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The John P. Robarts Research Institute, and Departments of Microbiology and Immunology, and Medicine, University of Western Ontario, London, Ontario, Canada N6A 5K8; and
Genetics Institute Inc., Cambridge, MA 02140
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-associated protein
(ZAP)-70-dependent tyrosine phosphorylation of CTLA-4 that is important
for cell surface retention of this molecule. However, CTLA-4 tyrosine
phosphorylation is not required for down-regulation of T cell
activation following CD3-CTLA-4 coengagement. Specifically, inhibition
of extracellular signal-regulated kinase (ERK) activation and of IL-2
production by CTLA-4-mediated signaling occurs in T cells expressing
mutant CTLA-4 molecules lacking the cytoplasmic tyrosine residues, and
in lck-deficient or ZAP-70-deficient T cells. Therefore,
CTLA-4 function involves interplay between two different levels of
regulation: phosphotyrosine-dependent cell surface retention and
phosphotyrosine-independent association with signaling
molecules. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism by which CTLA-4 mediates inhibition of T cell activation
is not understood. Examination of the CTLA-4 sequence reveals a
relatively short cytoplasmic tail with two potential Src homology
(SH)3-2 domain binding
sites centered at tyrosine residues 165 and 182, spaced by a
proline-rich stretch. Experiments on primary activated T cells showed
an association between CTLA-4 and phosphatidylinositol-3 kinase
(11) and SH-2 domain-containing protein (SHP)-2
phosphatase (12). These associations were explained by
interactions between the SH-2 domains in these molecules and
intracellular phosphotyrosine residues on CTLA-4. Recently, using an
epithelial cell transfection system, SHP-2 was found to be recruited to
a mutant CTLA-4 molecule lacking tyrosine residues 165 and 182
(13). This mutant CTLA-4 molecule associated with TCR
and SHP-2 in an lck-dependent manner, consistent with the reported
association between CTLA-4 and src-kinases
(14, 15). However, the functional relevance of these
findings has not been established.
The main obstacle in the development of suitable experimental systems to perform structure-function analysis of CTLA-4 arises from the complex regulation of CTLA-4 expression. This occurs on at least three different levels: gene transcription, protein production, and retention on the cell membrane. Upon T cell activation, there is a significant increase in the levels of CTLA-4 mRNA and protein (16, 17, 18, 19, 20, 21). However, most CTLA-4 molecules are not cell surface expressed but accumulate in an intracellular compartment from which they traffic to the cell surface and are rapidly internalized (19, 22, 23). Several studies showed that persistent expression of CTLA-4 on the cell surface is dependent on phosphorylation of a tyrosine residue (Y165) in the cytoplasmic domain of CTLA-4 (24, 25, 26, 27). Phosphorylation of this residue prevents the interaction of CTLA-4 with the clathrin-associated AP-2 internalization adapter (28) and hinders CTLA-4 internalization.
Generation of stable, surface CTLA-4 expressing T cell transfectants has not been reported to date. This may be due to the high levels of CTLA-4 protein required to obtain a relatively low level of expression at the membrane. To achieve transient high levels of CTLA-4 protein, we have used a regulatable system in which the expression of the CTLA-4 gene is under the control of an inducible trans-activator (29). Jurkat T cells were transfected with CTLA-4 molecules since previous reports have established that these cells are negative at the RNA and protein levels for CTLA-4 expression (16, 18). Here, we describe a panel of human T cell lines expressing high levels of wild type (WT) or tyrosine mutant human CTLA-4 on the cell surface.
Using this cell panel, we show that phosphorylation of tyrosine
residues 165 and 182 is not required for the negative regulatory
function of CTLA-4 on T cell responses. This is illustrated
functionally and biochemically by inhibition of IL-2 production and
extracellular signal-regulated kinase (ERK) activation, following
coengagement of CD3 with CTLA-4 molecules that lack cytoplasmic
tyrosine residues. Interestingly, CD3/CTLA-4 coligation in T cells
deficient in lck or -associated protein (ZAP)-70, the
two crucial tyrosine kinases for proximal TCR-mediated signaling, also
results in inhibition of ERK activation. Thus, CTLA-4-mediated
negative signaling occurs by a phosphotyrosine-independent mechanism.
