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Human Immunology 73 (2012) 448–455
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MUC1 is a novel costimulatory molecule of human T cells and functions in an
AP-1-dependent manner
Jeffrey D. Konowalchuk, Babita Agrawal*
Department of Surgery, Faculty of Medicine, Dentistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
A R T I C L E
I N F O
Article history:
Received 13 October 2011
Accepted 27 February 2012
Available online 6 March 2012
Keywords:
T cells
Costimulatory molecules
MUC1 mucin
A B S T R A C T
MUC1 mucin, primarily known as an epithelial antigen, has been demonstrated to be expressed on activated
human T cells. In the present study, we ﬁrst examined the expression of MUC1 on different subsets of T cells
(naive, effector, effector/memory). MUC1 appears to be strongly upregulated on activated CD4⫹ T cells in
comparison with CD8⫹ T cells. The cytoplasmic tail of MUC1 contains both immune tyrosine– based activation
and inhibitory motifs; therefore, we investigated whether MUC1 can also act as a costimulatory molecule on
human T cells. Nonpuriﬁed T-cell cultures from human peripheral blood exhibited enhanced proliferation and an
increase in cytokine production when CD3 and MUC1 were cross-linked and coligated. The intracellular mechanism of MUC1-mediated costimulation was determined to be mediated by the calcium-dependent NF-AT pathway. We further demonstrated that the cytoplasmic tail of MUC1 binds to the AP-1 transcription factors c-Fos and
c-Jun, with c-Fos binding constitutively and c-Jun binding only after MUC1 stimulation. Their nuclear migration is
then facilitated in a CD3-dependent manner. Our ﬁndings clearly demonstrate that MUC1 is a novel T-cell
costimulatory molecule involved in immune regulation. These studies delineate important mechanisms of T-cell
activation and regulation.
䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights
reserved.
1. Introduction
Mucin-1 (MUC1) is a large, ⬎200-kDa transmembrane glycoprotein expressed on the surface of most types of epithelial cells [1].
Its extracellular domain consists of a variable number of 20 amino
acid tandem repeats that are heavily glycosylated with o-linked
oligosaccharides, whereas its cytoplasmic domain contains many
signaling motifs [2] and is noncovalently linked to the extracellular
domain [3]. Recent studies have suggested that it migrates to the
nucleus upon extracellular ligation in epithelial cells, acting as a
shuttle protein for transcription factors such as ␤-catenin [4], and
can also generate both stimulatory and inhibitory responses [5].
MUC1, although most well characterized for its role on epithelial and tumor cells [6,7], has been demonstrated to also be expressed on activated T cells [8], dendritic cells [9], and monocytes
[10], whereas noncancerous B cells have been reported to have
extremely low expression [11–13] and natural killer cells [13] do
not express it at all. Previous assays performed on puriﬁed T cells
with anti-CD3 and anti-MUC1 antibodies have demonstrated that
MUC1 may act as an inhibitory protein because cross-linking severely inhibited the proliferation of T cells normally caused by the
anti-CD3 stimulus alone [8,14]. Further study has attributed this to
a reduced number of antigen-presenting cells cocultured with the T
cells [5].
* Corresponding author.
E-mail address: bagrawal@ualberta.ca (B. Agrawal).
Analysis of MUC1’s cytoplasmic domain has resulted in 2 putative sequences of interest being identiﬁed—1 resembling an immunotyrosine inhibitory motif and 1 resembling an immunotyrosine
activation motif (ITAM) [2], giving MUC1 a potential dual role in
immune stimulation/inhibition. Coimmunoprecipitation has also
revealed the binding of signal cascade proteins normally associated
with stimulation, including Lck [15], Grb-2 [16], and ZAP-70 [15]. In
lymphoma models, transfecting Jurkat T cells with a chimeric CD4
extracellular domain/MUC1 cytoplasmic protein, ERK1/2 can also
bind the cytoplasmic tail [17], potentially progressing the cell cycle
through phosphorylation of transcription factors [18].
In the original studies [8], it was demonstrated that MUC1 was
only expressed on a fraction of activated T cells at a given time.
Once matured with mitogens, MUC1 was determined to have coinhibitory capabilities in highly puriﬁed T-cell cultures [5,8,14], although whether this affected all T cells or a speciﬁc subset was
never elucidated. With this knowledge, along with the presence of
the ITAM motif in the cytoplasmic tail, we sought to investigate
whether MUC1 can act as a costimulatory molecule on T cells in
addition to being coinhibitory.
2. Subjects and methods
2.1. Isolation of nonadherent cells from human blood
Blood samples were obtained from individuals of both sexes
who were 30 – 60 years of age and had provided informed consent.
The use of human blood samples was approved by the institutional
0198-8859/12/$36.00 - see front matter 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.humimm.2012.02.024
J.D. Konowalchuk and B. Agrawal / Human Immunology 73 (2012) 448–455
health research ethics board at the University of Alberta, Canada.
The blood was layered on lymphocyte separation medium (Cellgro,
Herndon, VA) and centrifuged at 1,500 –2,000g for 30 minutes at
room temperature. The intermediate buffy layer containing the
peripheral blood mononuclear cells was removed, washed twice in
phosphate-buffered saline (PBS), and resuspended in RPMI 1640
medium with 0.2% penicillin–streptomycin, 0.2% sodium pyruvate
(Invitrogen, Carlsbad, CA), and 1% human AB serum (Sigma, St.
Louis, MO). Cells were plated at 3 ⫻ 107 cells/well and placed in an
incubator for 2 hours at 37oC and 5% CO2 (hereby just 37oC). The
nonadherent T, B, and natural killer cells (consisting of approximately ⬎60% T cells, based on ﬂow cytometric analysis [data not
shown]; hereby termed T cells) were collected and resuspended in
AIM-V medium (Invitrogen). T cells were stimulated with 1 ␮g/mL
of phytohemaglutinin A (PHA; Sigma) and incubated at 37oC for 3
days to induce optimal MUC1 expression. Cells used in all experiments were washed twice with PBS after PHA stimulation to remove the remaining PHA.
