Articles in PresS. Am J Physiol Cell Physiol (December 3, 2014). doi:10.1152/ajpcell.00262.2014
1
Krüppel-like factor KLF10 regulates Transforming growth factor receptor II expression
2
and TGF-β signaling in CD8+ T lymphocytes
3 4 5 6 7 8 9 10 11 12 13
Konstantinos A. Papadakis1, James Krempski1, Jesse Reiter1, Phyllis Svingen1, Yuning Xiong1, Olga F. Sarmento1, April Huseby2, Aaron J. Johnson2, Gwen A. Lomberk3,Raul A. Urrutia3,and William A. Faubion1. 1
Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA Division of Immunology and Neurology, Mayo Clinic, Rochester, Minnesota, USA 3 Epigenetics and Chromatin Dynamics Laboratory, Departments of Medicine and Biochemistry and Molecular Biology. Epigenetic Translational Program, Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota, USA 2
14 15 16 17
2
18
Hepatology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Tel.: 507-284-2468;
19
Fax: 507-255-6318; E-mail: [email protected].
To whom correspondence should be addressed: Dept. of Gastroenterology and
20 21 22
The abbreviations used are: KLF; krüppel-like factor, TGF-βRII; TGF-β receptor II
1 Copyright © 2014 by the American Physiological Society.
23
ABSTRACT
24
KLF10 has recently elicited significant attention as a transcriptional regulator of
25
TGF-β1 signaling in CD4+ T cells. In the current study, we demonstrate a novel
26
role for KLF10 in the regulation of TGF-βRII expression with functional relevance
27
in antiviral immune response. Specifically, we show that KLF10-deficient mice
28
have an increased number of effector/memory CD8+ T cells, display higher levels
29
of the T helper type 1 cell-associated transcription factor T-bet, and produce
30
more IFN-γ following in vitro stimulation. In addition, KLF10-/- CD8+ T cells show
31
enhanced proliferation in vitro and homeostatic proliferation in vivo. Freshly
32
isolated CD8+ T cells from the spleen of adult mice express lower levels of
33
surface TGF-βRII (TβRII). Congruently, in vitro activation of KLF10-deficient
34
CD8+ T cells up-regulate TGF-βRII to a lesser extent compared to wt CD8+ T
35
cells which results in attenuated Smad-2 phosphorylation following TGF-β1
36
stimulation compared to wt CD8+ T cells. Moreover, we demonstrate that KLF10
37
directly binds to the TGF-βRII promoter in T cells leading to enhanced gene
38
expression. In vivo viral infection with Daniel's strain TMEV also led to lower
39
expression of TGF-βRII among viral-specific KLF10-/- CD8+ T cells and a higher
40
percentage of IFN-γ-producing CD8+ T cells in the spleen. Collectively, our data
41
reveal a critical role for KLF10 in the transcriptional activation of TGF-βRII in
42
CD8+ T cells. Thus, KLF10 regulation of TGF-βRII in this cell subset may likely
43
play a critical role in viral and tumor immune responses for which the integrity of
44
the TGF-β1/TGF- βRII signaling pathway is crucial.
45
2
46
INTRODUCTION
47 48
Krüppel-like factors (KLFs) have emerged as important regulators of immune
49
function (2, 7, 8, 15, 18, 23, 24, 29). KLF proteins constitute a family of transcription
50
factors that regulate the expression of a large number of genes with established
51
relevance to cell proliferation, apoptosis, differentiation, and transformation (2, 12, 23,
52
29). One of these proteins, KLF10 (formerly TGF-β inducible early gene 1; TIEG1) was
53
identified as a transcript that is rapidly induced after TGFβ treatment in human
54
osteoblasts (17). Under normal conditions, KLF10 regulates the growth and
55
differentiation of many epithelial cell types and its disruption is associated to human
56
diseases (12). KLF10 is also critical for the homeostasis of mesenchymal cells,
57
including the cellular components of tendons, bone, and muscle (12). More relevant to
58
the current study, recent reports have identified a role for KLF10 in T lymphocytes and
59
innate immune cells (21, 28). We have previously shown that the genetic inactivation of
60
KLF10, epigenetically silences FOXP3 via EZH2 (Enhancer of Zeste 2)-mediated
61
trimethylation of Histone 3 K27 resulting in an impaired induction of this gene with a
62
concomitant inappropriate adaptive T regulatory cell differentiation in vitro and in vivo
63
(23).