In contrast, CD3 ligation alone is sufficient to induce phosphorylation
of CTLA-4 in a ZAP-70-dependent manner, and this phosphorylation
determines retention of CTLA-4 on the cell surface.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human CTLA-4 (hCTLA-4) cDNA was obtained from G. Freeman (Dana-Farber Cancer Institute, Boston, MA). Mutant CTLA-4 cDNAs were generated using the Chameleon site-directed mutagenesis kit (Strategene, La Jolla, CA) or PCR amplification with high fidelity Klentaq polymerase (Clontech, Palo Alto, CA), and the introduced mutation was confirmed by DNA sequencing. cDNAs were subcloned into the EcoRI site of pBIG2i, a vector that utilizes a hybrid bidirectional tetracycline-responsive promoter element to direct expression of both the CTLA-4 as well as the (rtTAN) tetracycline-responsive trans-activator cDNAs (29).
Cell lines and stable transfectants
Jurkat E6.1 and the lck-deficient JCaM1.6 line were obtained from American Type Culture Collection (Manassas, VA) (30). The ZAP-70-deficient Jurkat T cell line (P116) was provided by Ronald L. Wange (Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD) (31). Cells were transfected by DNA electroporation. Briefly, 10 µg of linearized plasmid DNA from the different pBIG2i constructs was electroporated in 5 x 106 Jurkat T cells resuspended in 0.5 ml of RPMI 1640 medium at 300 V and 950 µF capacitance using gene pulser (Bio-Rad, Hercules, CA), and stable transfectants were selected with hygromycin (Life Technologies, Gaithersburg, MD). CTLA-4 expression in these transfectants was induced by overnight incubation with 100 ng/ml doxycycline (Sigma, St. Louis, MO).
T cell functional assays
Ab-coated beads were prepared as described (5) with a constant amount of anti-CD3 (UCHT1; PharMingen, San Diego, CA) representing 20% (1 µg/107 beads) of the total protein bound to the beads. Anti-hCTLA-4 mAb (anti-CTLA-4-20A) or control mAb (anti-HLA class I mAb) were used to make up the remaining 80% (4 µg/107 beads) of protein. Ab-coated beads were added to doxycycline-induced Jurkat transfectants (ratio of 1 bead:1 cell) in the presence of soluble anti-CD28 (20 µg/ml; CD28.2; PharMingen). Supernatants were harvested at 72 h, and IL-2 was measured using a commercially available IL-2 ELISA kit (Genzyme, Framingham, MA).
T cell stimulation and biochemistry
Doxycycline-induced Jurkat T cells were stimulated with Ab-coated beads or with soluble anti-CD3 Ab (UCHT1) for the indicated times. Cell lysates were prepared and immunoblotted as described (32). Signal detection was performed by chemiluminescence (Roche Diagnostics, Laval, PQ, Canada). Detection of active ERK-1 and ERK-2 was conducted by Western blotting of cell lysates with the anti-active mitogen-activated protein kinase (MAPK) (Promega, Madison, WI) rabbit antiserum (32). Blots were reprobed with a rabbit antiserum to MAP kinase/ERK-1-CT (33). Immunoprecipitations were performed as described (34). In some experiments, a cross-linking reagent (DSP; Pierce, Rockford, IL) was used for CTLA-4 immunoprecipitates.
Immunoprecipitation of surface CTLA-4
Doxycycline-induced Jurkat transfectants were cultured in the presence of biotin (Pierce) (0.5 mg/ml) for 30 min at room temperature. Cell lysates were prepared and centrifuged for 15 min at 14,000 rpm and equalized for total CTLA-4 expression by titration of total protein and immunoblotting using anti-CTLA-4-11 mAb. Equalized lysates were divided in two aliquots and immunoprecipitated with anti-biotin (Jackson ImmunoResearch, West Grove, PA) or anti-CTLA-4-24 cross-linked beads. Abs were cross-linked to Sepharose beads using DSP and incubated for 30 min at room temperature. Reaction was quenched with 1 M Tris (pH 8.0) for 5 min at room temperature. Beads were washed, and a 50% slurry was made using PBS. Immunoprecipitates were eluted under nonreducing conditions, and 2-ME was added and immunoblotted with a mouse anti-hCTLA-4 (anti-CTLA-4-11) mAb. Signal intensity was quantitated using an imaging densitometer (model GS-700; Bio-Rad) and the Molecular Analist Software (version 1.0; Bio-Rad Laboratories).
