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(Received for publication, February 8, 1996, and in revised form, August 16, 1996)
From the Bristol-Myers Squibb Pharmaceutical Research Institute,
Seattle, Washington 98121
The monoclonal antibody (mAb) J393 induces
apoptosis in Jurkat T-cells. NH2-terminal amino acid
sequence analysis identified the 140-kDa surface antigen for mAb J393
as CD43/leukosialin, the major sialoglycoprotein of leukocytes. While
Jurkat cells co-expressed two discrete cell-surface isoforms of CD43,
recognized by mAb J393 and mAb G10-2, respectively, only J393/CD43
signaled apoptosis. J393/CD43 was found to be hyposialylated, bearing
predominantly O-linked monosaccharide glycans, whereas
G10-2/CD43 bore complex sialylated tetra- and hexasaccharide chains.
Treatment with soluble, bivalent mAb J393 killed 25-50% of the cell
population, while concomitant engagement of either the CD3·TcR
complex or the integrins CD18 and CD29 significantly potentiated this
effect. Treatment of Jurkat cells with mAb J393 induced tyrosine
phosphorylation of specific protein substrates that underwent
hyperphosphorylation upon antigen receptor costimulation. Tyrosine
kinase inhibition by herbimycin A diminished J393/CD43-mediated
apoptosis, whereas inhibition of phosphotyrosine phosphatase
activity by bis(maltolato)oxovanadium-IV enhanced cell death.
Signal transduction through tyrosine kinase activation may lead to
altered gene expression, as J393/CD43 ligation prompted decreases in
the nuclear localization of the transcriptional regulatory protein
NF- In T-lymphocytes CD43/leukosialin, the major sialoglycoprotein of
leukocytes (1, 2), is thought to serve a dual role in regulating
cellular immune responses. Due to the repulsive effect of its high
numbers of O-linked negatively charged sialic acid sugar
residues, CD43 acts as a ``barrier molecule'' by limiting
cell-cell/cell-ligand interactions (3, 4, 5, 6, 7), a property that may
negatively regulate T-cell activation (8). In addition, engagement of
CD43 by monoclonal antibodies has been shown to induce costimulatory
activity in T-cells by a mechanism analogous to a classic
ligand-receptor interaction (9, 10, 11). Alterations in O-glycan
structure and function of CD43 reportedly occur in the immunodeficiency
disorders Wiskott-Aldrich syndrome (12, 13) and AIDS (14, 15) as well
as in graft versus host disease (16), acute lymphocytic
leukemia (17), and permanent mixed-field polyagglutinability or Tn
antigen syndrome (18).
The molecular structure of human CD43 is mucin-like, consisting of an
extended rod-shaped extracellular portion bearing approximately 80 sialylated O-glycan sites and a single N-glycan
site, a highly conserved transmembrane region, and a long cytoplasmic
domain bearing potential serine/threonine phosphorylation sites (19).
Based on the exon/intron arrangement within its gene, the observed
molecular heterogeneity of CD43 in both mice and man is thought to
reflect differential post-translational modifications of a single gene
product (20, 21, 22). Linear protein epitopes in the native structure of
CD43 have been shown to be modified by glycosylation (23), allowing for
the development of isoform-specific antibodies (9, 11, 24). Following
T-cell activation, the O-linked oligosaccharides of CD43
change from tetrasaccharides (mAb1 G10-2
reactive) to more complex hexasaccharides (mAb T-305 reactive) due to
the activation-induced expression of core 2 There is little information correlating heterogeneity in
oligosaccharide structure with ligand specificity or the signal
transducing properties of CD43. In the thymus, thymocyte-thymic
epithelial cell interactions correlate with the preferential binding of
galectin-1 to the mAb T-305/CD43 isoform expressed in immature,
cortical thymocytes (27). In mature T-cells the binding of anti-CD43
antibodies, thought to mimic natural ligands, results in
CD28-independent costimulatory activity (11, 28, 29, 30, 31). Interestingly,
the L10 antibody directed against a neuraminidase-resistant epitope of
CD43 is a strong inducer of T-cell proliferation, whereas the B1B6
antibody directed against a neuraminidase-sensitive epitope is only
weakly mitogenic (10). Immunoprecipitations from T-lymphoblastoid cell
lysates have found CD43 to be associated with CD3/TcR and
p56LCK protein tyrosine kinase (11), providing both
physical and functional evidence for a role for CD43 in signal
transduction. The anti-CD43 antibody, mAb MEM-59, which is
costimulatory in T-lymphocytes has recently been reported to induce
programmed cell death or apoptosis in hematopoietic progenitor cells
(32). Clearly, CD43-mediated responses can differ significantly
depending on the cell type and the isoform being expressed. While
aberrant isoforms of CD43 have been associated with immunodeficiencies
resulting in lymphopenia (33, 34), there has been no direct evidence
linking CD43-mediated apoptosis with T-cell depletion.
Here we report the biochemical and functional characterization of an
anti-CD43 mAb designated J393 that recognizes a unique, alternatively
glycosylated isoform of CD43 expressed on the surface of the human
T-lymphoblastoid cell line, Jurkat. Treatment of Jurkat cells with mAb
J393 induces apoptosis in a CD43 isoform-specific manner. The level of
apoptosis may be enhanced by concomitant engagement of the TcR or
integrin molecules. Moreover, an isoform of CD43 is detected in
peripheral blood T-lymphocytes bearing a cryptic epitope for mAb J393.
These results describe a potentially novel mechanism for T-cell lineage
depletion involving the regulated expression of specific isoforms of
CD43.
All cell lines were obtained from the
American Type Culture Collection (Rockville, MD) and maintained in RPMI
1640 medium (Life Technologies, Inc.) supplemented with 10% fetal
bovine serum (Hyclone, Logan, UT) and 100 units/ml penicillin, 100 mg/ml streptomycin unless otherwise stated. The BMS-2 subclone of
Jurkat cells and the HPB-F6 subclone of HPB-ALL cells were developed at
Bristol-Myers Squibb, and the anti-CD43 mAb G10-2 and mAb G19-1,
anti-CD3 mAb G19-4, and G19-4-sFv fusion protein (35) and anti-CD18
( Both the level of cell-surface expression of CD43 and
the level of apoptosis (cell death) were determined by flow cytometry
(FACS) using a FACStar fluorescence activated cell sorter (Becton
Dickinson, Mountain View, CA) controlled by the Cicero automated data
acquisition and analysis system (Cytomation, Fort Collins, CO). Data
were collected for 1 × 104 cells. Cells were
phenotyped by indirect immunofluorescence as described previously (36).