2.2. Flow cytometry
Nonadherent T cells were seeded at approximately 1,000,000/
tube, resuspended into cold FACS buffer (PBS ⫹ 2% fetal bovine
serum), and kept at 4oC for the remainder of the experiment. Cells
were stained with ﬂuorescent antibodies against CD4-QR, CD8-QR,
MUC1 (B27.29 [Biomira, Edmonton, AB]; labeled with Alexa Fluor
647 protein-labeling kit [Invitrogen]), CD27–PE, CCR5–PE–Cy7,
CCR5–PE–Cy7, and CD45RA–FITC (eBioscience, San Diego, CA).
Cells were ﬁxed (PBS ⫹ 2% paraformaldehyde) and analyzed on a
FACSCanto (BD Biosciences, Franklin Lakes, NJ). Isotype control
antibody was used for each ﬂuorescent antibody and gates were set
to exclude 95% of the isotype-stained cells.
2.3. Proliferation assays
Nonadherent T cells from donors were incubated at 37oC for 3 days
with 1 ␮g/mL PHA. A 96-well plate was used for cell treatments,
seeding 2 ⫻ 105 cells/well, along with 10 ␮g/mL B27.29 (hereby antiMUC1), or 10 ␮g/mL mouse IgG1 isotype, 1 ␮g/mL goat antimouse
cross-linking antibody (Sigma), and 20 ␮g/mL OKT3 (anti-CD3
antibody). Plates were incubated at 37oC for 3 days, followed by the
addition of 0.5 ␮Ci/well [3H]thymidine (Amersham, Piscataway, NJ).
The cells were harvested after 18 hours and read on a Microbeta liquid
scintillation counter (PerkinElmer, Waltham, MA).
2.4. Enzyme-linked immunosorbent assay (ELISA) for cytokines
ELISA assays for interleukin (IL)-2, IL-10, tumor necrosis
factor-␣ (TNF-␣, and interferon-␥ (IFN-␥ Biosource, Carlsbad, CA)
were performed according to the manufacturer’s protocol. In brief,
plates were coated with anticytokine antibodies and cell supernatants were added in duplicate before the addition of a biotinylated
antibody. An enzyme–strepavidin conjugate was added along with
substrate. Plates were washed using the ELx405 ELISA plate washer
(Bio Tek, Winooski, VT) and analyzed on a Fluostar Optima ﬂuorimeter (BMG Labtech, Offenburg, Germany). Standard curves were
run between 15 and 2,000 pg/mL.
2.5. Microsphere preparation
Latex microspheres measuring 1 ␮m (Polysciences, Inc, Warrington, PA) were washed in 0.1 M borate buffer and coupled with
150 ␮g total of anti-MUC1, mouse immunoglobulin G (IgG) isotype,
and/or anti-CD3. The beads were left shaking overnight at room
temperature and washed 3 times for 30 minutes each in borate
buffer with 10 mg/mL bovine serum albumin. The beads were
stored at 4oC in PBS ⫹ 10 mg/mL bovine serum albumin ⫹ 0.1%
sodium azide ⫹ 5% glycerol. Beads were washed 3 times with PBS
before use.
449
2.6. Microsphere-based proliferation assays
Nonadherent T cells from donors were kept at 37oC for 3 days
either without PHA or with 1 ␮g/mL PHA to induce MUC1 expression [16]. Microspheres were resuspended in AIM-V to the required
concentrations. Cells were plated at 2 ⫻ 105 cells/well, followed by
microspheres at a ratio of 1,000 microspheres to 1 cell; for separately
ligated beads, the ratio was 500 microspheres to 1 cell per bead type.
Plates were incubated at 37oC for 3 days, followed by the addition of
0.5 ␮Ci/well [3H]thymidine. The following day the cells were harvested and counted on a Microbeta liquid scintillation counter.
2.7. Inhibition assay
Cyclosporine A, bisindolylmaleimide I, and SB203580 (Invitrogen) were purchased in solid form and resuspended in DMSO.
Cyclosporine A, bisindolylmaleimide, and SB203580 were diluted
in PBS and then AIM-V medium in each treatment well to a ﬁnal
concentration of 42 nM, 30 nM, and 1 ␮M, respectively. Proliferation assays were then performed as described.
2.8. Confocal microscopy
Glass slides were coated with poly-L-lysine (Sigma). T cells stimulated with PHA for 3 days were added to the slides at 1–2 ⫻ 107 cells
per slide. Nonadherent T cells were adhered for 30 minutes and stimulated with 20 ␮g anti-CD3 or no antibody. The slides were ﬁxed in 4%
paraformaldehyde ⫹ 120 ␮M glucose, followed by the addition of 0.1%
Triton X in PBS. Slides were treated with 1 ␮g of CT2 (a gift from Dr.
Sandra Gendler, Mayo Clinic, Scottsdale, AZ), an antibody against the
cytoplasmic tail of MUC1, for 1 hour. Slides were washed and a Cy3
conjugate (Leinco Technologies, St. Louis, MO) was added at 1 ␮g to
each slide. Slides were washed and mounted with a 60:40 ratio of
glycerol:PBS, 2% of the antifadant 1,4-diazabicyclo(2)octane (Sigma),
and 1 ␮l of DAPI dye (Invitrogen) per 500 ␮L of solution. Slides were
analyzed via confocal microscopy (Zeiss LSM-510 confocal microscope, Zeiss, Ontario, Canada).
2.9. Lysates
T cells stimulated with PHA for 3 days were washed twice with
PBS and stimulated with antibody-bound plates (20 ␮g/mL antiCD3, 20 ␮g/mL anti-CD3, and 10 ␮g/mL anti-MUC1 or 20 ␮g/mL
anti-CD3 and 10 ␮g/mL mouse IgG isotype) for 45 minutes at 37oC
in AIM V medium. Cells were lysed to obtain cytoplasmic and
nuclear fractions as previously described [19]. All lysates were
stored at ⫺80oC until used.
2.10. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and
western blotting
Approximately 10 ␮g of protein from each of the nuclear and
cytoplasmic fractions was run on a 10% resolving gel and transferred
to nitrocellulose overnight. The membrane was blocked and incubated with anti-NFATc1, anti-c-Fos, or anti-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA) in 0.1% Tween 20 in PBS for 1 hour. After
membranes were washed, a secondary horseradish peroxidase antibody (Novus Biologicals, Littleton, CO) was added for 2 hours. After
another washing, an enhanced chemiluminescence substrate (Fisher,
Pittsburgh, PA) was added, followed by imaging on X-ray ﬁlm.