Flow cytometry
Jurkat T cells (0.5 x 106) were stained with saturating concentrations of PE-anti-CD3 (UCHT1; PharMingen) or -anti-CD28 (CD28.2; PharMingen), or biotin-anti-CTLA-4 (anti-CTLA-4-24)-conjugated mAbs. Anti-CTLA-4-stained cells were incubated with PE-labeled streptavidin (Southern Biotechnology Associates, Birmingham, AL). Cells were analyzed in a FACScan Flow Cytometer (Becton Dickinson, Mountain View, CA). Statistical analyses were performed with CellQuest computer software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat T cells do not express CTLA-4 in resting conditions or
after activation (16, 18). Also, CTLA-4 mRNA is not
detectable by PCR in samples prepared from Jurkat T cells (data not
shown). Following transfection with WT CTLA-4 cDNA, these cells
expressed high levels of surface CTLA-4 upon induction with doxycycline
(Fig. 1
A). Mutation of
tyrosine 165 into a phenylalanine (Y165F), to prevent interaction of
CTLA-4 with AP-2, caused a significant increase in the levels of CTLA-4
expression on the cell surface. In contrast, mutation in tyrosine
residue 182 (Y182F) did not cause a similar increase in CTLA-4 surface
expression. The effects of Y165F mutation were dominant since
transfectants with Y165F/Y182F showed high levels of surface CTLA-4
expression similar to those of CTLA-4 Y165F mutant.
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B). In both sublines, we detected
low CTLA-4 expression compared with the more heterogeneous profile seen
in WT CTLA-4 E6.1 transfectants. This finding suggests that these two
kinases contribute to the regulation of cell surface expression of
CTLA-4.
Expression of CTLA-4 was confirmed by Western blotting (Figs. 2A and
3B). In addition, surface
levels were compared with the total levels of CTLA-4 using cell surface
biotinylation, followed by immunoprecipitation with anti-biotin
(indicative of surface CTLA-4) or with anti-CTLA-4 (indicative of
total CTLA-4), and subsequent blotting for CTLA-4. As shown in Fig. 2B, a higher ratio of surface to total CTLA-4 molecules was
detected in the Y165F/182F mutant cells as compared with the WT
transfectants. In the absence of these two tyrosine residues, almost
all CTLA-4 produced is exported and retained on the cell surface.
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CTLA-4 is expressed on the surface of primary T cells upon T cell
activation (35, 36). Current evidence suggests that CTLA-4
surface expression is regulated by phosphorylation of at least one of
the tyrosine residues in its cytoplasmic tail (24, 25, 26, 27).
Thus, we hypothesized that TCR-mediated signaling could induce
phosphorylation of the two tyrosine residues of CTLA-4 and up-regulate
its surface expression. T cells transfected with WT CTLA-4 showed a
significant level of tyrosine phosphorylation of CTLA-4 in resting
conditions (Fig. 3A). Anti-CD3 stimulation induced a
significant increase in CTLA-4 phosphorylation. A similar effect was
observed in Y165 mutant and in Y182 mutant CTLA-4 transfectants (Fig. 3B). Note that this experiment was performed with
anti-CTLA-4 Ab cross-linked to beads as immunoprecipitating Ab to
get rid of the L chain band after Western blotting; under these
conditions, there are lower recovery levels of protein. No CTLA-4
phosphorylation was observed in Y165F/Y182F CTLA-4-transfected cells.
Thus, CTLA-4 phosphorylation is dependent on TCR engagement and does
not require CTLA-4 engagement.
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B). Thus, these two kinases are involved in
the phosphorylation-dependent regulation of CTLA-4 surface expression.
Lack of detection of phospho-CTLA-4 under basal or stimulated
conditions in the lck-deficient and ZAP-70-deficient T cells
is not due to lower expression of total CTLA-4 since tyrosine
phosphorylated CTLA-4 is detected in the Y182F CTLA-4 mutant cell line
despite lower total and surface CTLA-4 expression.
To test whether TCR-induced phosphorylation of CTLA-4 caused its
retention on the cell surface rather than an increase in export to the
membrane, we looked at the effect of CD3 ligation on surface levels of
CTLA-4 in WT and Y165F/Y182F transfectants. We found that CTLA-4
surface levels increased after CD3 ligation in doxycycline-induced WT
CTLA-4-expressing Jurkat cells (Fig. 3C). However, CD3
ligation had no effect on surface CTLA-4 levels in the Y165F/Y182F
transfectants (Fig. 3D). Thus, CD3 ligation-induced tyrosine
phosphorylation of CTLA-4 correlates with retention of CTLA-4 on the
cell surface.
