|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
IMMUNOBIOLOGY
From the Laboratory of Lymphocyte Biology, National
Heart, Lung, and Blood Institute, Laboratory of Cellular and Molecular
Biology, National Cancer Institute, National Institutes of Health,
Bethesda, MD.
Activation of T cells can be initiated through cell surface
molecules in addition to the T-cell receptor-CD3 (TCR-CD3) complex. In
human T cells, ligation of the CD2 molecule by mitogenic pairs of
anti-CD2 monoclonal antibodies activates T cells via biochemical signaling pathways similar but not identical to those elicited on TCR
engagement. This study describes a key role for the p36/38 membrane
adapter protein linker for T cell activation (LAT) in CD2-mediated
T-cell activation. Following ligation of CD2 on the surface of the
Jurkat T-cell line and human purified T cells, LAT was tyrosine
phosphorylated and shown to associate in vivo with a number of other
tyrosine phosphorylated proteins including PLC Engagement of the T-cell antigen receptor (TCR) by
specific antigen-major histocompatibility complexes (MHC) or anti-TCR
monoclonal antibody (mAb) triggers an intracellular cascade of
biochemical events that culminate in T-cell activation.1-3
Many of these events are now well characterized, although incompletely
understood. One of the earliest biochemical responses elicited by the
TCR is the activation of protein tyrosine kinases (PTK) of the Src family, Lck and/or Fyn, that mediate the phosphorylation of the immunoglobulin receptor family tyrosine-based activation motifs (ITAMs)
on the intracellular tails of the CD3 and Linker for T-cell activation (LAT) is a recently identified 36- to
38-kd membrane adapter protein expressed in T cells, natural killer
(NK) cells, mast cells, and megakaryocytes, but not in B cells or
monocytes.10,11 Following ligation of the TCR-CD3 complex,
ZAP70 or Syk kinase mediates the tyrosine phosphorylation of LAT that,
in turn, is required for Ras activation as for the induction of nuclear
factor of activated T cells (NFAT) and AP1 transcriptional
activation.10,12,13 LAT tyrosylphosphorylation results in
the recruitment and SH2-dependent binding of the adapter molecule Grb2
and thus binding of mSos (an upstream activator of Ras), Cbl, and a
complex of proteins including SLP-76 and Vav (an upstream activator of
Rac). Moreover, in vivo, tyrosine phosphorylation of LAT results in its
association with PLC Our present studies were undertaken to determine whether LAT
plays a role in T-cell activation induced through ligation of the
CD2 molecule. CD2 is a 50- to 55-kd glycoprotein expressed on a
majority of thymocytes, T cells, and NK cells. Through physiologic interactions with its ligands CD58 (lymphocyte function-associated antigen-3 [LFA-3]), CD48, and CD59, the CD2 molecule plays a role in
T-cell signaling and promotes lymphocyte adhesion. Specific pairs of
CD2-specific mAbs were demonstrated to be able to induce interleukin
(IL)-2 production and T-cell proliferation in the absence of engagement
of the TCR-CD3 complex.16,17 In our studies, biochemical
and functional data support a critical role for LAT in CD2-mediated
T-cell activation. In particular, we show, on CD2 stimulation of human
purified T cells and of Jurkat T cells, the time- and
activation-dependent tyrosine phosphorylation of LAT and its
association in vivo with several phosphotyrosine-containing proteins,
including PLC Cells and reagents
For all the cell lines, surface expression of both CD3 and CD2 was
routinely evaluated by direct immunofluorescence; cells were incubated
on ice for 20 minutes with phycoerythrin (PE)-conjugated anti-CD3 mAb,
clone UCHT1 (Beckman Coulter Inc, Fullerton, CA), and fluorescein
isothiocyanate (FITC)-conjugated anti-CD2 mAb, clone RPA-2.10 (BD
PharMingen, San Diego, CA) or appropriate PE- and/or FITC-conjugated
IgG isotype control (Beckman Coulter). Cells then were washed and
analyzed using a Coulter Epics flow cytometer (Beckman Coulter). CD3
and CD2 expressions were equivalent between wild-type Jurkat, P116,
P116-ZAP70, and JCaM1.6-Lck and comparable between ANJ3 and ANJ3-LATwt
(not shown). CD3 and CD2 cell surface expression remained constant with time.
