergistic activation
of NF-kappa B by functional cooperation between vav and PKCtheta in T
lymphocytes. The Journal of biological chemistry 275:24547-24551.
Dong, C., R. J. Davis, and R. A. Flavell. 2002. MAP kinases in the immune response.
Annu Rev Immunol 20:55-72.
Rincon, M. 2001. MAP-kinase signaling pathways in T cells. Curr Opin Immunol
13:339-345.
Myung, P. S., G. S. Derimanov, M. S. Jordan, J. A. Punt, Q. H. Liu, B. A. Judd, E. E.
Meyers, C. D. Sigmund, B. D. Freedman, and G. A. Koretzky. 2001. Differential
requirement for SLP-76 domains in T cell development and function. Immunity
15:1011-1026.
Clements, J. L., B. Yang, S. E. Ross-Barta, S. L. Eliason, R. F. Hrstka, R. A.
Williamson, and G. A. Koretzky. 1998. Requirement for the leukocyte-specific
adapter protein SLP-76 for normal T cell development. Science 281:416-419.
36
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
Pivniouk, V., E. Tsitsikov, P. Swinton, G. Rathbun, F. W. Alt, and R. S. Geha. 1998.
Impaired viability and profound block in thymocyte development in mice lacking the
adaptor protein SLP-76. Cell 94:229-238.
Maltzman, J. S., L. Kovoor, J. L. Clements, and G. A. Koretzky. 2005. Conditional
deletion reveals a cell-autonomous requirement of SLP-76 for thymocyte selection. J
Exp Med 202:893-900.
Yablonski, D., M. R. Kuhne, T. Kadlecek, and A. Weiss. 1998. Uncoupling of
nonreceptor tyrosine kinases from PLC-gamma1 in an SLP-76-deficient T cell.
Science 281:413-416.
Fang, N., D. G. Motto, S. E. Ross, and G. A. Koretzky. 1996. Tyrosines 113, 128, and
145 of SLP-76 are required for optimal augmentation of NFAT promoter activity. J
Immunol 157:3769-3773.
Musci, M. A., D. G. Motto, S. E. Ross, N. Fang, and G. A. Koretzky. 1997. Three
domains of SLP-76 are required for its optimal function in a T cell line. J Immunol
159:1639-1647.
Wu, J., D. G. Motto, G. A. Koretzky, and A. Weiss. 1996. Vav and SLP-76 interact
and functionally cooperate in IL-2 gene activation. Immunity 4:593-602.
Raab, M., A. J. da Silva, P. R. Findell, and C. E. Rudd. 1997. Regulation of VavSLP-76 binding by ZAP-70 and its relevance to TCR zeta/CD3 induction of
interleukin-2. Immunity 6:155-164.
Onodera, H., D. G. Motto, G. A. Koretzky, and D. M. Rothstein. 1996. Differential
regulation of activation-induced tyrosine phosphorylation and recruitment of SLP-76
to Vav by distinct isoforms of the CD45 protein-tyrosine phosphatase. The Journal of
biological chemistry 271:22225-22230.
Liu, Q., D. Berry, P. Nash, T. Pawson, C. J. McGlade, and S. S. Li. 2003. Structural
basis for specific binding of the Gads SH3 domain to an RxxK motif-containing SLP76 peptide: a novel mode of peptide recognition. Molecular cell 11:471-481.
Berry, D. M., P. Nash, S. K. Liu, T. Pawson, and C. J. McGlade. 2002. A highaffinity Arg-X-X-Lys SH3 binding motif confers specificity for the interaction
between Gads and SLP-76 in T cell signaling. Curr Biol 12:1336-1341.
Liou, J., F. Kiefer, A. Dang, A. Hashimoto, M. H. Cobb, T. Kurosaki, and A. Weiss.
2000. HPK1 is activated by lymphocyte antigen receptors and negatively regulates
AP-1. Immunity 12:399-408.
Fu, C., C. W. Turck, T. Kurosaki, and A. C. Chan. 1998. BLNK: a central linker
protein in B cell activation. Immunity 9:93-103.