Inhibition of IL-2 production by CD3-CTLA-4 coengagement does not require the cytoplasmic tyrosine residues in CTLA-4
Next, we examined IL-2 production by Jurkat T cells transfected
with WT CTLA-4 or with mutant CTLA-4 upon activation with beads coated
with anti-CD3 and anti-HLA class I Abs or with anti-CD3 and
anti-CTLA-4 Abs. Soluble anti-CD28 was added to provide
costimulation signals that are required for detectable IL-2 production
by Jurkat cells (5). Different clones for each
transfectant were characterized, and results from a representative
clone are shown (Fig. 4). We consistently
found that coligation of CD3/CTLA-4 resulted in inhibition of IL-2
production to a similar extent (87% or more) among the various types
of CTLA-4 molecules (Fig. 4A).
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B). This effect is specific to
CTLA-4 engagement since no inhibition was obtained when Abs other than
anti-CTLA-4 Abs (e.g., anti-CD4 Abs) were coating the beads
along with anti-CD3 Abs. Similar results were obtained with
different clones of either the single tyrosine mutants or the double
mutants. Thus, once expressed on the cell surface, CTLA-4-mediated
inhibition of IL-2 production is independent of tyrosine residues 165
and 182 and may involve a direct interaction with signaling molecules
through modules other than phosphotyrosine-binding motifs. Inhibition of ERK-1 and ERK-2 activation after CD3-CTLA-4 coligation does not require lck or ZAP-70 kinases
It has been reported that CTLA-4 ligation may interfere with
activation of the ras-MAPK and the c-Jun N-terminal kinase (jnk)
pathways (37). Thus, we examined ERK-1 and ERK-2
activation following coligation of CD3 and CTLA-4 (Fig. 5A). Stimulation of parental
(non-CTLA-4-expressing) Jurkat T cells with
anti-CD3/anti-CTLA-4-coated beads had no effect on total dually
phosphorylated ERK-1 and ERK-2, a modification that correlates with
active forms of ERKs. However, when a similar experiment was performed
with Jurkat T cells expressing surface WT CTLA-4, a consistent and
significant decrease in dually phosphorylated ERK-1 and ERK-2 was
observed. This effect did not require the cytoplasmic tyrosine residues
of CTLA-4 since similar inhibition was seen in cells transfected with
the double tyrosine mutant form of CTLA-4 (Fig. 5B).
Preliminary data indicate that kinetics of inhibition of ERK activation
by engagement of double tyrosine mutant CTLA-4 molecules may be much
faster than for WT CTLA-4-mediated inhibition.
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A). This effect could not be
correlated with inhibition of IL-2 production since JcaM1.6 and P116
fail to produce any detectable IL-2 under these experimental
conditions. Together, our results show that negative regulation of T
cell function by CTLA-4, once expressed on the cell surface, is
independent of CTLA-4 tyrosine phosphorylation or the two main kinases
involved in proximal TCR-mediated signaling. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTLA-4 cell surface expression is tightly regulated by clathrin-associated endocytosis of nonphosphorylated, surface CTLA-4. This process involves the protein adapter AP-2 recognizing at least one of the tyrosine residues in the cytoplasmic tail of CTLA-4. Phosphorylation of this residue prevents binding of AP-2 to CTLA-4 and subsequent internalization of CTLA-4 (24, 25, 26, 27). Our data indicate that tyrosine 165 is the primary regulator of CTLA-4 surface expression in T cells. In addition, we demonstrate that phosphorylation of Y165 and Y182 occurs in vivo and upon TCR ligation, in an lck- and ZAP-70-dependent manner. Within the current framework of TCR-mediated signaling, these two kinases act sequentially on tyrosine phosphorylation of TCR subunits and on the linker for T cell activation (LAT) (38, 39), leading to the formation of multimolecular complexes that activate several intracellular signaling pathways (40). Based in our results, we postulate that the requirement for lck may illustrate the need for activation of ZAP-70 and/or another downstream kinase that phosphorylates CTLA-4. In this sense, a recent report by Schneider et al. has implicated Rlk/Txk kinase in the phosphorylation of CTLA-4 and induction of its association with other signaling molecules (41).