Resting human peripheral blood lymphocytes (PBLs) were isolated from
adult healthy donors by density-gradient centrifugation on Ficoll-Paque
PLUS (Amersham Pharmacia Biotech, Uppsala, Sweden) and plastic
adherence, resuspended in complete RPMI medium, and used within 24 hours of isolation.
The anti-CD2 mAbs T112 and T113 were the kind
gift of Ellis Reinherz (Dana-Farber Cancer Institute, Boston, MA); the
murine antihuman CD3 Cell stimulation and immunoprecipitation
For LAT immunoprecipitations, 2 µL of anti-LAT polyclonal Ab and 20 µL of protein A-agarose beads were added together to the clarified cell lysates and incubated at 4°C for 2 hours. The lysates were not precleared by preincubation with beads alone. Therefore clarified cell lysates from the same conditions of stimulation incubated with 20 µL protein A-agarose beads alone served as the control for carryover of stimulating mAbs, as indicated. For immunoprecipitation of phosphotyrosine-containing proteins, 20 µL of anti-pTyr (PY99) directly conjugated to agarose beads were used. The beads were then washed 3 times with washing buffer (0.1% Brij97, 150 mmol/L NaCl, 25 mmol/L Tris [pH 7.5], 1 mmol/L Na3VO4). Proteins were boiled in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF; Millipore, Bedford, MA), and probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Polypeptides recognized in the Western blot were detected using the enhanced chemiluminescence (ECL) methods according to manufacturer's instructions (Amersham Pharmacia Biotech Inc, Piscataway, NJ). Quantification of band density was determined by densitometry using the Imagequant software (Molecular Dynamics, Sunnyvale, CA) when indicated. Transient transfection and luciferase assay Jurkat cells (2 × 107) were removed from culture, washed, resuspended in 0.5 mL RPMI 1640 to which 10% FCS had been added (termed 10% FCS-RPMI) and incubated with 10 µg of a reporter plasmid p3xNFAT-luc19 carrying the luciferase gene driven by 3 tandem repeats of the distal NFAT sequences derived from the IL-2 promoter and 1 µg of pRL-TK vector (Promega, Madison, WI), which provides constitutive expression of Renilla luciferase, for 15 minutes at room temperature. Cells were then electroporated at 250 V, 800 microfarads, low (Life Technologies,
Inc); incubated for 15 minutes at room temperature; and then
transferred to complete RPMI medium and incubated at 37°C.
Twenty-four hours after the transfection, 1 × 106
cells/test were left unstimulated or stimulated with plate-bound OKT3,
1 µL of T112 plus 1 µL of T113, each either
alone or in combination with PMA (10 ng/mL) or ionomycin (1.5 µmol/L), or PMA plus ionomycin for 6 hours. Cells were then washed
with PBS, and samples were prepared using the Dual-Luciferase Reporter
Assay System (Promega) according to the manufacturer's instructions. Results are reported as the activity of firefly luciferase normalized to that of Renilla luciferase (to correct for the efficiency
of transfection) in the lysates, and expressed as the percentage of
maximal response obtained with PMA plus ionomycin.
Reverse transcriptase-polymerase chain reaction To examine the induction of IL-2 transcription, Jurkat cells (1 × 106/test) were either left unstimulated or stimulated with plate-bound OKT3, 1 µL of T112 plus 1 µL of T113, each either alone or in combination with PMA (10 ng/mL), or PMA plus ionomycin for 4 hours. Total RNA was then extracted from cells with TRIAZOL (GibcoBRL, Life Technologies) according to the manufacturer's instructions. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using the human IL-2-specific primers 5'-CATTGCACTAAGTCTTGCACTTGTCA-3' and 5'-CGTTGATATTGCTGATTAAGTCCCTG-3' using the QUIAGEN OneStep RT-PCR kit (QUIAGEN Inc, Valencia, CA) according to the manufacturer's instructions.