2.11. Statistical analyses
Statistics were performed using 1-way analysis of variance with
Tukey’s test for post hoc analysis or independent-sample t test
using SPSS 16.0 software (SPSS, Inc, Chicago, IL). An asterisk in the
ﬁgures represents a signiﬁcant difference at the p ⬍ 0.05 level to the
closest appropriate control group. All error bars are indicative of
standard error.
3. Results
+
+
3.1. MUC1 expression increases on both CD4 and CD8 T cells after
mitogen (PHA) stimulation
To investigate MUC1 expression proﬁles, expression was analyzed on naive, memory, memory/effector, and effector CD4⫹ and
CD8⫹ T cells before and after mitogen (PHA) stimulation. CD8⫹ T
cells were gated for CD45RA⫹/CD27⫹CCR5⫺ (naive), CD45RA⫺/
CD27⫹/CCR5⫹ (memory), CD45RA⫺/CD27Low/CCR5⫺ (memory/effector), and CD45RA⫹/CD27⫺/CCR5⫺ (effector), whereas CD4⫹ T
cells were gated for CD45RA⫹/CD27⫹/CCR7⫹ (naive), CD45RA⫺/
CD27⫹/⫺/CCR7⫹ (memory), CD45RA⫺/CD27⫺/CCR7⫺ (memory/effector), and CD45RA⫺/CD27⫹/CCR7⫺ (effector), as previously described [20,21]. It was determined that a low percentage of CD4⫹ T
cells expressed MUC1 (⬃5%) after isolation (Fig. 1A). CD8⫹ T cells
exhibited a higher percentage of MUC1⫹ cells (15– 40%), primarily
in naive and memory subsets (Fig. 1B). Three days after PHA stimulation, MUC1 expression on both CD4⫹ and CD8⫹ T cells increased,
primarily the CD4⫹ T cells (2- to 6-fold increase), with naive CD4⫹ T
cells having the largest increase, likely demonstrating a progression of unstimulated cells into a matured phenotype. Expression on
CD8⫹ T cells did not increase substantially (20 –30% increase across
all groups), although no CD8⫹ memory cells were observed to
express MUC1 postmitogenic stimulus.
3.2. CD3 and MUC1 coligation and cross-linking in T-cell cultures
causes enhanced cellular proliferation
To investigate whether MUC1 can act as a costimulatory molecule in addition to its purported coinhibitory properties, T cells
were ﬁrst stimulated with PHA for 72 hours to induce MUC1 expression. These T cells were then treated with antibodies against
CD3/MUC1/IgG isotype control and a cross-linking antibody. After
3 days, the cells in the MUC1 costimulated group proliferated more
60
Non-Stimulated
PHA-Stimulated
40
em
or
y
M
or
ec
t
r
ec
to
em
or
y/
Ef
f
M
Ef
f
ve
0
B)
Percentage of CD8+ T cell
Subsets Expressing
MUC1
250
200
*
Anti-CD3
Anti-CD3 + Anti-MUC1
Anti-CD3 + IgG Isotype
150
100
50
0
Fig. 2. Proliferation was measured in 3-day phytohemaglutinin A (PHA)-stimulated
T cells treated with 10 ␮g/mL of soluble anti-CD3 antibody (black bar), 10 ␮g/mL
anti-CD3, and 10 ␮g/mL of the anti-MUC1 (hatched bar) or 10 ␮g/mL anti-CD3 and
10 ␮g/mL of mouse immunoglobulin G (IgG) isotype control (dashed bar). A significant difference existed between the anti-CD3 plus anti-MUC1-treated group and
the other treatment groups, with p ⬍ 0.05. Data are representative of ⬎10 experiments on ⬎10 different donors.
than those in the anti-CD3 group and the isotype, with p ⬍ 0.01 (Fig.
2). A control test was also performed using anti-MUC1, anti-MUC1
with a cross-linking antibody, and isotype with a cross-linking
antibody in the absence of anti-CD-3, with resultant counts per
minute ⬍ 7,000 per treatment per experiment (data not shown).
This experiment provides the ﬁrst evidence that cross-linking
MUC1 is able to provide costimulation to enhance the proliferation
generated by the anti-CD3 stimulus. Cultures with anti-MUC1 and
cross-linking antibody only did not demonstrate signiﬁcant proliferation over background (data not shown).
3.3. CD3 and MUC1 costimulation leads to an increase in CD4+
memory, CD8+ memory, and memory/effector cells
The apparent costimulatory effects of MUC1 stimulation on T
cells in the presence of CD3 stimulation encouraged us to determine what T-cell subsets, within either CD4⫹ or CD8⫹ cells, were
increased after anti-CD3 and anti-MUC1 costimulation. T cells costimulated with anti-CD3 and anti-MUC1 antibody were stained for
markers of memory, memory/effector, and effector T cells in both
CD4⫹ and CD8⫹ T-cell subsets. Analysis demonstrated that there
was a signiﬁcant increase in CD4⫹ memory cells (⬃70%) and CD8⫹
memory cells (⬃35%) after MUC1 costimulation compared with
isotype (Fig. 3). All other subsets did not differ from 0% and, thus,
did not demonstrate a signiﬁcant change after MUC1 costimulation
compared with isotype.
20
N
aï
Percentage of CD4+ T cell
Subsets Expressing
MUC1
A)
Thymidine Incorporation (cpm x 103)
J.D. Konowalchuk and B. Agrawal / Human Immunology 73 (2012) 448–455
3H
450
3.4. MUC1-mediated costimulation requires CD3 and
MUC1 coligation
ND
Fig. 1. T cells were isolated from fresh human blood and either stained immediately
or treated with 1 ␮g/mL of phytohemaglutinin A (PHA) for 3 days and then stained
afterward. A gate was set on the lymphocyte population for analysis. CD4⫹ and CD8⫹
T cells were analyzed for naive, memory, memory/effector, and effector phenotypes.