Apoptosis within a culture was determined by incubating cell samples
with 10 µg/ml propidium iodide (PI) for 10 min at room temperature
prior to analysis. Dead cells exhibiting size reduction and PI
fluorescence were electronically gated into quadrants 1-3 and counted
as a percentage of the total cell population. FACS analysis of DNA
fragmentation was carried out after incubating cell samples in lysis
buffer (0.03% sodium citrate, 0.01% Triton X-100) containing 50 µg/ml PI. Samples were analyzed by forward light scatter
versus fluorescence to generate a histogram representing the
size and relative amount of fragmented DNA.
The antigen for mAb
J393 was purified by affinity chromatography from detergent lysates of
a high-expresser subclone of the Jurkat cell line, BMS-2. The affinity
column was prepared by covalently attaching purified mAb J393 to
GammaBind Plus protein G-Sepharose (Pharmacia Biotech Inc.) using
maleimide as the cross-linking reagent. Non-covalently attached
antibody was removed by alternating rinses in pH 4.0 and pH 9.0 Tris
buffers. BMS-2 cell pellets were solubilized at 4 °C in Nonidet P-40
lysis buffer (1% Nonidet P-40, 140 mM NaCl, 20 mM Tris, 10 mM EDTA) containing protease
inhibitors (PMSF, leupeptin, soybean trypsin inhibitor, pepstatin, and
antipain) and the protein concentration adjusted to 2 mg/ml. The lysate
was clarified by centrifugation at 100,00 × g for 90 min and consecutively passaged through a 15-ml GammaBind Plus column, a
10-ml GammaBind Plus column with control mAb P3X attached, and a 2-ml
GammaBind Plus column with mAb J393 attached. The mAb J393 affinity
column was flushed with lysis buffer, pH 8.0, followed by another rinse
with lysis buffer containing 0.1% Nonidet P-40, 0.1% deoxycholate,
and 20 mM MOPS. Antigen was eluted using glycine buffer (50 mM glycine, 0.1% deoxycholate, pH 11.5) and the pH
adjusted to 8.0 with 2 M Tris. The antigen preparation was
dialyzed/concentrated by filtration using 10 mM Tris
buffer, pH 8.0, containing 0.1% deoxycholate prior to amino acid
sequence analysis.
The affinity-purified antigen for mAb
J393 was prepared for protein sequencing by subjecting the sample to
polyacrylamide gel electrophoresis using a SDS-Tricine buffer system
(Bio-Rad) on a minigel apparatus (80 × 80 × 0.5 mm) with a
10% acrylamide resolving gel and a 4% acrylamide stacking layer run
under reducing conditions. The separated proteins were then
electroblotted (37) onto polyvinylidene difluoride membrane
(Immobilon-P, Millipore Corp., Bedford, MA), and the location of the
mAb J393 antigen was confirmed by Western blotting. The Coomassie
Brilliant Blue stainable band of protein corresponding to the 140-kDa
mAb J393 antigen was subjected to NH2-terminal amino acid
sequence analysis (38). Automated sequence analysis was performed in a
pulsed-liquid protein sequencer (model 476A, Applied Biosystems, Inc.)
using manufacturer-released cycle programs as described previously
(39).
Cells were
metabolically labeled with [3H]glucosamine as described
previously (40). Briefly, cells were incubated in glucose-free RPMI
1640 medium supplemented with 10% dialyzed fetal bovine serum and 2%
standard medium. Cells were labeled for 24 h at 37 °C with 20 µCi/ml [3H]glucosamine (40 Ci/mmol; DuPont NEN). Cells
were then harvested and washed twice with PBS before disruption in
ice-cold lysis buffer (PBS containing 1% Nonidet P-40, 1 mM PMSF, and 1 mg/ml each of leupeptin and aprotinin). The
lysates were clarified by high speed centrifugation and the
supernatants collected. Radiolabeled CD43 was immunoprecipitated from
these supernatants with 10 µg/ml anti-CD43 mAb and the resulting
immune-complex recovered by binding to protein A-Sepharose.
O-Linked oligosaccharides were released from the
immunoprecipitates by Radiolabeled CD43 was prepared from
immunoprecipitates of [3H]glucosamine-labeled
T-lymphoblastoid cells as described above. Glycopeptides were generated
by digestion of labeled mAb J393 antigen with 5 mg/ml nonspecific
protease (Pronase) in 0.1 M Tris-HCl buffer containing 1 mM CaCl2, pH 8.0, for 24 h at 60 °C
under a toluene atmosphere. The digestion was terminated by boiling for
10 min. Epitope integrity of glycopeptides was evaluated by
immunoaffinity chromatography. Glycopeptides were suspended in 50 µl
of TBS buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5) and applied to a 1-ml mAb
J393-Sepharose column (0.3 × 14 cm) coupled with 6.3 mg of
antibody and equilibrated in TBS buffer at 4 °C. Glycopeptides have
been shown to optimally interact with carbohydrate binding proteins
coupled to Sepharose at this protein density and in the cold (43).
Fractions of 1 ml were collected at a flow rate of 1 ml/h with all
steps being carried out at 4 °C. After 5 ml of TBS buffer had been
applied and collected, bound material was eluted with 200 mM glycine buffer, pH 3.0. Intact, undigested antigen was
also examined under identical chromatographic conditions.
Anti-CD43 immunoblotting was performed on
either whole cell lysates or on immunoprecipitates from whole cell
lysates as described previously (44). Briefly, lysates were prepared by
solubilizing 1-10 × 107 cells in a 1-ml volume of
Nonidet P-40 lysis buffer. Immune complexes were recovered by mixing
GammaBind G-Sepharose (Pharmacia Biotech, Inc.) with lysate at 4 °C.
The immunoprecipitates were solubilized in Tris-glycine SDS sample
buffer containing 2 mM dithiothreitol and subjected to 6%
SDS-polyacrylamide gel electrophoresis fractionation. Proteins were
electrotransferred onto polyvinylidene difluoride membrane, and
antibody binding was detected by enhanced chemiluminescence (ECL,
Amersham Corp.) according to manufacturer's directions.