In contrast to the regulatory effect of phosphorylation on CTLA-4 surface expression, inhibition of IL-2 production by TCR-CTLA-4 coengagement is not dependent on CTLA-4 tyrosine phosphorylation. This finding precludes a mechanism based on interaction between phosphotyrosines in CTLA-4 and SH-2 domains of signaling molecules. This particularly applies to SHP-2, an SH-2 domain-containing phosphatase (42) implicated in negative signaling through CTLA-4 (12, 13). However, it does not exclude the possibility of interactions between SHP-2 with neighboring residues other than phosphotyrosines, as reported for the interaction between SLAM-associated protein and signaling lymphocyte-activation molecule (43). In contrast, negative signaling through CTLA-4 likely involves an association between this receptor and a signaling molecule (e.g., a phosphatase or a negative regulatory kinase) through a non-phosphotyrosine-dependent interaction, either directly or through an adapter protein (13). However, our findings do not support the previous claims that tyrosine phosphorylation of CTLA-4 (44) and lck itself (13) enhances CTLA-4-mediated down-regulatory signals. These contrasting results may be secondary to some of the effects attributed to lck being related to the phosphorylation-dependent retention of CTLA-4 on the surface, which is a prerequisite for its engagement and subsequent negative signaling.
The inhibition of ERK activation by coligation of CD3 and CTLA-4 in lck-deficient and in ZAP-70-deficient Jurkat T cells argues against an essential role of these two kinases in the regulatory function of CTLA-4 on T cell responses. We and others have previously demonstrated that the ras-MAPK pathway can be activated in the absence of proximal activation of these two kinases as a result of TCR engagement with partial agonist ligands or dimerization of CD3 (32, 45). This contrasts with the severe deficit of phosphorylation in lck-deficient Jurkats (30). This difference may reflect the level of TCR oligomerization achieved using immobilized Abs (like in our studies) compared with the use of soluble anti-CD3 mAbs, and may translate in the ability to activate ERK in cells lacking lck or ZAP-70. Our data emphasize that negative signaling through CTLA-4 acts upon an intracellular pathway that is strictly dependent on TCR engagement. We cannot rule out that, in the absence of lck, another src kinase such as fyn may take over and play similar role in activating this pathway.
The specific mechanism by which CTLA-4 exerts its negative regulatory
role on TCR-mediated cell signaling remains an open question. Data in
this report demonstrate that CTLA-4 phosphorylation correlates with
cell surface retention, and this is regulated by a TCR-mediated,
lck- and ZAP-70-dependent mechanism that does not require
CTLA-4 engagement. However, once CTLA-4 is expressed on the cell
surface and is engaged, its phosphorylation is not required for
down-regulation of T cell responses (Fig. 6). We cannot formally exclude functional
differences between signaling resulting from CTLA-4 ligation with
immobilized Abs and B7-induced CTLA-4 ligation. Our observations point
toward a mechanism other than direct SH-2 domain-dependent recruitment
of a signaling molecule to explain CTLA-4 mediated inhibition of T cell
responses. Attention as to how this occurs has mostly been concentrated
on effects at a proximal level of TCR-mediated signaling, such as
dephosphorylation of TCR -chains through recruitment and activation
of the SH-2 domain-containing phosphatase SHP-2 (13). To
date, we have failed to detect such an event using our cell lines after
CD3 ligation (M.L.B. and J.M., data not shown). Rather, our findings
support an effect on inhibition of a TCR-dependent pathway such as the
ras-MAPK, as previously suggested (37). It is not known
whether this occurs through activation of some of its regulators such
as rap1 or CrkL (46, 47, 48) or by activation of a new
signaling cascade.
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While this paper was under review, similar conclusions were reported using a mouse T cell clone (49).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joaquín Madrenas, The John P. Robarts Research Institute, Room 2.05, Post Office Box 5015, 100 Perth Drive, London, Ontario, Canada N6A 5K8. E-mail address:
3 Abbreviations used in this paper: SH-2, Src homology-2, ERK, extracellular signal-regulated kinase, MAPK, mitogen-activated protein kinase; ZAP, -associated protein; SHP, SH-2 domain-containing protein tyrosine phosphatase; WT, wild type; hCTLA-4, human CTLA-4.
Received for publication August 5, 1999. Accepted for publication October 13, 1999.