The percentages of the gated T-cell phenotypes were then compared between
non-stimulated (white) and 3-day PHA-stimulated (black) CD4⫹ T cells (A) and
CD8⫹ T cells (B). Results are representative ﬁgures of 3 separate experiments on 3
different donors. ND, not detectable.
Most costimulatory molecules of T cells require CD3 within
close proximity because of the sharing of kinases and phosphatases
[22]. We hypothesized that MUC1 may function in a similar
manner. Using 1-␮m latex microspheres coligated with anti-CD3
and either anti-MUC1 or isotype or beads ligated separately with
anti-CD3 and anti-MUC1 or anti-CD3 and isotype, it was determined that T cells exhibited enhanced proliferation with the
anti-CD3 and anti-MUC1 coligated group compared with the
other groups (p ⬍ 0.05; Fig. 4). There was no signiﬁcant enhancement of proliferation when cells were treated with the separately ligated beads rather than the coligated beads (p ⬎ 0.05)
compared with the isotype.
J.D. Konowalchuk and B. Agrawal / Human Immunology 73 (2012) 448–455
A)
CD3 +
MUC1
Stimulus
Donor 1
CD3 +
Isotype
Stimulus
2.65%
5.32%
4.41%
4.71%
3.02%
Donor 2
CD27 PE
3.30%
CD3 +
MUC1
Stimulus
*
451
CD3 +
Isotype
Stimulus
18.30%
13.06%
28.65%
11.56%
15.07%
12.98%
Donor 3
CCR5 PE-Cy7
CD8+ Memory Cells
B)
CCR7 PE-Cy7
CD4+ Memory Cells
90%
Percent Difference Between CD3/MUC1
Costimulated and CD3/Isotype-Treated
Lymphocytes
80%
CD4 Cells
*
CD8 Cells
70%
60%
50%
40%
*
30%
20%
10%
0%
Memory
Memory/Effector
Effector
Fig. 3. T cells were stimulated with 1 ␮g/mL of phytohemagultinin A (PHA) for 3 days to induce MUC1 expression before being stimulated for 3 more days with 10 ␮g/mL
anti-CD3, 10 ␮g/mL anti-MUC1, and 1 ␮g/mL goat antimouse immunoglobulin G (IgG) or 10 ␮g/mL anti-CD3, 10 ␮g/mL IgG isotype control, and 1 ␮g/mL goat antimouse IgG.
A gate was set on the lymphocyte population for analysis. CD8⫹ and CD4⫹ T cells were analyzed for memory, memory/effector, and effector phenotypes, ﬁrst by gating for
CD4/8 and CD45RA, followed by subsequent gating with CD27 and CCR7/CCR5, according to the subpopulation. Plots illustrating the CD4/CD8 memory cells gated are
presented in (A). The percentages of cell phenotypes in the anti-MUC1-stimulated cells were then subtracted from the IgG isotype-stimulated results, divided by the IgG
isotype-stimulated results, and graphed, demonstrating the percentage difference between anti-MUC1 and isotype stimulation in CD4⫹ and CD8⫹ T-cell subsets (B). Statistics
were performed by comparing the values with a 0% change between the compared groups. Results are the average of 3 individual experiments on 3 different donors.
3H
Thymidine Incorporation (cpm x 103)
452
J.D. Konowalchuk and B. Agrawal / Human Immunology 73 (2012) 448–455
functions independently of the p38 MAPK pathway but involves
the nuclear factor of activated T cells (NF-AT) pathway.
Anti-CD3 + Anti-MUC1 (Coligated)
120
Anti-CD3 + Anti-MUC1 (Separate)
*
3.6. MUC1 costimulation is unaffected by an inhibitor of the nuclear
factor ␬B pathway, but not the NF-AT or p38 MAPK pathways
Anti-CD3 + IgG Isotype (Coligated)
Anti-CD3 + IgG Isotype (Separate)
90
60
30
0
Fig. 4. Three-day phytohemaglutinin A (PHA)-stimulated T cells were treated with
microspheres ligated separately with anti-CD3 and either anti-MUC1 or isotype or,
alternatively, coligated with anti-CD3 and either anti-MUC1 or isotype. Microspheres were added to a ﬁnal amount of 2 ⫻ 108 per 2 ⫻ 105 cells; for beads with
separate antibodies bound, 1 ⫻ 108 beads of both types were added to the culture to
achieve the ﬁnal amount of 2 ⫻ 108. The coligated anti-CD3 plus anti-MUC1-treated
group exhibited a signiﬁcant increase in proliferation over the other groups (p ⬍
0.05). Data are representative of 2 separate experiments on 2 different donors.
3.5. MUC1-based costimulation increases the expression and release
of TNF-␣, IFN-␥, and IL-2, but not IL-10
Activations of the calcineurin, PKC␪, and p38 mitogen-activated
protein kinase (MAPK) pathways have been demonstrated to induce unique cytokine production proﬁles. To further delineate the
pathway involved in MUC1 costimulation, supernatants were collected from MUC1 costimulation cultures (Fig. 2 and similar experiments) and analyzed via ELISA. The MUC1 costimulated group
produced increased TNF-␣, IFN-␥, and IL-2 into the supernatant at a
signiﬁcant level compared with the controls (p ⬍ 0.05; Fig. 5). For
IL-10, however, the MUC1 costimulated group and the control did
not differ. This further suggests that MUC1 costimulation likely
*
pg/ml
8000
A) IL-2
To determine the pathway utilized by MUC1-mediated costimulation resulting in enhanced proliferation, intracellular inhibitors of
different signaling pathways were added to MUC1-costimulated
cultures. These inhibitors included cyclosporine A (inhibitor of
calcineurin), bisindolylmaleimide I (inhibitor of the PKC family),
and SB203580 (inhibitor of p38 MAPK). Optimal concentrations of
inhibitors were determined (42 nM cyclosporine A, 30 nM bisinolylmaleimide I, 1 ␮M SB203580), based on an amount that would
inhibit proliferation but not result in cell death. The T cells were
stimulated with PHA for 3 days to induce MUC1 and then washed
and treated with inhibitors for 10 minutes. Afterward, they were
treated with anti-CD3 alone, CD3 and MUC1, or CD3 and isotype,
along with the cross-linking antibody. After 3 days, the MUC1costimulated group and the controls with cyclosporine A or SB203580
did not differ (Fig. 6). There was a signiﬁcant difference between the
MUC1-costimulated group compared with the controls with bisindolylmaleimide I (p ⬍ 0.01), indicating a reversal/negation of the
inhibitory effect by MUC1 costimulation. The p38 MAPK inhibitor,
however, had a high rate of proliferation regardless of the antibodies
treated, likely as a result of inhibition of the IL-10 production by the
p38 MAPK pathway. This demonstrates that MUC1-mediated costimulation functions independent of the PKC-dependent pathways,
likely involving the calcineurin pathway.