BMS-2 cells were
adjusted to 1 × 107 cells/ml in RPMI 1640 containing
10% fetal bovine serum held at 37 °C. Cells were aliquoted in 1-ml
volumes in Eppendorf tubes containing all test reagents and placed in a
37 °C heat block for the desired length of time. Reactions were
terminated by spinning the tubes for 30 s at 4 °C, decanting,
and solubilizing the cell pellets in 1% Nonidet P-40 containing
protease, and phosphatase inhibitors and immunoprecipitates were
analyzed for phosphotyrosine content by immunoblotting as described
previously (44).
Nuclear extracts
were prepared from approximately 1 × 107 cells using
a modification of the procedure of Dignam et al. (45).
Briefly, cells were lysed for 5 min at 4 °C in 10 mM
Hepes, 1.5 mM MgCl2, 10 mM NaCl,
0.25% Nonidet P-40, pH 7.5, followed by centrifugation and salt
extraction of nuclei in 20 mM Hepes, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.25% Nonidet P-40, pH 7.5, for 30 min at
4 °C. The nuclear extract was centrifuged at 14,000 rpm for 5 min,
and the supernatants were used for the electrophoretic gel mobility
shift assay. The electrophoretic gel mobility shift assay was
essentially performed according to the procedure of Sen and Baltimore
(46). Double-stranded oligonucleotide probes for the recognition
sequence of NF- BMS-2 cells and PHA-activated
peripheral blood T-cells were analyzed for expression and cellular
localization of the J393 antigen. BMS-2 cells were labeled with
PE-streptavidin/biotinylated mAb J393 and FITC-conjugated mAb
G10-2. Cells were washed, fixed on ice in 4% paraformaldehyde/PBS for
20 min, washed, and resuspended in PBS with 0.02% NaN3.
Peripheral blood T-cells were isolated from normal volunteers by
Ficoll-Hypaque density gradient centrifugation and sheep red blood cell
rosetting as described previously (48). Activated T-cells (>95%
CD3+) cultured in PHA for 72 h were fixed on ice in
4% paraformaldehyde/PBS for 20 min. The cells were washed in PBS and
permeabilized in 0.1% saponin/PBS. Samples were washed and incubated
for 30 min on ice with either mAb J393 or FLOPC-21 (IgG3
isotype-matched control) at 10 µg/ml followed by a similar incubation
with FITC-conjugated F(ab Mice immunized with the human T-lymphoblastoid cell line,
Jurkat, produced an antibody termed J393 that induced homotypic
adhesion and then death of these cells in culture. Within the first
hour of treatment Jurkat cells underwent pronounced homotypic adhesion,
forming large cellular aggregates. Over a period of 4-6 h a certain
proportion of the cell population began to die. Under the phase
contrast microscope, morphologic changes were observed that were
characteristic of the type of programmed cell death referred to as
apoptosis (membrane blebbing, cellular shrinkage, and nuclear
condensation). As an indicator of death, cells were monitored for the
uptake of the fluorescent compound propidium iodide (PI). Samples were
analyzed by flow cytometry as a function of size versus
fluorescence intensity to determine the level of killing and the degree
of nuclear damage. By comparison of Fig. 1A
with Fig. 1B, the percent of dead or dying cells detected by
PI permeability (quadrants 2 and 3) or decrease in forward light
scatter (quadrants 1 and 2) increased from 1.9 to 52.9% as a result of
mAb J393 treatment (sum of quadrants 1, 2, and 3), whereas the number
of PI-impermeable cells or viable cells decreased accordingly (quadrant
4). It should be noted that quadrants 1 and 2 represent a population of
cells with decreased forward light scattering properties, reflecting a
decrease in cell size (shrinkage). The level of killing of Jurkat cells
by mAb J393 was both concentration- and time-dependent,
reaching a maximum of 25-50% dead after a 24-h treatment with 5 µg/ml of antibody (data not shown).
Apoptosis may be associated with the breakdown of DNA as a result of
increased endonuclease activity. To further assess whether cell death
induced by mAb J393 was characteristic of apoptosis, PI was allowed to
intercalate with the DNA in Jurkat cell lysates and the nuclear
staining analyzed cytofluorimetrically (Fig. 1C). Treatment
with mAb J393 resulted in an increase in the population of hypodiploid
cells as measured by a 3-fold increase in fluorescence intensity below
that of intact G0/G1 chromatin (26% of the
total DNA in treated cultures versus 8% in the untreated
population). Since apoptosis has been regarded as fundamentally
important to homeostasis of the immune system, it was of interest to
identify the antigen for mAb J393.
The antigen for mAb J393 was isolated from detergent
lysates of Jurkat cells by affinity chromatography and purified as
described under ``Experimental Procedures.'' The 140-kDa protein was
then examined for its primary structure. NH2-terminal amino
acid sequence analysis of the mAb J393 antigen resulted in 13 of the
first 15 NH2-terminal amino acid positions to be assigned
(STTAVQTPT(X)GE(X)LV). This sequence was searched
in the Swiss-Prot data base for homology to known proteins. Excluding
the two unassigned positions, the protein sequence obtained for the mAb
J393 antigen was 100% homologous to the NH2-terminal
region of human CD43/leukosialin (19, 20). Since no intron structures
exist in the coding region of the human CD43 gene allowing for
alternative splicing of exons (21), we take this partial sequence
information to be adequate in identifying the mAb J393 antigen as
CD43.
Since several antibodies to
CD43 have been previously described, we questioned whether they were
functionally similar to mAb J393. Cultures of Jurkat cells were treated
with soluble forms of the anti-CD43 mAbs G10-2, G19-1, and J393 and
monitored for apoptosis as described under ``Experimental
Procedures.'' While all three anti-CD43 antibodies induced observable
homotypic adhesion, only mAb J393 induced apoptosis (data not shown).
We observed that cells not killed by mAb J393 were growth-arrested,
whereas cells treated with mAb G10-2 or mAbG19-1 continued to divide.
Jurkat cells were double-stained with the fluorescent conjugates
PE-J393 and FITC-G19-1 and examined using confocal microscopy. In Fig.
2, the two-color staining pattern that was observed
revealed that the epitopes reactive with these two antibodies resided
on distinct molecules that segregated independently in the plane of the
membrane. This indicated that single cells in the population were
co-expressing two distinct alternatively glycosylated isoforms of CD43;
however, only the J393/CD43 isoform was involved in signaling an
apoptotic response. Therefore, we looked for structural differences
between these isoforms.