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References |
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phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 267:515. and ZAP70. J. Exp. Med. 186:1645.This article has been cited by other articles:
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  M. G. Kirchhof, L. A. Chau, C. D. Lemke, S. Vardhana, P. J. Darlington, M. E. Marquez, R. Taylor, K. Rizkalla, I. Blanca, M. L. Dustin, et al. Modulation of T Cell Activation by Stomatin-Like Protein 2 J. Immunol., August 1, 2008; 181(3): 1927 - 1936. [Abstract] [Full Text] [PDF]
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P. J. Darlington, M. G. Kirchhof, G. Criado, J. Sondhi, and J. Madrenas Hierarchical Regulation of CTLA-4 Dimer-Based Lattice Formation and Its Biological Relevance for T Cell Inactivation J. Immunol., July 15, 2005; 175(2): 996 - 1004. [Abstract] [Full Text] [PDF]
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K. I. Mead, Y. Zheng, C. N. Manzotti, L. C. A. Perry, M. K. P. Liu, F. Burke, D. J. Powner, M. J. O. Wakelam, and D. M. Sansom Exocytosis of CTLA-4 Is Dependent on Phospholipase D and ADP Ribosylation Factor-1 and Stimulated during Activation of Regulatory T Cells J. Immunol., April 15, 2005; 174(8): 4803 - 4811. [Abstract] [Full Text] [PDF]
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J. Madrenas, L. A. Chau, W. A. Teft, P. W. Wu, J. Jussif, M. Kasaian, B. M. Carreno, and V. Ling Conversion of CTLA-4 from Inhibitor to Activator of T Cells with a Bispecific Tandem Single-Chain Fv Ligand J. Immunol., May 15, 2004; 172(10): 5948 - 5956. [Abstract] [Full Text] [PDF]
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E. Siu, B. M. Carreno, and J. Madrenas TCR subunit specificity of CTLA-4-mediated signaling J. Leukoc. Biol., December 1, 2003; 74(6): 1102 - 1107. [Abstract] [Full Text]
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F. Bennett, D. Luxenberg, V. Ling, I-M. Wang, K. Marquette, D. Lowe, N. Khan, G. Veldman, K. A. Jacobs, V. E. Valge-Archer, et al. Program Death-1 Engagement Upon TCR Activation Has Distinct Effects on Costimulation and Cytokine-Driven Proliferation: Attenuation of ICOS, IL-4, and IL-21, But Not CD28, IL-7, and IL-15 Responses J. Immunol., January 15, 2003; 170(2): 711 - 718. [Abstract] [Full Text] [PDF]
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C. A. Chambers, J. Kang, Y. Wu, W. Held, D. H. Raulet, and J. P. Allison The lymphoproliferative defect in CTLA-4-deficient mice is ameliorated by an inhibitory NK cell receptor Blood, May 29, 2002; 99(12): 4509 - 4516. [Abstract] [Full Text] [PDF]
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M. L. Baroja, L. Vijayakrishnan, E. Bettelli, P. J. Darlington, T. A. Chau, V. Ling, M. Collins, B. M. Carreno, J. Madrenas, and V. K. Kuchroo Inhibition of CTLA-4 Function by the Regulatory Subunit of Serine/Threonine Phosphatase 2A J. Immunol., May 15, 2002; 168(10): 5070 - 5078. [Abstract] [Full Text] [PDF]
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C. Guntermann and D. R. Alexander CTLA-4 Suppresses Proximal TCR Signaling in Resting Human CD4+ T Cells by Inhibiting ZAP-70 Tyr319 Phosphorylation: A Potential Role for Tyrosine Phosphatases J. Immunol., May 1, 2002; 168(9): 4420 - 4429. [Abstract] [Full Text] [PDF]
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D. K. Newman, C. Hamilton, and P. J. Newman Inhibition of antigen-receptor signaling by Platelet Endothelial Cell Adhesion Molecule-1 (CD31) requires functional ITIMs, SHP-2, and p56lck Blood, April 15, 2001; 97(8): 2351 - 2357. [Abstract] [Full Text] [PDF]
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T. F. Gajewski, F. Fallarino, P. E. Fields, F. Rivas, and M.-L. Alegre Absence of CTLA-4 Lowers the Activation Threshold of Primed CD8+ TCR-Transgenic T Cells: Lack of Correlation with Src Homology Domain 2-Containing Protein Tyrosine Phosphatase J. Immunol., March 15, 2001; 166(6): 3900 - 3907. [Abstract] [Full Text] [PDF]
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B. M. Carreno, F. Bennett, T. A. Chau, V. Ling, D. Luxenberg, J. Jussif, M. L. Baroja, and J. Madrenas CTLA-4 (CD152) Can Inhibit T Cell Activation by Two Different Mechanisms Depending on Its Level of Cell Surface Expression J. Immunol., August 1, 2000; 165(3): 1352 - 1356. [Abstract] [Full Text] [PDF]
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