64
TGF-β acting through transforming growth factor-β (TGF-β) receptor I (TGF-βRI)
65
and II (TGF-βRII) plays a critical role also in the control of CD8+ T cell differentiation in
66
lymphoid and peripheral organs (26, 27). Indeed, recent studies have shown that TGF-
67
β-signaling promotes IL7Rα expression and CD8+ T cell differentiation (14). Moreover,
68
TGF-β-signaling inhibits the migration of effector CD8+ T cells from the spleen to the gut
3
69
by dampening the expression of the integrin α4β7 (26). T cell-specific deletion of TGF-
70
βRII receptor early in development (Tgfbr2f/f CD4-cre) leads to an early onset lethal
71
autoimmune disease (9, 11). Notably, however, the signals that control the expression
72
and regulation of TGF-βR and hence TGF-β1 signaling in T cells remain largely
73
unidentified (27). Our laboratory has focused on better understanding the functional role
74
of the transcription factor KLF10 in regulating TGF-β signaling in CD4+ T cells. Both our
75
group and Cao et. al., have previously shown that KLF10 constitutes an important
76
component of T regulatory cell suppressive function and CD4+CD25- T cell activation
77
through distinct mechanisms involving transforming growth factor (TGF)-beta1 and
78
Foxp3 (1, 23). Interestingly, KLF10-/- T reg cells have reduced suppressor function,
79
independent of Foxp3 expression, with decreased expression and elaboration of TGF-
80
beta1 (1). In response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and
81
Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-
82
intrinsic control of T cell activation (1, 23).
83
Thus, given the established importance of KLF10 in TGF-β signaling in CD4+ T
84
cells, in the current study, we hypothesize that this protein controls CD8+ T cell
85
responses by transcriptionally regulating genes encoding key signaling proteins within
86
this pathway. We hypothesized that the TGF βRII promoter is a good candidate for a
87
KLF10 target in T cells. We were guided by previous studies, performed in pancreatic
88
epithelial cells, which revealed the existence of several functional KLF cis-regulatory
89
sites within the TGF-βRII promoter (20). Indeed, our results show that KLF10 is critically
90
involved in the regulation of CD8+ T cell phenotype by transcriptionally regulating TGF-
91
βRII expression. Thus, insights into the KLF10-mediated mechanisms of TGF-βR
4
92
regulation are critical for understanding CD8+ T cell differentiation and function and thus
93
provide important therapeutic applications for vaccine development and the treatment of
94
certain autoimmune diseases and cancer.
95 96 97 98
5
99
MATERIALS AND METHODS
100
Mouse Strains
101
C57BL/6 mice were purchased from the Jackson Laboratory. KLF10−/− mice were
102
kindly provided by Thomas C. Spelsberg (Mayo Clinic, Rochester, MN)(16). The
103
CAR transgenic mouse was obtained through the NIAID Exchange Program of
104
the National Institutes of Health: Balb/cJ[Tg]CARdelta1-[Tg]DO11.10 mouse line
105
4285 (22).All of the mice used in experiments were of 6–8 weeks in age and have
106
been maintained under SPF conditions. The mice were age-matched in
107
experiments comparing wild type with KLF10−/−. All animal experiments were
108
performed per the recommendations outlined in the Guide for Care and Use of
109
Laboratory Animals from the National Institutes of Health as required by Mayo
110
Clinic. These guidelines were incorporated into the current study protocol
111
(IACUC No. A13313), which was reviewed and approved by the Institutional
112
Animal Care and Use Committee (IACUC), at Mayo Clinic, Rochester, MN.
113 114
Isolation of primary murine CD8+ T Cells and T cell Stimulation
115
Murine CD8+ splenocytes were isolated using a CD8+ T cell isolation kit (Miltenyi
116
Biotec, San Diego, CA). In vitro activation of murine T cells was done by plate-
117
bound anti-CD3, (clone 145-2C11, BD Biosciences) at 2 μg/ml. 100 units/ml of
118
IL-2 was added to the cultures throughout the incubation period. Recombinant
119
human TGFβ-1 (AUSTRAL Biologicals, San Ramon, CA ) at a concentration of 5
120
ng/ml was used to induce CD103 expression and SMAD2 phosphorylation.
121
6
122
Flow cytometry
123
Fluorescent dye–labeled Abs against murine CD8α, CD4, CD3, CD45.1, CD45.2,
124
CD62L, CD44, CD103 (integrin αE) and T-bet were purchased from BioLegend
125
(San Diego, CA). Anti-IFN-γ and anti-IL-17 Abs were from BioLegend.