Diversity
among human CD43 molecules has been ascribed to post-translational
modifications of a single gene product (20, 21, 22). Therefore, we examined
the carbohydrate structure of the mAb J393 antigen in comparison with
that of other mAb-specific isoforms of CD43. Analysis was limited to
O-linked sugars since CD43 is known to contain only one
potential N-linked glycosylation site and 80 potential
O-linked sites (19). Different isoforms of CD43 were
immunoprecipitated from cellular lysates of T-lymphoblastoid cells
using mAb J393, mAb T-305, and mAb G10-2, and the attached radiolabeled
carbohydrate was analyzed according to ``Experimental Procedures.''
As shown in Table I, 82% of the total
serine/threonine-linked carbohydrate of the J393/CD43 isoform in Jurkat
cells were of the GalNAc monosaccharide class and 12% were of the
Gal
Structures and relative amounts of the O-linked oligosaccharides found
on CD43
Volume 271, Number 44,
Issue of November 1, 1996
pp. 27686-27695
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
B and proteins binding the interferon-inducible regulatory
element. Since peripheral blood T-lymphocytes express cryptic epitopes
for mAb J393, these findings demonstrate the existence of a tightly
regulated CD43-mediated pathway for inducing apoptosis in human T-cell
lineages.
1
6
N-acetylglucosaminyltransferase (17, 25). This change in
oligosaccharide structure results in a shift from 115 to 140 kDa (9,
17, 25, 26).
Cells and Reagents
2-integrin) mAb 60.3 were kindly provided by Dr. Jeffrey Ledbetter
(Bristol-Myers Squibb PRI (BMSPRI), Seattle, WA). The anti-MHC class I
mAb W6/32 was obtained from the ATCC (Rockville, MD). The anti-CD28 mAb
2E12 was produced by Dr. Robert Mittler (BMSPRI). The anti-CD43 mAb
T-305 was a gift from Dr. Minoru Fukuda (La Jolla Cancer Research
Foundation, La Jolla, CA). Anti-CD49d (
4-integrin) mAb P4C2 and
anti-CD29 (
1-integrin) mAb P4C10 were provided as ascites
preparations by Dr. Paul Gladstone (BMSPRI). The phosphotyrosine
phosphatase inhibitor, bis-(maltolato)oxovanadium-IV (BMLOV), was
kindly provided by Dr. Gary Schieven (BMSPRI). Herbimycin A was
purchased from Life Technologies, Inc. The anti-CD95/Fas monoclonal
antibody (IgM isotype) was purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY), and the CD95/Fas neutralizing mAb ZB4 was purchased
from Immunotech Inc. (Westbrook, ME). All glycolytic enzymes except
Vibrio cholerae neuraminidase (Calbiochem) and
Clostridium perfringens neuraminidase
(Sigma) were purchased from Oxford GlycoSystems Inc.
(Rosedale, NY). Nonspecific protease XIV from Streptomyces
griseus (Pronase) was purchased from Sigma.
Protease inhibitors were obtained from Boehringer Mannheim.
FITC-conjugated goat anti-mouse IgG was purchased from TAGO, Inc.
(Burlingame, CA). PE- and FITC-conjugated streptavidin and propidium
iodide were purchased from Molecular Probes, Inc. (Eugene, OR).
-elimination as described previously (41),
desalted on a Sephadex G-10 column, dried, taken up in water, and
analyzed by high performance liquid chromatography on a column
(0.4 × 30 cm) of amino-bonded silica (AX-10, Varian). The mobile
phase contained a mixture of 15 mM
KH2PO4, pH 4.5, and acetonitrile. One-ml
fractions were collected at a flow rate of 1 ml/min over a linear
gradient of decreasing acetonitrile concentration (80-50%), and
aliquots were sampled for radioactivity by liquid scintillation
counting. CD43 oligosaccharides were obtained from HL60 and K562 cells
as described previously (42) and used as standards.
B (5
-GATCCGAGGGGACTTTCCGCTGGGGACTTTCCAGG-3
),
octamer (5
-TGTCGAATGCAAATCACTAGAA-3
), and AP-1
(5
-CGCTTGATGAGTCAGCCATG AA-3
) were obtained from Promega, Madison,
WI, and IRE (5
-AAGTACTTT CAG TTTCATATTACTCTA-3
) from Santa Cruz
Biotechnology, Santa Cruz, CA, and radiolabeled at the 5
-end as
recommended by the manufacturer. Equal amounts of nuclear extract
protein (3 µg) were incubated with 32P-labeled
oligonucleotide probes and analyzed on a native 6% polyacrylamide gel.
Gels were dried and radioactivity quantitated by autoradiography
(47).
)2 goat anti-mouse IgG. Finally,
the cells were washed and resuspended in culture medium prior to
analysis using the Bio-Rad MRC 1024 confocal microscope.
mAb J393-induced Apoptosis in Cultured T-lymphoblastoid
Cells
Fig. 1.
Apoptosis in T-lymphoblastoid cells is
induced by mAb J393. Actively growing Jurkat cells (1 × 106/ml) were treated for 24 h (A) without
or (B) with 5 µg/ml mAb J393 followed by cytofluorimetric
analysis of propidium iodide (PI) uptake as described under
``Experimental Procedures.'' The percent of total population falling
within each quadrant is given. A decrease in cells within quadrant 4 indicates an increase in cell death. C, cells treated with
(hatched) or without (solid) 5 µg/ml mAb J393
for 24 h were exposed to Triton X-100 lysis buffer containing PI,
and the nuclei were cytofluorimetrically analyzed as described under
``Experimental Procedures.'' Fluorescence intensity below that of
intact G0/G1 chromatin indicates the presence
of hypodiploid cells in the original cell population.
[View Larger Version of this Image (29K GIF file)]
Fig. 2.
T-lymphoblastoid cells co-express
antigenically distinct isoforms of CD43. Actively growing Jurkat
cells were immunostained with phycoerythrin-labeled mAb J393
(red) and FITC-conjugated mAb G19-1 (green) and
analyzed for two-color fluorescence (red/green) by confocal
microscopy as described under ``Experimental Procedures.'' Both red
and green fluorescence appear to be differentially distributed at the
cell surface. Yellow fluorescence depicts regions of
colocalization.
[View Larger Version of this Image (86K GIF file)]
1-3GalNAc disaccharide class. The remaining 6% of the
O-linked sugars of the J393/CD43 molecule contained sialic
acid and were of the NeuNAc
2-3Gal
1-3GalNAc trisaccharide class.