CD2 stimulation induced LAT tyrosine
phosphorylation and association with PLC
LAT was found to coprecipitate with additional proteins following
stimulation. Longer exposures of LAT immunoprecipitation membrane
revealed the association of LAT with a number of other phosphotyrosine-containing proteins, including proteins of
approximately 135, 120, 97, and 76 kd molecular weight (Figure 2A,
lower panel); proteins of similar size have previously been shown to
coprecipitate with LAT on TCR-CD3 engagement.10 Control
precipitations using protein A-agarose alone, in the absence of
anti-LAT antisera, did not reveal any of these phosphorylated proteins
(not shown). The membrane was stripped and reprobed with specific
antibodies; this led to the identification of the 135-kd protein as
PLC To further explore the kinetics of LAT tyrosine phosphorylation on CD2
stimulation, wild-type Jurkat T cells were either left unstimulated or
stimulated via either CD2 or the TCR-CD3 complex for different times,
and the pattern of tyrosine phosphorylated proteins in clarified
postnuclear lysates was analyzed (Figure 3, left panel). Following CD2
stimulation, LAT phosphorylation returned to basal levels after
approximately 30 minutes but was still detectable at 120 minutes. The
time course of apparent LAT dephosphorylation was similar following CD2
as CD3 stimulation (Figure 3, left panel).
To determine whether either CD2 or TCR-CD3 engagement resulted in LAT
tyrosine phosphorylation in peripheral human T cells, human PBLs were
either left unstimulated or stimulated for different time points with
either anti-CD3 mAb, mitogenic pairs of anti-CD2 mAbs, or a combination
of anti-CD3 and pairs of anti-CD2 mAbs to evaluate additivity of
costimulation. Cell lysates were prepared and subjected to LAT
immunoprecipitation or incubated with protein A-agarose alone, as
indicated (Figure 4). As observed in the
Jurkat human T-cell line, either TCR-CD3 or CD2 engagement of resting human PBLs induced the time-dependent tyrosine phosphorylation of LAT
(Figure 4A, upper panel). LAT phosphorylation was observed at 5 minutes, increased slightly at 15 minutes, and was still present at 30 minutes. Furthermore, costimulation via the CD2 and the CD3 receptors
marginally increased LAT tyrosine phosphorylation. LAT phosphorylation
was quantified by densitometry (Figure 4C).
As with Jurkat T cells, a number of other tyrosine phosphorylated proteins were shown to coprecipitate with LAT on stimulation of peripheral blood T cells; in particular, phosphotyrosine-containing proteins of molecular weight between 70 and 203 kd were revealed in a longer exposure of the membrane (Figure 4A, middle panel). Stimulation-dependent coprecipitation of Grb-2 with tyrosine phosphorylated LAT was also observed (Figure 4B, lower panel). The amount of coprecipitated Grb-2 correlated with the tyrosine phosphorylation of LAT, as quantified by densitometry (Figure 4B,C). Moreover, both TCR-CD3 and CD2 engagement of peripheral blood T cells led to induction of downstream events such as Erk activation, as shown by probing the electrophoresis of PNLs with antiphospho-Erk-specific antibody (Figure 4D). Coligation of both CD2 and TCR-CD3 demonstrated some additivity in the biologic response, even when the specific stimulating antibodies were used at optimal concentrations (Figure 4D,E). A correlation between Erk phosphorylation and LAT/Grb-2 association was observed and quantified by densitometry (compare Figure 4E with 4C). Taken together, our data suggest a role for the LAT protein as an adapter molecule in signaling via either CD2 or CD3 in peripheral blood human T cells. On CD2 stimulation, the tyrosine phosphorylation of
PLC  -1 or SLP-76
required LAT, as had been previously shown for CD3
engagement.13 Either ANJ3 or ANJ3 cells reconstituted with
a construct expressing the myc-tagged wild-type LAT
(ANJ3-LATwt) were left unstimulated or stimulated for the indicated
periods of time via either the CD2 or TCR-CD3 molecules, and the
pattern of tyrosine phosphorylated proteins was analyzed (Figure
5, upper panel). Absent in the p-Tyr
immunoprecipitates of the LAT-deficient cell line were proteins of
~135 and ~75 kd, both of which are tyrosine phosphorylated on CD2
stimulation in wild-type Jurkat cells (Figure 1 and data not shown) and
in the LAT-reconstituted cells (Figure 5). Stripping the membrane and reprobing with specific antibody allowed the identification of the
135-kd protein as PLC-1 and the 75-kd protein as SLP-76 (Figure 5,
lower panels). These results demonstrate the requirement for LAT in the
activation of PLC-1 and SLP-76 not only on TCR engagement but also