Goitsuka, R., Y. Fujimura, H. Mamada, A. Umeda, T. Morimura, K. Uetsuka, K. Doi,
S. Tsuji, and D. Kitamura. 1998. BASH, a novel signaling molecule preferentially
expressed in B cells of the bursa of Fabricius. J Immunol 161:5804-5808.
Wienands, J., J. Schweikert, B. Wollscheid, H. Jumaa, P. J. Nielsen, and M. Reth.
1998. SLP-65: a new signaling component in B lymphocytes which requires
expression of the antigen receptor for phosphorylation. J Exp Med 188:791-795.
Qiao, F., and J. U. Bowie. 2005. The many faces of SAM. Sci STKE 2005:re7.
Pawson, T., and P. Nash. 2003. Assembly of cell regulatory systems through protein
interaction domains. Science 300:445-452.
Pawson, T. 2004. Specificity in signal transduction: from phosphotyrosine-SH2
domain interactions to complex cellular systems. Cell 116:191-203.
37
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
Schaffhausen, B. 1995. SH2 domain structure and function. Biochim Biophys Acta
1242:61-75.
Zarrinpar, A., R. P. Bhattacharyya, and W. A. Lim. 2003. The structure and function
of proline recognition domains. Sci STKE 2003:RE8.
Kim, C. A., and J. U. Bowie. 2003. SAM domains: uniform structure, diversity of
function. Trends Biochem Sci 28:625-628.
Aviv, T., Z. Lin, S. Lau, L. M. Rendl, F. Sicheri, and C. A. Smibert. 2003. The RNAbinding SAM domain of Smaug defines a new family of post-transcriptional
regulators. Nature structural biology 10:614-621.
Green, J. B., C. D. Gardner, R. P. Wharton, and A. K. Aggarwal. 2003. RNA
recognition via the SAM domain of Smaug. Molecular cell 11:1537-1548.
Hall, T. M. 2003. SAM breaks its stereotype. Nature structural biology 10:677-679.
Sharrocks, A. D. 2001. The ETS-domain transcription factor family. Nature reviews
2:827-837.
Watson, D. K., M. J. McWilliams-Smith, M. F. Nunn, P. H. Duesberg, S. J. O'Brien,
and T. S. Papas. 1985. The ets sequence from the transforming gene of avian
erythroblastosis virus, E26, has unique domains on human chromosomes 11 and 21:
both loci are transcriptionally active. Proc Natl Acad Sci U S A 82:7294-7298.
Orlando, V. 2003. Polycomb, epigenomes, and control of cell identity. Cell 112:599606.
Peterson, A. J., M. Kyba, D. Bornemann, K. Morgan, H. W. Brock, and J. Simon.
1997. A domain shared by the Polycomb group proteins Scm and ph mediates
heterotypic and homotypic interactions. Mol Cell Biol 17:6683-6692.
Peterson, A. J., D. R. Mallin, N. J. Francis, C. S. Ketel, J. Stamm, R. K. Voeller, R. E.
Kingston, and J. A. Simon. 2004. Requirement for sex comb on midleg protein
interactions in Drosophila polycomb group repression. Genetics 167:1225-1239.
McBurney, M. W., L. C. Sutherland, C. N. Adra, B. Leclair, M. A. Rudnicki, and K.
Jardine. 1991. The mouse Pgk-1 gene promoter contains an upstream activator
sequence. Nucleic Acids Res 19:5755-5761.
Araki, K., M. Araki, and K. Yamamura. 2006. Negative selection with the Diphtheria
toxin A fragment gene improves frequency of Cre-mediated cassette exchange in ES
cells. J Biochem (Tokyo) 140:793-798.
Yanagawa, Y., T. Kobayashi, M. Ohnishi, T. Kobayashi, S. Tamura, T. Tsuzuki, M.
Sanbo, T. Yagi, F. Tashiro, and J. Miyazaki. 1999. Enrichment and efficient
screening of ES cells containing a targeted mutation: the use of DT-A gene with the
polyadenylation signal as a negative selection maker. Transgenic research 8:215-221.
Sarkadi, B., A. Tordai, L. Homolya, O. Scharff, and G. Gardos. 1991. Calcium influx
and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargintreated human T lymphoblasts. The Journal of membrane biology 123:9-21.