By comparison, 80% of the O-linked sugars attached to the
G10-2/CD43 isoform in HPB-ALL cells were made up of sialylated tri-,
tetra-, and hexasaccharides with the remaining 20% being predominantly
monosaccharides. The T-305/CD43 isoform in HPB-ALL cells displayed an
oligosaccharide structure similar to that of the G10-2/CD43 isoform;
however, it contained less tetrasaccharide and more hexasaccharide
moieties consistent with previous reports (17).
CD43 oligosaccharides
mAb J393a
mAb
G10-2b
mAb T-305b
%
GalNAcOH
82
15
19
Gal
1-3GalNAcOH12
5
0
NeuNAc
2-3Gal
1-3GalNAcOH6
0
10
NeuNAc
2
6
NeuNAc
2-3Gal
1-3GalNAcOH0
30
11
NeuNAc
2-3Gal
1-4GlcNAc
1
6
NeuNAc
2-3Gal
1-3GalNAcOH0
50
60
a
Immunoprecipitated from Jurkat cells (mAb G10-2 and
mAb T-305 did not immunoprecipitate in NP-40 lysis buffer).
b
Immunoprecipitated from HPB-ALL cells (mAb J393 did not
immunoprecipitate in NP-40 lysis buffer).
Antibodies for CD43 have been characterized as to their
epitope requirement for terminal sialic acid sugar residues. The
anti-CD43 mAb T-305 has been described as being sialic
acid-independent, since the epitope is resistant to digestion by
neuraminidase (9), while the immunoreactivities of several other
anti-CD43 antibodies have been shown to be sensitive to neuraminidase
treatment (10, 11). The histograms presented in Fig. 3
were generated by immunostaining Jurkat and HPB-ALL cells with mAb
G10-2 or mAb J393 before and after V. cholerae neuraminidase
treatment. The mAb G10-2 demonstrated an absolute requirement for
sialic acid residues in maintaining immunoreactivity as shown in Fig.
3A. Mean channel fluorescence decreased from a value of 245 to a value of 11 as a result of neuraminidase treatment. By contrast,
the presence of sialic acid was not required for antigen recognition by
mAb J393 (Fig. 3, B and C). Indeed, the
immunoreactivity of mAb J393 increased following neuraminidase
treatment, which was most notable in HPB-ALL cells. Enzymatic digestion
by neuraminidases from either C. perfringens or A. ureafaciens sources gave similar results (data not shown).
) 0.05 units/ml Vibrio cholerae neuraminidase as described under
``Experimental Procedures.'' Changes in fluorescence intensity as
compared with isotype-matched control antibodies (· · ·) were
detected by flow cytometry. A, HPB-ALL cells immunostained
with mAb G10-2. B, HPB-ALL cells immunostained with mAb
J393. C, Jurkat cells immunostained with mAb J393.
Protease-sensitive Property of the mAb J393 Epitope
Jurkat
cells are known to possess a deficiency in
1,3-galactosyltransferase
activity (49) resulting in the expression of an alternatively
glycosylated isoform of CD43 bearing the GalNAc-Ser/Thr cluster
structure of Tn antigen (50). The carbohydrate analysis of the mAb J393
antigen indicated structural similarity with this Tn antigen-containing
isoform of CD43; however, we were unable to demonstrate immunostaining
by mAb J393 of Tn antigen-positive CEM cells and certain Tn-expressing
human carcinoma cell lines (data not presented). This result suggested
that the epitope for mAb J393 differed from that described for other Tn
antibodies. In order to characterize the nature of the epitopic
structure recognized by mAb J393, [3H]glucosamine-labeled
J393 antigen was proteolytically digested into glycopeptides and tested
for reactivity on a mAb J393-Sepharose affinity column under conditions
that promote the retention of glycopeptides (see ``Experimental
Procedures''). Many anti-carbohydrate antibodies bind to carbohydrate
antigens under these experimental conditions (51, 52). As demonstrated
in Fig. 4, 87% of the radioactivity associated with
intact CD43 required elution at acidic pH, indicating a strong
antigen-antibody interaction with the column substrate. By contrast,
93% of the radioactivity associated with CD43 glycopeptides eluted at
neutral pH, indicating very weak interaction with the column substrate
and loss of antigenic valency. A minor proportion (7%) of CD43 digest
that eluted under acid conditions may represent larger glycoprotein
fragments resulting from incomplete proteolysis. Intact
[3H]glucosamine-labeled mAb G10-2 antigen isolated from
the HPB-ALL cell line failed to interact with the mAb J393 affinity
substrate, demonstrating the specificity for antigen (data not
presented). These results suggest that specific protein and
carbohydrate domains of CD43 are important for high-avidity recognition
of epitope by mAb J393.
) or without (
) nonspecific protease as described under
``Experimental Procedures.'' Glycopeptides generated by proteolysis
eluted from the mAb J393-Sepharose immunoaffinity column at pH 7.5, predominantly in fractions 1-5 and prior to applying an isocratic
shift to pH 3.0. Under identical conditions, undigested antigen was
retained at neutral pH and eluted in fractions 5-10 following the
isocratic shift to pH 3.0. Chromatographies were carried out at 4 °C
to enhance retention of low valency glycopeptide.
Cell-surface Interactions That Enhance J393/CD43-Mediated Apoptosis
It has previously been shown that certain isoforms of
CD43 can function as accessory molecules in CD3/TcR-stimulated T-cell
activation (11, 28, 29, 30, 31) and may also be involved in cell-cell
interactions by binding to the integrin family adhesion molecule ICAM-1
(4). Therefore, we investigated the effect of ligating additional
surface molecules on the ability of mAb J393 to induce apoptosis. As
represented in Fig. 5, concomitant treatment of Jurkat
cells with mAb J393 plus mAbs specific for either CD3, CD43, CD18
(
2-integrin), or CD29 (
1-integrin) increased the level of
apoptosis from 42% to as much as 65% after 24 h. Costimulation
with either anti-MHC class I, anti-CD28, or anti-CD49d (
4-integrin)
reduced the level of apoptosis induced by mAb J393, demonstrating that
only certain surface molecules interact to potentiate J393/CD43
signaling events. A synergistic enhancement in cell killing was
observed at suboptimal concentrations of mAb J393 in the presence of
anti-CD43 mAb G10-2 (Fig. 6A) or anti-CD3 mAb
G19-4 (Fig. 6B). Apoptosis was enhanced over 2-fold by the
simultaneous ligation of both isoforms of CD43 expressed in Jurkat
cells. However, the greatest killing was observed by the simultaneous
ligation of J393/CD43 and CD3/TcR. In these experiments 10% of the
cell population were killed with 0.9 µg/ml mAb J393; however, in the
presence of soluble anti-CD3 mAb this same concentration of mAb J393
induced 60% killing, a 6-fold increase in the level of apoptosis. To
address whether the costimulation responsible for the increase in
apoptosis was due to the activation-induced production of the ligand
for CD95/Fas (53), Jurkat cells were costimulated with mAb J393 and mAb
G19-4 (anti-CD3) in the presence of antibodies that block
CD95/Fas-induced apoptosis or in the presence of soluble CD95/Fas-Ig
fusion protein. Neither CD95/Fas blocking antibodies nor CD95/Fas-Ig
affected the synergistic killing observed with costimulation of
CD43/CD3 (data not shown).