on CD2 stimulation. Notably, the apparent basal phosphorylation of
proteins identified as ZAP70 and the TCR  chain was increased in
cells lacking LAT expression compared with cells reconstituted with the
epitope-tagged LAT construct (Figure 5), as has been observed
previously.13
ZAP70/Syk kinase expression and Src family kinase activity are required for optimal CD2-dependent LAT phosphorylation By cotransfection studies, LAT has been shown to be a substrate of the ZAP70 and Syk protein tyrosine kinases,10 a conclusion supported by the lack of LAT tyrosine phosphorylation on TCR engagement in a ZAP70/Syk-deficient Jurkat cell line, P116.26 We wished to determine whether ZAP70/Syk kinases were also required for LAT phosphorylation following CD2 stimulation. In the absence of Syk and ZAP70, minimal but detectable tyrosine phosphorylation of LAT was observed on CD2 stimulation (Figures 6 and 7). Despite modest LAT phosphorylation, neither PLC-1 nor SLP-76 was observed to be
phosphorylated following stimulation via either CD2 or the TCR-CD3
complex (data not shown). Engagement of CD2 and TCR-CD3 concurrently
did not bypass the requirement for ZAP70/Syk kinase expression for LAT
phosphorylation (Figure 7). In contrast, reconstitution of the
ZAP70/Syk-deficient cell line P116 with a construct expressing wild-type ZAP70 restored both CD3- and CD2-mediated LAT phosphorylation (Figure 7).
ZAP70 kinase, unlike Syk, requires activation by Src family members,
Lck or Fyn.4 To explore the role of Src family members in
the CD2-mediated tyrosine phosphorylation of LAT, wild-type Jurkat T
cells were either left untreated or treated with the specific Src
kinases inhibitor PP127 prior to stimulation via either
CD2 or TCR-CD3 molecule. LAT tyrosine phosphorylation was then analyzed
on LAT immunoprecipitates (Figure 8,
upper panel). A significant, but incomplete, inhibition of CD2- or
CD3-dependent LAT tyrosine phosphorylation was observed on treatment
with PP1. Coprecipitation of other tyrosine phosphorylated proteins
known to associate with phospho-LAT on stimulation was impaired (data not shown). The residual LAT phosphorylation in PP1-treated cells may
have been due to incomplete inhibition of Src kinase activity by the
concentration (20 µM) of PP1 used in this experiment.
To explore the specific role of Lck in CD2-mediated LAT tyrosine
phosphorylation, we used the CD2+ Lck-reconstituted clone
of the Lck-deficient Jurkat cell line, JCaM1.6, in which Lck kinase was
expressed under the control of a tetracycline-repressible TetR-VP16
fusion protein.18 Treatment of these cells with 1 µg/mL
tetracycline for 4 days induced a complete inhibition of Lck expression
as shown by an anti-Lck immunoblot of lysates derived from equivalent
numbers of either untreated or tetracycline-treated cells (Figure
9A). Surface expression of either CD2 or
CD3 was not changed by tetracycline treatment (Figure 9B). In contrast
to untreated cells, neither CD2 nor CD3 stimulation induced appreciable
LAT phosphorylation in the tetracycline-treated cells that lacked Lck
expression (Figure 9C). Our data suggest an essential role for Lck in
mediating the (ZAP70-dependent) CD2-induced tyrosine phosphorylation of
LAT.
LAT is required for Erk1/2 activation on CD2 stimulation Stimulation of Jurkat T cells through either the TCR-CD3 or the CD2 receptor induces Erk activation.28-30 We wished to determine whether LAT expression was required for Erk activation on CD2 engagement. A specific antiphospho-Erk1/2 mAb was used to probe PNLs derived from either unstimulated, OKT3-, or T112 + T113-stimulated ANJ3 and LAT-reconstituted ANJ3 cells. No Erk activation was observed in ANJ3 cells either on TCR-CD3, as previously shown,13 or CD2 stimulation (ANJ3, Figure 10, upper panel). Reconstitution of ANJ3 with LAT restored both TCR-CD3- and CD2-mediated Erk activation (ANJ3-LATwt, Figure 10, upper panel). Comparable expression of Erk1/2 protein between ANJ3 and LAT-reconstituted ANJ3 cells was confirmed by stripping and reblotting the membrane with an anti-Erk1/2-specific antibody (Figure 10, lower panel). As expected, although no Erk activation was observed in the ZAP70/Syk-deficient P116 cells, reconstitution of ZAP70 expression restored Erk phosphorylation, comparable with that observed in wild-type Jurkat cells, following either TCR-CD3 or CD2 stimulation (Figure 11, upper panel). Erk1/2 expression (Figure 11, middle panel) is comparable in each cell line used, despite differences in ZAP70 expression (Figure 11, lower panel). These data demonstrate that LAT tyrosine phosphorylation by ZAP70/Syk kinases is required for coupling CD2 to Ras pathway activation.