Testi, R., J. H. Phillips, and L. L. Lanier. 1989. Leu 23 induction as an early marker
of functional CD3/T cell antigen receptor triggering. Requirement for receptor crosslinking, prolonged elevation of intracellular [Ca++] and stimulation of protein kinase
C. J Immunol 142:1854-1860.
Testi, R., D. D'Ambrosio, R. De Maria, and A. Santoni. 1994. The CD69 receptor: a
multipurpose cell-surface trigger for hematopoietic cells. Immunology today 15:479483.
38
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
Waldmann, T. A. 1989. The multi-subunit interleukin-2 receptor. Annual review of
biochemistry 58:875-911.
He, X., and D. J. Kappes. 2006. CD4/CD8 lineage commitment: light at the end of the
tunnel? Curr Opin Immunol 18:135-142.
Yankee, T. M., T. J. Yun, K. E. Draves, K. Ganesh, M. J. Bevan, K. Murali-Krishna,
and E. A. Clark. 2004. The Gads (GrpL) adaptor protein regulates T cell homeostasis.
J Immunol 173:1711-1720.
Liu, S. K., D. M. Berry, and C. J. McGlade. 2001. The role of Gads in hematopoietic
cell signalling. Oncogene 20:6284-6290.
Zhong, X. P., E. A. Hainey, B. A. Olenchock, M. S. Jordan, J. S. Maltzman, K. E.
Nichols, H. Shen, and G. A. Koretzky. 2003. Enhanced T cell responses due to
diacylglycerol kinase zeta deficiency. Nat Immunol 4:882-890.
Villalba, M., J. Hernandez, M. Deckert, Y. Tanaka, and A. Altman. 2000. Vav
modulation of the Ras/MEK/ERK signaling pathway plays a role in NFAT activation
and CD69 up-regulation. European journal of immunology 30:1587-1596.
Sasaki, T., J. Irie-Sasaki, R. G. Jones, A. J. Oliveira-dos-Santos, W. L. Stanford, B.
Bolon, A. Wakeham, A. Itie, D. Bouchard, I. Kozieradzki, N. Joza, T. W. Mak, P. S.
Ohashi, A. Suzuki, and J. M. Penninger. 2000. Function of PI3Kgamma in thymocyte
development, T cell activation, and neutrophil migration. Science 287:1040-1046.
Cantley, L. C., and B. G. Neel. 1999. New insights into tumor suppression: PTEN
suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT
pathway. Proc Natl Acad Sci U S A 96:4240-4245.
Kane, L. P., and A. Weiss. 2003. The PI-3 kinase/Akt pathway and T cell activation:
pleiotropic pathways downstream of PIP3. Immunol Rev 192:7-20.
Malinin, T. I., and V. P. Perry. 1967. A review of tissue and organ viability assay.
Cryobiology 4:104-115.
Mannering, S. I., J. S. Morris, N. L. Stone, K. P. Jensen, V. A. N. E. PM, and L. C.
Harrison. 2004. CD4+ T cell proliferation in response to GAD and proinsulin in
healthy, pre-diabetic, and diabetic donors. Annals of the New York Academy of
Sciences 1037:16-21.
Mannering, S. I., J. S. Morris, K. P. Jensen, A. W. Purcell, M. C. Honeyman, P. M.
van Endert, and L. C. Harrison. 2003. A sensitive method for detecting proliferation
of rare autoantigen-specific human T cells. Journal of immunological methods
283:173-183.
Zhu, M., S. Koonpaew, Y. Liu, S. Shen, T. Denning, I. Dzhagalov, I. Rhee, and W.
Zhang. 2006. Negative regulation of T cell activation and autoimmunity by the
transmembrane adaptor protein LAB. Immunity 25:757-768.
Zhu, M., I. Rhee, Y. Liu, and W. Zhang. 2006. Negative regulation of Fc epsilonRImediated signaling and mast cell function by the adaptor protein LAX. The Journal of
biological chemistry 281:18408-18413.
Shimomura, T., S. Araga, E. Esumi, and K. Takahashi. 1991. Decreased serum
interleukin-2 level in patients with chronic headache. Headache 31:310-313.
39