2-integrin
(60.3), anti-CD29/
1-integrin (P4C10), anti-CD3 (G19-4),
anti-CD49d/
4-integrin (P4C2), anti-CD28 (2E12), and anti-MHC class I
(W6/32). Under these conditions none of the antibodies tested, with the
exception of mAb J393, were capable of inducing apoptosis as soluble,
single reagents (data not shown).
) or presence of 10 µg/ml anti-CD43/mAb G10-2 (A) (
) or 10 µg/ml
anti-CD3/mAb G19-4 (B) (
) to induce apoptosis. The
percent of cell death was determined by flow cytometry as described
under ``Experimental Procedures.'' Under these conditions mAb G10-2
and mAb G19-4 induced cell death <5% when used as soluble, single
reagents (data not shown).
mAb J393/CD43-Induced Protein Tyrosine Phosphorylation
Protein tyrosine phosphorylation plays an
essential role in the signal transduction pathways of several T-cell
surface molecules including the CD3/TcR, CD4, CD8, and CD28 (54).
Although CD43 does not possess tyrosine kinase activity or contain
tyrosine phosphorylation sites within the cytoplasmic domain (20), its
association with other membrane receptor-tyrosine kinase complexes such
as CD3/TcR have been reported (11). Therefore, we examined early
tyrosine phosphorylation following mAb J393 or mAb J393 plus anti-CD3
stimulation of Jurkat cells. Cells were treated with mAb J393 in the
presence or absence of the anti-CD3 single-chain variable fragment,
G19-4-sFv (35). Following costimulation, anti-phosphotyrosine
immunoprecipitates were analyzed by immunoblotting with
anti-phosphotyrosine antibodies. The immunoblot presented in Fig.
7 shows that tyrosine phosphorylation was induced
rapidly within 1 min. The pattern of phosphorylation observed depended
on the type of stimulus. The most significant increase in
tyrosine-phosphorylated proteins induced by mAb J393 was found in the
range of 90-kDa and below; in particular those in the range 50 to
34-kDa. By comparison, G19-4-sFv-induced tyrosine phosphorylation of
proteins above 100-kDa as well as those below 90-kDa but did not induce
phosphorylation of proteins in the range of 50-55-kDa range.
Costimulation with both antibodies resulted in the hyperphosphorylation
of multiple substrates in the range of 150 to 34-kDa range. In
contrast, neither G10-2 nor G19-1 induced an increase in protein
tyrosine phosphorylation (data not shown).
Regulation of J393/CD43-induced Apoptosis by Phosphotyrosine Kinase/Phosphatase Inhibitors
It was apparent that mAb
J393-induced tyrosine phosphorylation peaked at 5 min and rapidly
diminished thereafter. Since the dephosphorylation of specific
substrates might be involved in down-regulating CD43-induced signals,
we examined the effect of tyrosine phosphatase inhibition on mAb
J393-induced apoptosis. Jurkat cells pretreated overnight with the
phosphotyrosine phosphatase (PTPase) inhibitor, BMLOV (55), were found
to be more responsive to the induction of apoptosis by mAb J393. As
shown in Fig. 8A, BMLOV was not toxic to
Jurkat cells; however, it enhanced mAb J393-induced killing in a
dose-dependent manner. The level of cell death increased
from 32 to 65% as a result of treatment with 45 µM
BMLOV. Inhibition of PTPase activity by BMLOV had less effect on
anti-CD3-induced apoptosis, whether used alone or in combination with
mAb J393. The effect of inhibition of tyrosine kinase activity on
apoptosis was also examined. In Fig. 8B, the level of
apoptosis induced by mAb J393 was reduced from 35 to 16% in the
presence of the tyrosine kinase inhibitor herbimycin A. Furthermore,
apoptosis induced by treating Jurkat cells with mAb J393 plus anti-CD3
was inhibited 95% by herbimycin A. These results strongly suggested a
role for protein tyrosine phosphorylation/dephosphorylation events in
regulating CD43-mediated signals leading to an apoptotic response.
- -
) or in combination with mAb G19-4
(
--
) had minimal effect on apoptosis; however, BMLOV acted in a
dose-dependent manner to enhance cell death induced by mAb
J393 (
-
) or mAb J393 + mAb G19-4 (
--
). B, Jurkat
cells were treated for 24 h with 5 µg/ml mAb J393 and 10 µg/ml
G19-4 in the presence of 3 µM of the protein tyrosine
kinase inhibitor herbimycin A following a 6-h pretreatment with the
inhibitor. Herbimycin A treatment alone had little effect on apoptosis;
however, herbimycin A acted to decrease the level of cell death induced
by mAb J393 or mAb J393 + mAb G19-4.
mAb J393-induced Alterations in the Nuclear Localization of Transcriptional Regulatory Proteins
In order to determine whether
mAb J393-induced signal transduction events might lead to downstream
alterations in gene activation, we examined the effects of anti-CD43
mAb J393, anti-CD3 mAb G19-4, and a combination of both antibodies on
the nuclear localization of the transcriptional factors NF-
B, AP-1
(fos/jun), octamer, and interferon-inducible regulatory element (IRE)
DNA-binding proteins. Jurkat cells (1 × 107) were
aliquoted in 1-ml volumes containing either mAb G19-4 (anti-CD3), mAb
J393 (anti-CD43), each at 10 µg/ml, a combination of both, or left
untreated. The cells were then incubated at 37 °C for 1 h and
pelleted by centrifugation, and nuclear extracts were prepared for
analysis as described under ``Experimental Procedures.'' As shown in
Fig. 9, BMS-2 cells had constitutively elevated levels
of the four transcription factors within the nucleus. Treatment of the
cells with mAb J393 had a marked effect on reducing the nuclear levels
of both NF-
B and IRE binding proteins but not AP-1 or Octamer.