LAT tyrosine phosphorylation couples CD2 stimulation to NFAT and IL-2 transcriptional activation To analyze whether deficient LAT expression or failure of LAT tyrosine phosphorylation translated into a defect in NFAT transcriptional activation, wild-type Jurkat, the ZAP70/Syk kinase-deficient Jurkat cell line P116, the ZAP70-reconstituted P116 cells, the LAT-deficient Jurkat cells ANJ3, and the LAT-reconstituted Jurkat cells ANJ3-LATwt were transiently transfected with a reporter luciferase gene driven by 3 copies of the distal NFAT site derived from the IL-2 promoter.19 Transfected cells were either left unstimulated or stimulated with plate-bound OKT3, mitogenic pairs of anti-CD2 mAbs (T112 + T113), either alone or in combination with either PMA or ionomycin, or PMA plus ionomycin, as indicated. As expected, treatment of each of these cell lines with PMA and ionomycin, agents that bypass surface and proximal signaling events, resulted in equivalent NFAT transcriptional activation (data not shown). However, in the absence of either ZAP70/Syk kinases (P116, Figure 12A) or LAT (ANJ3, Figure 12B), no NFAT transcriptional activation was observed on either CD2 or CD3 stimulation, either alone or in combination with PMA or ionomycin. These latter data demonstrate that activating PKC (with PMA) or modifying internal calcium concentrations (with ionomycin) did not bypass the lack of ZAP70/Syk kinase or LAT expression. Appropriate LAT reconstitution (ANJ3-LATwt, Figure 12B) and ZAP70 reconstitution (P116-ZAP70, Figure 13) restored NFAT transcriptional activity on engagement of either CD3 or CD2. Correlation of NFAT transcriptional activation with IL-2 transcription was documented by RT-PCR in appropriately stimulated wild-type and ZAP70/Syk deficient Jurkat cells (Figure 12C).
In this study, we analyzed the role of LAT and Syk family PTKs in T-cell activation induced through ligation of the CD2. We provide biochemical and functional evidence that LAT expression and its tyrosine phosphorylation by ZAP70/Syk kinases are critical, not only in TCR-mediated T-cell activation, but also on CD2 engagement. Biochemical studies have demonstrated that CD2 stimulation can initiate a multitude of intracellular signaling events, similar to those elicited on TCR engagement. The involvement of Src family PTK in CD2-mediated signaling has been suggested by the demonstration of CD2 coassociation with both Lck and Fyn31-35; CD2 signaling was also shown to be defective in the Lck-deficient Jurkat cell line, JCaM1,24 and in cell lines deficient in the expression of CD45,36-38 a transmembrane protein-tyrosine phosphatase believed to regulate the kinase activities of Src family members.39,40 Here we extend those observations to shown defective CD2-dependent LAT tyrosine phosphorylation in the absence of Lck expression (Figure 9). The expression of Fyn did not bypass the requirement for Lck in CD2-induced LAT phosphorylation, at least in the cell lines used here. However, Lck expression is not an absolute requirement for CD2 signaling as an Lck-independent pathway of CD2 signaling involving Jun kinase has been described.41 The role of ZAP70/Syk family PTKs in CD2 signaling is less clear. The time-dependent activation of ZAP70 kinase has been reported following CD59 cross-linking42; however, other studies24 have failed to demonstrate ZAP70/Syk family activation on CD2 engagement. The overlapping roles of Syk and ZAP70, both of which are capable of expression in T lymphocytes, has confounded the analysis of these kinases. Recently, Ueno and colleagues43 demonstrated that ZAP70 was required for calcium mobilization, but was dispensable for mitogen-activated protein kinase (MAPK) family activation induced by CD2 engagement of human T cells. The analysis was conducted on a human T-cell line derived from a patient with a genetic deficiency of ZAP70 expression. Syk kinase, expressed in these cells, may have compensated for the lack of ZAP70. Our results using the Jurkat P116 cell line support a key role of the ZAP70/Syk kinase family in CD2-mediated T-cell activation. Deficient in expression of both ZAP70 and Syk kinase, the Jurkat P116 cell line displayed severe defects in CD2-induced