3.7. The cytoplasmic tail of MUC1 migrates into the cytoplasm and
nucleus upon CD3 stimulation in T cells
In tumor cells, the cytoplasmic tail of MUC1 has been demonstrated to translocate into the nucleus with transcription factors, as
well as the mitochondria [23]. We hypothesized that MUC1 on T
cells may play a similar role. Cells stained for the nucleus and
cytoplasmic tail were analyzed by confocal microscopy, with ap-
20000
6000
15000
4000
10000
2000
5000
ND
ND
*
B) TNF-α
0
0
6000
*
C) IFN-γ
300
4500
225
3000
150
1500
75
0
0
D) IL-10
Anti-CD3
Anti-CD3 + Anti-MUC1
Anti-CD3 + IgG Isotype
Fig. 5. Supernatants taken at 3 days from Fig. 2 and similar experiments were analyzed via enzyme-linked immunosorbent assay for the cytokines: (A) interleukin (IL)-2, (B)
tumor necrosis factor-␣ (TNF-␣, (C) interferon-␥ (IFN-␥ and (D) IL-10. The anti-CD3 plus anti-MUC1 antibody treatment produced statistically higher amounts of the
proliferation-inducing cytokine IL-2 as well as the proinﬂammatory cytokines TNF-␣ and IFN-␥ (p ⬍ 0.05). Amounts of the inhibitory cytokine IL-10, however, were not
signiﬁcant among the groups (p ⬎ 0.05). All cytokine amounts are in picograms per milliliter. IL-2 data are representative of 2 experiments performed on 2 different donors,
whereas the other cytokines are representative of 3 experiments performed on 3 different donors. ND, not detectable.
453
could be seen translocating into both the cytoplasm and the
nucleus.
3.8. The cytoplasmic tail of MUC1 binds to the transcription factors
c-Jun and c-Fos
**
**
3H
Thymidine Incorporation (cpm x 103)
J.D. Konowalchuk and B. Agrawal / Human Immunology 73 (2012) 448–455
Controls
Cyclosporine
A
Bisindolylmaleimide I
SB203580
Anti-CD3
Anti-CD3 + Anti-MUC1
Anti-CD3 + IgG Isotype
Fig. 6. T cells were stimulated with phytohemaglutinin A (PHA) for 3 days before
being treated with intracellular inhibitors along with 10 ␮g/mL anti-CD3 (white
bars), 10 ␮g/mL anti-CD, and 10 ␮g/mL anti-MUC1 (black bars) or 10 ␮g/mL anti-CD3
and 10 ␮g/mL isotype (hatched bars). Each group was also given 1 ␮g/mL of goat
antimouse antibody to cross-link. There was no signiﬁcant difference between
groups in the data for the intracellular inhibitors cyclosporine A or SB203580.
However, there was a signiﬁcant increase in proliferation in the anti-CD3 plus
anti-MUC1-treated group given the PKC inhibitor bisindolylmaleimide I (p ⬍ 0.05)
compared with the inhibitor-treated control groups. Data are representative of 4
separate experiments on 4 different donors. Unstimulated cells provided ⬍5,000
counts per minute for all experiments.
proximately 300 cells being analyzed per experimental group and
representative pictures taken. Without CD3 stimulation, the cytoplasmic tail remained at the cell membrane and clustered in the
staining proﬁle of lipid rafts [24] (Fig. 7). However, with CD3 stimulation, regardless of MUC1 costimulation, the cytoplasmic tail
CT – Cy3
The cytoplasmic tail of MUC1 migrated to the cytoplasm and
nucleus after CD3 stimulation, leading us to believe the cytoplasmic
tail of MUC1 would carry transcription factors into the nucleus of T
cells. With earlier results supporting the calcineurin-dependent
NF-AT pathway in MUC1 costimulation, we blotted the transcription factors NF-ATc1, c-Jun, and c-Fos after immunoprecipitation of
the cytoplasmic tail of MUC1 from T-cell lysates. For NF-ATc1, only
the positive control (whole nuclear lysate) exhibited a band at the
appropriate molecular weight (data not shown). For c-Jun, bands of
the appropriate molecular weight of 35–39 kDa were observed in
both the control group (whole nuclear lysate) and the MUC1costimulated cytoplasmic and nuclear fractions. No other pretreatment had a band indicative of c-Jun coimmunoprecipitation with
the cytoplasmic tail of MUC1 (Fig. 8A). For c-Fos, a band between 60
and 70 kDa representative of the 62-kDa weight of c-Fos was
observed in the positive control group and all CD3-stimulated cytoplasmic and nuclear fractions, as well as small amounts in the
unstimulated groups (Fig. 8B). However, there was a visible increase in c-Fos in the nuclear fraction of the MUC1-costimulated
group.
4. Discussion
The role of MUC1 in T-cell activation, regulation, and homeostasis as an activation-induced glycoprotein in T lymphocytes has
been recognized recently [5,8,14,25]. In these studies, cross-linking
MUC1 has been determined to inhibit the proliferation of T cells
when given a CD3-based stimulation [8,14], in the absence of a
sufﬁcient number of antigen-presenting/accessory cells [5]. However, the presence of a putative ITAM domain and evidence of
signaling proteins binding to its cytoplasmic tail suggest that MUC1
may have dual costimulatory and coinhibitory functions in T cells.