Treatment of the cells with mAb G19-4 had little effect on nuclear
levels of these factors, whereas combined antibody treatment inhibited
nuclear localization the greatest degree.
B and IRE
transcription factors. Actively growing Jurkat cells were treated
with 5 µg/ml anti-CD43/mAb J393, 10 µg/ml antiCD3/mAb G19-4, or a
combination of the two antibodies for 1 h at 37 °C. These cells
were used in the preparation of nuclear extracts for the detection of
specific DNA-binding proteins by electrophoretic gel mobility shift
assay as described under ``Experimental Procedures.'' The above
phosphorimages of nondenaturing polyacrylamide gel electrophoresis show
the high Mr shift in radiolabel due to binding
of specific 32P-labeled oligonucleotides to nuclear
proteins. Treatment by mAb J393 alone or in combination with mAb G19-4
resulted in a decrease in the amount of NF-
B and IRE in the nucleus
but did not affect AP-1 or Oct levels.
Expression of mAb J393 Epitopes in T-lymphocytes
Using flow
cytometry we examined several different cell types of hematopoietic
origin and found the expression of J393/CD43 to be highly restricted
(data not shown). All lineages known to express CD43 were reactive with
mAb G10-2. By contrast, mAb J393 failed to react with human
erythrocytes, platelets, neutrophils, eosinophils, monocytes, T- and
B-lymphocytes, freshly isolated CD34+ bone marrow-derived
precursor cells, and freshly prepared thymocytes. However, immunoblot
analysis of whole cell lysates revealed the presence of mAb J393
epitopes in both resting and activated peripheral blood T-lymphocytes.
As shown in Fig. 10, resting T-cells predominantly
expressed the 120-kDa mAb G10-2-reactive isoform of CD43 that appeared
to decrease following cellular activation. By comparison, resting
T-cells expressed a very low level of mAb J393 reactivity that was
elevated following activation. The 140-kDa isoform observed in Jurkat
cells was not detected in either resting or activated T-lymphocytes;
however, a higher 160-kDa band of reactivity specific for mAb J393 was
present in activated cells. It should be noted that in activated
T-cells mAb J393 and mAb G10-2 cross-reacted with isoforms of CD43 that
were of 97 kDa or smaller.
As a further demonstration of mAb J393 reactivity with human
T-lymphocytes, PHA-activated peripheral T-cells were fixed,
permeabilized, and immunostained as described under ``Experimental
Procedures.'' Examination of this preparation by confocal microscopy
revealed localization of mAb J393 antigen at or near the plasma
membrane (Fig. 11).
The existence of a CD43-mediated pathway for signaling apoptosis in T-cell lineages has not been previously described. Our observation that the BMS-2 Jurkat cell line co-expressed two antigenically distinct isoforms of CD43 on its surface provided us with a good model for examining structure-function relationships between these two molecules. While both mAb J393 and mAb G10-2 induced homotypic adhesion, only mAb J393 induced apoptosis. This implied that the apoptotic response was independent of the homotypic adhesion phenomenon and was associated with a specific CD43 isoform. The reactivity of mAb G10-2 required terminal sialic acid moieties, whereas reactivity of mAb J393 was sialic acid-independent. Other investigators have observed sialic acid-independent CD43 antibodies to elicit stronger cellular responses than their sialic acid-dependent counterparts (10). We found the carbohydrate structure of J393/CD43 in Jurkat cells to be deficient in oligosaccharide complexity and sialic acid content when compared with that of the G10-2/CD43 isoform expressed in HPB-ALL cells. The majority of serine/threonine residues in the extracellular portion of the J393 antigen contained only terminal GalNAc monosaccharides, similar to the Tn antigen-bearing CD43 molecule expressed in Jurkat cells (50). Glycopeptides generated from intact mAb J393 antigen lost functional epitope as determined by immunoaffinity chromatography run under conditions that favor interaction of low valency glycopeptides with the antibody. Low valency for antibodies has been reported for glycopeptides containing the cluster antigens of Tn when compared with intact glycoprotein (56). Although mAb J393 likely recognizes the Tn-containing isoform of CD43, it does not react with the surface of certain cell types expressing Tn epitopes. Therefore, we propose that mAb J393 recognizes a unique epitope on CD43 that has not been previously characterized.
Little is known about how CD43 functions as a signaling molecule. We find that soluble, bivalent antibody is sufficient to initiate a CD43 signal, unlike the requirement for trimeric ligation by members of the TNF receptor superfamily including CD95/Fas (57). In T-lymphocytes, CD43 has been shown to be constitutively phosphorylated in resting cells and hyperphosphorylated following cellular activation (58). Because CD43 contains no catalytic region or tyrosine residues within the cytoplasmic domain, its phosphorylation is thought to reflect its association with serine/threonine-specific protein kinases (59, 60, 61). However, investigators characterizing CD43 as a CD28-independent costimulatory molecule have described the physical association of CD43 with CD3 in a complex containing the SRC family protein tyrosine kinases lck and fyn (11), suggesting an involvement of CD43 with tyrosine phosphorylation events. Indeed, pretreatment of Jurkat cells with the tyrosine kinase inhibitor herbimycin A significantly interfered with mAb J393-induced cell death. We found that treatment of Jurkat cells with mAb J393 induced a rapid increase in overall protein tyrosine phosphorylation and, in particular, for proteins in the 50-55-kDa range. Interestingly, this group of proteins was not phosphorylated following engagement of CD3/TcR, suggesting a degree of specificity in the pattern of CD43-induced tyrosine phosphorylation. Moreover, concomitant ligation of J393/CD43 and CD3/TcR resulted in the hyperphosphorylation of these CD43-dependent substrates, providing a biochemical correlate for the synergy observed for these two receptor molecules in mediating apoptosis. Collectively, these findings suggest that protein tyrosine phosphorylation of specific substrates is important in signaling CD43-mediated apoptosis.