signaling functions, including protein tyrosine phosphorylation and NFAT-driven and IL-2 transcriptional activation (Figure 12A,C and data not shown). The activation of Erk1/2 MAP kinases, downstream effectors of the Ras pathway, was also defective on CD2 stimulation of the ZAP70/Syk-deficient cell line (Figure 11). Importantly, these defects were overcome by the reconstitution of the P116 cells with ZAP70, demonstrating that the expression of ZAP70 alone, in the absence of Syk, is sufficient to mediate such downstream events. We suggest that, although Syk kinase appears capable of transducing CD2-induced MAPK family activation43 in T cells, ZAP70 kinase is capable of transducing CD2 signaling leading not only to Erk1/2 MAPK activation (Figure 11), but also to calcium mobilization43 and NFAT transcriptional activation (Figure 12). We were unable to address directly the role of Syk kinase in our system because Syk-reconstituted P116 cells are not stable and transient expression of Syk kinase alone conferred NFAT transcriptional activity.44 A deficiency of ZAP70 expression in the P116 cell line did not affect
the expression or activation of the Src family members Lck and Fyn on
TCR/CD3 stimulation; the tyrosine phosphorylation, however, of certain
proteins such as SLP-76 and PLC The mechanism by which LAT is coupled to CD2 remains uncertain. LAT has not been shown to associate directly with the TCR-CD3 complex, but does associate with CD4 and CD8 molecules.45 Thus, the CD4 and CD8 receptors appear to recruit not only the Src family members but also LAT to the TCR-CD3 complex, in proximity, following activation, to ZAP70. Preliminary observations suggest that CD2 and LAT coprecipitate in lysates prepared in Brij97 detergent (Martelli and Bierer, unpublished observations) but there is no evidence to date that the association is direct. These data are consistent with the recent observation that the CD2 molecule is localized to the GEMs of the plasma membrane,46 a domain to which LAT preferentially localizes. The molecular basis of CD2/LAT association is the subject of current investigation. Recent identification of the CD2-associated molecule CD2AP47 and its human homologous CMS48 has renewed interest in the relationship between CD2 and cytoskeletal elements. The CD2AP interaction with the cytoplasmic domain of CD2 appears to regulate CD2 clustering at the site of the T cell-antigen-presenting cell contact, cytoskeletal rearrangement, and T-cell polarization.47 It is tempting to speculate that the CD2AP/CD2 regulation of T-cell cytoskeletal rearrangements is responsible for the ability of CD2 to lower the threshold of T-cell activation,49 rendering T cells responsive to low antigen densities. Although the signaling intermediates for CD2-dependent cytoskeletal rearrangements have not been defined, CMS has been shown to regulate Rac, a small GTPase that itself regulates cytoskeletal rearrangement. Vav, a GTP exchange factor for Rac, is in turn regulated by SLP76, a protein that we have shown is dependent on LAT following CD2 activation. Taken together, these disparate observations would support a model by which the adapter protein LAT and CD2AP/CMS are both involved in the CD2-dependent regulation of Rac pathways leading to cytoskeletal rearrangement and lymphocyte adhesion. A number of immune functions, such as IL-2 production and T-cell
proliferation, can be elicited via stimulation of either the TCR-CD3 or
the CD2 receptor; signaling pathways involving the ZAP70/Syk kinase
family and the adapter protein LAT appear to be shared between these
cell surface receptors and may be involved in eliciting downstream
actions. CD2 has been implicated in a number of specific T-cell
functions, such as the regulation of the responsiveness of activated
human T cells to IL-1250 and interferon- |
Top
Abstract
Introduction
mAb OKT3 (American Type Culture Collection,
Rockville, MD) was used as purified ascites fluid; the
antiphosphotyrosine mAb 4G10 was a kind gift of Thomas Roberts
(Dana-Farber Cancer Institute); the SLP-76 antiserum was a kind gift of
Gary Koretzky (University of Pennsylvania, Philadelphia, PA). The
rabbit polyclonal anti-LAT antiserum was previously
described.