DAPI
Overlay
No Stim
Anti-CD3
Anti-CD3 +
Anti-MUC1
Fig. 7. T cells stimulated with phytohemaglutinin A (PHA) for 3 days were given no treatment, 10 ␮g/mL anti-CD3, or 10 ␮g/mL anti-CD3, 10 ␮g/mL anti-MUC1, and 1 ␮g/mL
goat antimouse IgG antibody for 30 minutes before being stained against the cytoplasmic tail of MUC1 (Cy3, in red) and the nucleus (DAPI, in blue). Images are presented in
single colors and overlaid. Pictures are taken from a single illustrative slide each from the same donor, representative of 3 separate experiments on 3 individual donors.
Approximately 300 cells were analyzed in each group and pictures are representative of those observations.
A)
CD3 + Isotype, Nucleus
CD3 + Isotype, Cytoplasm
CD3 + MUC1, Nucleus
CD3 + MUC1, Cytoplasm
CD3, Nucleus
CD3, Cytoplasm
No Stim, Nucleus
kDa
50
Control
J.D. Konowalchuk and B. Agrawal / Human Immunology 73 (2012) 448–455
No Stim, Cytoplasm
454
CT2 IP: c-Jun IB
22
B)
50
CT2 IP: c-Fos IB
Fig. 8. Western blots using anti-MUC1 cytoplasmic tail (CT2) to precipitate and (A)
anti-c-Jun or (B) anti-c-Fos to blot. For c-Jun, bands of the appropriate molecular
weight (⬃39 kDa) appeared for the positive control (cellular lysate run without a
precipitating antibody) and both anti-CD3 plus anti-MUC1 treatment’s cytoplasmic
and nuclear fractions. For c-Fos, bands of the appropriate molecular weight (⬃62
kDa) appeared for the positive control (pure cellular lysate run without a precipitating antibody) and all treatment groups in both the cytoplasmic extracts and the
nuclear extracts. In the untreated (nonstimulated) group, however, only a small
amount of c-Fos was detected bound to MUC1 compared with the other groups.
We determined that MUC1 expression increases signiﬁcantly on
CD4⫹ T cells after mitogen stimulation, with naive T cells having the
largest increase. This supports the hypothesis that MUC1 plays a
role in T-cell immunoregulation because naive T cells begin to
express maturation markers after mitogen stimulation [20]. Treating T cells with antibodies against CD3 and MUC1, under crosslinking conditions, led to enhanced proliferation. This is the ﬁrst
evidence obtained in characterizing MUC1 as a costimulatory protein of T cells. Earlier studies had utilized puriﬁed T-cell populations (⬎80% CD3⫹ T cells) and demonstrated coinhibition mediated
by MUC1 [5,8,14]. In the current study, a T-cell population consisting of ⬎60% CD3⫹ T cells was used to demonstrate costimulatory
effects. However, in partially puriﬁed T-cell (⬃80 –95% CD3⫹ T
cells) cultures, the addition of irradiated autologous CD3⫺ accessory cells resulted in a costimulatory effect proportional to the
amount of accessory cells added [5]. These experiments suggest
that for MUC1 to function as a costimulatory molecule, an additional signal/interaction is required.
After determining the conditions that result in a costimulatory
response, we examined its effects on different CD4⫹ and CD8⫹
T-cell subsets. With CD4⫹ T cells, the percentage of memory cells
increased greatly, whereas with CD8⫹ T cells, the percentage of
naive, memory, and memory/effector cells increased after CD3 and
MUC1 costimulation. Interestingly, we observed that mitogen
stimulation results in higher MUC1 expression on naive CD4⫹ T
cells, whereas MUC1 costimulation encourages memory CD4⫹ Tcell expansion. In contrast, MUC1 costimulation allows the expansion of memory, memory/effector, and effector CD8⫹ T cells. This
ﬁnding suggests that MUC1 costimulation causes a speciﬁc subset
of cells to proliferate/generate, potentially allowing for regulation
of T-cell responses. Like other costimulatory proteins, we discovered that MUC1 is required to be bound and cross-linked in close
proximity to extracellular CD3, likely because of shared kinases/
phosphatases [26]. The intracellular interaction of these molecules
will be characterized in the future.
Three major intracellular signaling pathways used by T cells are
the NF-AT, nuclear factor ␬B, and p38 MAPK pathways. The
calcium-dependent NF-AT pathway is activated by CD3 stimulation
and results in an increase in IFN-␥, TNF-␣, and IL-2 [27]; the nuclear
factor ␬B pathway requires both CD3 and CD28 costimulation and
produces the cytokine IL-2 [28]; and the p38 MAPK pathway results
in, after T-cell activation, production of IL-4, IL-13, and IL-10 [29]. In
our results, proliferation did not differ with MUC1-mediated costimulation compared with controls in p38 MAPK- and NF-ATinhibited groups, suggesting that either pathway could be used. By
examining cytokines from T cells given MUC1 costimulation, it was
determined that MUC1 functions through the calcium-dependent
NF-AT pathway. These data are also supported by our observation
that MUC1 costimulation increases the number of memory CD4⫹ T
cells, which produce IFN-␥ and memory and memory/effector
CD8⫹ T cells, which produce IFN-␥ and TNF-␣ [30].
Proliferation is enhanced at the nuclear level by transcription
factors, several of which MUC1 binds to in tumor cells [3,4,31]. The
primary factor, ␤-catenin, is not expressed by mature T cells [26].
Thus, the most likely transcription factors were the NF-AT family
members NF-ATc1, c-Fos, and c-Jun. Indeed, we determined that
c-Jun and c-Fos bind to the cytoplasmic tail of MUC1 and enter the
nucleus, whereas NF-ATc1 does not (data not shown). Both c-Fos
and c-Jun are imperative in the NF-AT pathway, dimerizing together after phosphorylative activation to produce the transcription factor AP-1 [32]. However, our data indicate that c-Fos is
constitutively bound to the cytoplasmic tail of MUC1, whereas
c-Jun is only bound after MUC1 costimulation. Because we determined that CD3 stimulation alone is sufﬁcient to translocate the
cytoplasmic tail into the cytoplasm and nucleus, this provides a role
for MUC1 stimulation: phosphorylation of c-Fos and/or c-Jun, allowing it to form the AP-1 dimer and be brought into the nucleus.