We found that preventing dephosphorylation of phosphotyrosyl residues by blocking phosphatase activity resulted in a significant enhancement in mAb J393-induced cell death. Thus, the PTPase inhibitor BMLOV mimicked the action of CD3/TcR in potentiating CD43-mediated cell death, seemingly consistent with a hyperphosphorylated state. These results indicate that the catalytic activity of BMLOV-sensitive phosphatases negatively regulates CD43-mediated apoptosis. It is likely that the potentiation of CD43-mediated apoptosis by CD3/TcR engagement involves CD45 phosphatase activity since CD45 is expressed in high abundance in Jurkat cells and is required to activate TcR-associated SRC family kinases allowing for competent antigen-induced signal transduction (62, 63). Therefore, in this context CD45 may be positively regulating CD43-mediated apoptosis by directly enhancing kinase activity. Notably, BMLOV treatment of Jurkat cells does not interfere with TcR-induced tyrosine phosphorylation (55) which may explain why BMLOV treatment did not block the synergism we observed for TcR/CD43-induced apoptosis. Conversely, the catalytic activity of SH2 PTPases may be expected to exert a negative regulatory effect on CD43-mediated apoptosis, since PTP1C has been shown to negatively regulate antigen receptor signaling in B-lymphocytes as confirmed in PTP1C-deficient mice expressing the motheaten phenotype (64). Further investigation is necessary to identify which enzymes and substrate sites are involved in this CD43-mediated pathway.
It was recently reported that CD3/TcR-induced apoptosis in T-cells is
mediated by the autocrine production of the ligand for CD95/Fas (FasL),
thereby activating the CD95/Fas receptor signaling pathway (65).
Interestingly, a requirement for tyrosine kinase activation in
CD95/Fas-mediated programmed cell death has been described in
conjunction with a requirement for ceramide-initiated RAS activation
(66, 67); however, these signals alone were not sufficient for
subsequent apoptosis. We found that neither a blocking antibody to
CD95/Fas nor the competing fusion protein CD95/Fas-Ig prevented
J393/CD43-induced apoptosis, making it unlikely that FasL was mediating
this response. Moreover, we observed that combined engagement of
J393/CD43-CD3/TcR leads to a rapid reduction in the nuclear
localization of NF-
B and IRE regulatory proteins normally associated
with transcriptional activation of the FasL gene (68). Likewise, since
sphingomyelinase-dependent NF-
B activation has been
reported to lead to apoptosis signaled by the TNF receptor-1 ``death
domain'' (69), it is unlikely that TNF receptor-1 is involved in
J393/CD43-mediated killing. Therefore, engagement of CD43 may define a
distinct signaling pathway for programmed cell death that differs from
that of the TNF-nerve growth factor receptor family. Perhaps in the
BMS-2 Jurkat cells, constitutive activation and nuclear localization of
NF-
B and IRE DNA-binding proteins are critical for continued
proliferation; hence, a decrease in nuclear localization of these
transcriptional factors may lead to molecular events that promote
growth arrest and eventual suicide. Recent findings in CD43-deficient
T-cells generated by homologous recombination indicate that CD43 acts
as a negative regulator of T-cell activation and proliferation (8),
which may reflect our observation that CD43 signaling can suppress the
activity of transcriptional regulators of early T-cell activation
genes.
While mAb J393 reacted strongly with Jurkat cell surfaces, it failed to
immunostain the surface of either resting or activated peripheral blood
T-lymphocytes. However, immunoblot analysis revealed the presence of
several mAb J393-reactive proteins in the lysates of activated T-cells,
possibly representing intracellular intermediates of CD43 in the
process of O-glycan elongation. Interestingly, in activated
T-cells both mAb J393 and mAb G10-2 cross-reacted with a 97-kDa isoform
of CD43. Investigators have previously shown that isoform-specific
antibodies may cross-react with linear protein epitopes on the
deglycosylated molecule and that these epitopes may be modified by
glycosylation in conferring antigenic specificity (23). This suggested
that the antigenic specificity for these two antibodies developed as a
consequence of activation-induced changes in the glycosylation of the
core protein. Furthermore, the mAb J393-specific 140-kDa isoform
present in Jurkat cells was replaced with a 160-kDa isoform in
activated T-cells. Presumably, the J393/CD43 intermediate in
T-lymphocytes contains less sialic acid than its counterpart in Jurkat
cells, thus contributing to its reduced electrophoretic mobility (14).
Unlike T-cells from normal individuals, Jurkat cells have been shown to
be deficient in
1,3-galactosyltransferase activity, a key enzyme in
the formation of complex O-glycan structures (49). Thus, we
postulate that the epitope for mAb J393 is tightly regulated in T-cell
lineages, normally being masked at the cell surface by the presence of
complex carbohydrate chains attached to the CD43 protein.
Our finding that apoptosis can be induced through a CD43-mediated pathway may have physiologic significance in peripheral lymphoid tissues such as the thymus and lymph nodes, since the inhibition of O-glycan elongation in T-cells potentiates the apoptotic effect of galectin-1, an endogenous ligand for CD43 expressed at these sites (70). Concomitant engagement of specific integrin molecules may serve to modulate CD43induced responses as our data suggest, in addition to the specific regulation of integrin expression mediated by CD43 as observed by others (71). We propose that the truncated O-glycan structure of the J393/CD43 molecule represents a molecular phenotype with an altered affinity or specificity for natural ligands involved in cell-cell interactions and that following its ligation an apoptotic response may be triggered in appropriate cell types.
As mentioned previously, abnormalities in O-glycan biosynthesis are found in a variety of pathologic conditions involving hematopoietic and immunologic disorders, often correlating with the appearance of Tn antigen and autoantibodies to cell-surface molecules including CD43 (14, 18, 72). These findings suggest that the expression of alternatively glycosylated isoforms of CD43 may contribute to the progression of such diseases by promoting T-cell deficiency and lymphopenia as a consequence of programmed cell death.
To whom correspondence should be addressed: 3005 First Ave.,
Seattle, WA 98121. Tel.: 206-727-3541; Fax: 206-727-3600.
B, nuclear factor kappa
B; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; MOPS,
3-[N-morpholino]propanesulfonic acid; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PTPase,
phosphotyrosine phosphatase; IRE, interferon-inducible regulatory
element.
We express our appreciation to Drs. Jeffrey Ledbetter, Gary Schieven, Paul Gladstone, Jacques Garrigues, and Irv Bernstein (Fred Hutchinson Cancer Research Center, Seattle, WA) for helpful discussions and reagents, Patti Moran-Davis and Alison Wallace for expert technical assistance, and Teresa Nelson for assistance in the preparation of this manuscript.