This theory is supported by previous observations by Gendler and
co-workers, who demonstrated that transfection of a tumor cell
line with a MUC1 analogue resulted in an intracellular increase in
AP-1 [33]. Additionally, mitochondrial costaining will be performed in the future to determine whether, like tumor cells [23], an
association exists with the mitochondria based on the granularity
of the staining pattern displayed by MUC1 in Fig. 7.
AP-1 is vital in the early immune response, enhancing cytokine
production, cellular activation, and proliferation [34]. Normally,
c-Jun is expressed after the initial CD3 response, with c-Jun dimers
leading to c-Fos production after CD28 costimulation [35]. AP-1
dimers then form and migrate into the nucleus, binding promoter
regions and resulting in the production of proinﬂammatory and
proliferation-inducing cytokines [36]. Without AP-1, anergyinducing genes are transcribed, resulting in T-cell nonresponse to
stimuli [37]. Regarding the absence of NF-ATc1 on MUC1’s cytoplasmic tail, it is likely that the cytoplasmic tail dissociates from
AP-1 before it binds to DNA, possibly because of size restriction,
although future studies will be performed to determine whether
this is correct.
Previously, it has been demonstrated that c-Fos has a weaker
nuclear localization sequence than c-Jun; small quantities of c-Fos
migrate into the nucleus in the absence of c-Jun, meaning it is
dependent on c-Jun to adequately enter the nucleus [38]. By binding the cytoplasmic tail of MUC1, c-Fos may circumvent this regulation because it is provided with an alternate pathway of nuclear
translocation. One obstacle lies in the fact that PKC␪ is required for
c-Jun phosphorylation and subsequent dimerization [36]. Because
we observed that MUC1 costimulation was unaffected by generalized PKC inhibition, the cytoplasmic tail of MUC1 either has phosphorylative abilities or is able to recruit other proteins that are able
to phosphorylate c-Jun/c-Fos in a PKC-independent manner.
ERK1/2, having been demonstrated to bind MUC1’s cytoplasmic
domain in T cells [13], is able to phosphorylate these proteins [14],
providing a potential mechanism that will be researched further.
These results provide evidence that MUC1 is a novel costimulatory protein on T cells. With both CD3 and MUC1 costimulation,
MUC1’s cytoplasmic tail binds c-Jun and c-Fos, followed by nuclear
translocation. By enhancing the amount of AP-1 entering the nucleus in a PKC-independent manner, MUC1 costimulation is able to
further activate genes that cause the production of proinﬂamma-
J.D. Konowalchuk and B. Agrawal / Human Immunology 73 (2012) 448–455
tory and proliferation-inducing cytokines, resulting in an enhanced
proliferative response. MUC1 could exist on T cells in the same
manner as OX40, which maintains the immune response after a
primary activation, allowing them to become active again when
presented with their antigen [39]. Alternatively, it could enhance
lower levels of CD3 stimulation, increasing nuclear AP-1 when
normally the amount was too low. These are possibilities that will
be tested in future research.
In conclusion, our study establishes MUC1 as a novel T-cell
activation molecule with a signiﬁcant role as a costimulatory molecule. Our results point toward a novel paradigm by which MUC1
adopts a costimulatory function in T cells. Further characterization
of the costimulatory abilities of MUC1 may prove useful in the
treatment of diseases of immune inhibition, such as in many tumor
microenvironments or in diseases of immune hyperactivity, such as
autoimmune disorders.
Acknowledgments
The authors acknowledge Dorothy Kratochwii-Otto for her help
with ﬂow cytometry, Honey Chan for her aid regarding the confocal
microscope, and Dr. Colin Anderson for his comments and criticisms regarding the manuscript. This work was supported by
grants from the Canadian Institutes of Health Research and the
Canadian Foundation for Innovation. BA is a recipient of a senior
scholar award from the Alberta Heritage Foundation for Medical
Research (now Alberta Innovates-Health Solutions).
References
[1] Gendler SJ. MUC1, the renaissance molecule. J Mammary Gland Biol Neoplasia
2001;6:339 –53.
[2] Zrihan-Licht S, Baruch A, Elroy-Stein O, Keydar I, Wreschner DH. Tyrosine
phosphorylation of the MUC1 breast cancer membrane proteins. Cytokine
receptor-like molecules. FEBS Lett 1994;356:130 – 6.
[3] Singh PK, Hollingsworth MA. Cell surface-associated mucins in signal transduction. Trends Cell Biol 2006;16:467–76.
[4] Wen Y, Caffrey TC, Wheelock MJ, Johnson KR, Hollingsworth MA. Nuclear
association of the cytoplasmic tail of MUC1 and beta-catenin. J Biol Chem
2003;278:38029 –39.
[5] Konowalchuk JD, Agrawal B. MUC1 mucin is expressed on human T-regulatory
cells: function in both co-stimulation and co-inhibition. Cell Immunol 2012;
272:193–9.
[6] Samuel J, Longenecker BM. Development of active speciﬁc immunotherapeutic
agents based on cancer-associated mucins. Pharm Biotechnol 1995;6:875–90.
[7] Reis CA, David L, Seixas M, Burchell J, Sobrinho-SimÖes M. Expression of fully
and under-glycosylated forms of MUC1 mucin in gastric carcinoma. Int J
Cancer 1998;79:402–10.
[8] Agrawal B, Krantz MJ, Parker J, Longenecker BM. Expression of MUC1 mucin on
activated human T cells: implications for a role of MUC1 in normal immune
regulation. Cancer Res 1998;58:4079 – 81.
[9] Cloosen S, Thio M, VanclÊe A, van Leeuwen EBM, Senden-Gijsbers BLMG, Oving
EBH, et al. Mucin-1 is expressed on dendritic cells, both in vitro and in vivo. Int
Immunol 2004;16:1561–71.
[10] Cheong SK, Leong CF, Raudhawati O, Sivagengei K, Hamidah HN. Epithelial
membrane antigen (EMA) or MUC1 expression in monocytes and monoblasts.
Pathology 2003;35:422–7.
[11] Vasir B, Avigan D, Wu Z, Crawford K, Turnquist S, Ren J, et al. Dendritic cells
induce MUC1 expression and polarization on human T cells by an IL-7dependent mechanism. J Immunol 2005;174:2376 – 86.
[12] Correa I, Plunkett T, Vlad A, Mungul A, Candelora-Kettel J, Burchell JM, et al.
Form and pattern of MUC1 expression on T cells activated in vivo or in vitro
suggests a function in T-cell migration. Immunology 2003;108:32– 41.
455
[13] Wykes M, MacDonald KPA, Tran M, Quin RJ, Xing PX, Gendler SJ, et al. MUC1
epithelial mucin (CD227) is expressed by activated dendritic cells. J Leukoc Biol
2002;72:692–701.
[14] Agrawal B, Longenecker BM. MUC1 mucin-mediated regulation of human T
cells. Int Immunol 2005;17:391–9.
[15] Mukherjee P, Tinder TL, Basu GD, Gendler SJ. MUC1 (CD227) interacts with lck
tyrosine kinase in Jurkat lymphoma cells and normal T cells. J Leukoc Biol
2005;77:90 –9.
[16] Pandey P, Kharbanda S, Kufe D. Association of the Df3/Muc1 breast-cancer
antigen with Grb2 and the Sos/Ras exchange protein. Cancer Res 1995;55:
4000 –3.
[17] Wang HH, Lillehoj EP, Kim KC. MUC1 tyrosine phosphorylation activates the
extracellular signal-regulated kinase. Biochem Biophys Res Commun 2004;
321:448 –54.
[18] Turjanski AG, Vaque JP, Gutkind JS. MAP kinases and the control of nuclear
events. Oncogene 2007;26:3240 –53.
[19] Andrews NC, Faller DV. A rapid micropreparation technique for extraction of
DNA-binding proteins from limiting numbers of mammalian cells. Nucleic
Acids Res 1991;19:2499 –505.
[20] Kurosawa K, Kobata T, Tachibana K, Agematsu K, Hirose T, Schlossman SF, et al.
Differential regulation of Cd27 expression on subsets of Cd4 T-cells. Cell Immunol 1994;158:365–75.
[21] Kobayashi N, Takata H, Yokota S, Takiguchi M. Down-regulation of CXCR4
expression on human CD8⫹ T cells during peripheral differentiation. Eur
J Immunol 2004;34:3370 – 8.
[22] Howie D, Simarro M, Sayos J, Guirado M, Sancho J, Terhorst C. Molecular
dissection of the signaling and costimulatory functions of CD150 (SLAM):
CD150/SAP binding and CD150-mediated costimulation. Blood 2002;99:
957– 65.
[23] Benjamin JB, Jayanthi V, Devaraj H. MUC1 expression and its association with
other aetiological factors and localization to mitochondria in preneoplastic
and neoplastic gastric tissues. Clin Chim Acta 2010;411:2067–72.
[24] Greene WC, Fenard D, Yonemoto W, de Noronha C, Cavrois M, Williams SA. Nef
is physically recruited into the immunological synapse and potentiates T cell
activation early after TCR engagement. J Immunol 2005;175:6050 –7.
[25] Fattorossi A, Battaglia A, Malinconico P, Stoler AB, Andreocci L, Parente D, et al.
Constitutive and inducible expression of the epithelial antigen MUC1 (CD227)
in human T cells. Exp Cell Res 2002;280:107–18.
[26] Trepel JB, Chung EJ, Hwang SG, Nguyen P, Lee S, Kim JW, et al. Regulation of
leukemic cell adhesion, proliferation, and survival by beta-catenin. Blood
2002;100:982–90.
[27] Im SH, Rao A. Activation and deactivation of gene expression by Ca2⫹/
calcineurin-NFAT-mediated signaling. Mol Cells 2004;18:1–9.
[28] Schmitz ML, Bacher S, Dienz O. NF-kappaB activation pathways induced by T
cell costimulation. FASEB J 2003;17:2187–93.
[29] Cook R, Wu CC, Kang YJ, Han J. The role of the p38 pathway in adaptive
immunity. Cell Mol Immunol 2007;4:253–9.
[30] Seder RA, Ahmed R. Similarities and differences in CD4(⫹) and CD8(⫹) effector
and memory T cell generation. Nat Immunol 2003;4:835– 42.
[31] Singh PK, Behrens ME, Eggers JP, Cerny RL, Bailey JM, Shanmugam K, et al.
Phosphorylation of MUC1 by Met modulates interaction with p53 and MMP1
expression. J Biol Chem 2008;283:26985–95.
[32] Gentz R, Rauscher FJ, Abate C, Curran T. Parallel association of Fos and Jun
leucine zippers juxtaposes DNA-binding domains. Science 1989;243:1695–9.
[33] Thompson EJ, Shanmugam K, Hattrup CL, Kotlarczyk KL, Gutierrez A, Bradley
JM, et al. Tyrosines in the MUC1 cytoplasmic tail modulate transcription via the
extracellular signal-regulated kinase 1/2 and nuclear factor-kappaB pathways.
Mol Cancer Res 2006;4:489 –97.
[34] Serﬂing E, Berberich-Siebelt F, Chuvpilo S, Jankevics E, Klein-Hessling S,
Twardzik T, et al. The role of NF-AT transcription factors in T cell activation and
differentiation. Biochim Biophys Acta 2000;1498:1–18.
[35] Kiefer F, Vogel WF, Arnold R. Signal transduction and co-stimulatory pathways.
Transpl Immunol 2002;9:69 – 82.
[36] Hayashi K, Altman A. Protein kinase C theta (PKCtheta): a key player in T cell
life and death. Pharmacol Res 2007;55:537– 44.
[37] Waldmann I, Walde S, Kehlenbach RH. Nuclear import of c-Jun is mediated by
multiple transport receptors. J Biol Chem 2007;282:27685–92.
[38] Chida K, Nagamori S, Kuroki T. Nuclear translocation of Fos is stimulated by
interaction with Jun through the leucine zipper. Cell Mol Life Sci 1999;55:297–
302.
[39] Croft M. Costimulation of T cells by OX40, 4 –1BB, and CD27. Cytokine Growth
Factor Rev 2003;14:265–73.