126
Fluorescent dye–labeled antibody to TGF-βRII was from R & D Systems
127
(Minneapolis, MN). For intracellular cytokine staining, CD8+ T cells from wt or
128
KLF10-/- mice were stimulated with plate-bound anti-CD3 145-2C11
129
Biosciences, Franklin Lakes, NJ) in the presence of Golgi-stop (BD Biosciences)
130
for 4 h, followed by fixing and permeabilization according to the manufacturer’s
131
instructions (BD Biosciences). For cell division assay, purified CD8+ T cells were
132
stained with CFSE (Life Technologies, Grand Island, NY) and cultured in the
133
presence of anti-CD3 or IL-15 (30 ng/ml; R & D Systems) for 2–4 d. The cells
134
were analyzed on an LSRII or FACSCalibur (Becton Dickinson, Franklin Lakes,
135
NJ), and data were analyzed with FlowJo software (Tree Star, Ashland, OR).
(BD
136 137
Western blot
138
CD8+ T cells were activated with plate-bound (pb) anti-CD3 antibodies for 72
139
hours and incubated with TGF-β1 for different time points. The cells were lysed in
140
RIPA buffer (1X lysis buffer-150 with protease inhibitors) for p-SMAD2
141
measurement or Laemmli buffer for total SMAD2 and β-actin measurements.
142
Proteins were separated by 10% SDS/PAGE, transferred to membrane, blocked
143
with 5% milk and probed overnight with primary antibodies at the following
144
dilutions: p-SMAD2 (1:5000; Cell Signaling, Danvers, MA), SMAD2 (1:1000; Cell
7
145
signaling), and β-actin (1:1000). Blots were washed and incubated with HRP-
146
conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology Inc., Dallas,
147
TX) and developed with chemiluminescence.
148
Transfection and Luciferase Assays
149
Two million Jurkat cells were transfected using the Amaxa® Cell Line
150
Nucleofector® Kit V from Lonza (Köln Germany) according to the optimized
151
protocol provided with the kit. Two ug of plasmid DNA for TβRIIluc promoter,
152
KLF10, or empty vector (EV) were added to the cells. The nucleofection was
153
done using a Nucleofector®II device. Cells were divided into triplicate and
154
allowed to sit for 24 hours. Luciferase assays were done following the
155
manufacturer's recommendations (Promega, Madison, WI).
156
For primary CD8+ T cells transfection experiments, mouse CD8+ T cells form
157
spleens were negatively selected using the CD8+ T Cell Isolation kit from Miltenyi
158
Biotec. Ten million cells were transfected using the Amaxa Mouse T Cell
159
Nucleofector kit. For siRNA nucleofection, 600nM total scrambled or KLF10
160
targeted siRNA was used (ON-TARGET plus siRNA;Thermo Scientific
161
Dharmacon, Lafayette, CO). In order to quantify nucleofected cells, 2.5μg of
162
pMAXGFP® was added to each sample.
163 164
ChIP Assays
165
ChIP assays were performed as previously described (23). Two million murine
166
CAR-expressing CD8+ T cells were transfected with KLF10-His adenovirus or 8
167
empty vector. After 48 hours cells were treated with 1% formaldehyde to cross-
168
link histones to DNA. Fixed cells were sonicated to yield chromatin fragments of
169
200–1000 bp. Antibody used in the ChIP assays was Omni-probe (D-8) (catalog
170
# sc7270) from Santa Cruz Biotechnology (Santa Cruz, CA). DNA was recovered
171
by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation with the
172
addition of an inert carrier. Options for critically relevant control samples include
173
total IgG or pre-enriched chromatin (input). We chose to control with pre-enriched
174
chromatin because nonspecific IgG frequently does not control adequately for
175
nonspecific cross-reactivity. Furthermore, the chromatin input generates a more
176
accurate estimation of biases introduced through sonication of chromatin and
177
subsequent PCR (23). The TGF-βRII ChiP primers #1 to #4 have been
178
previously described (27).
179
Viral Infection
180
Recipient Thy1.1 mice were partially irradiated (400 rads) on day 1. Splenocytes
181
from B6-UBC-GFP and KLF10-/- (thy1.2) were isolated on day 2 and 7.2 X 106
182
total CD8+ T cells (1/1 mix of GFP and KLF10-/-) were adoptively transferred into
183
the thy1.1 recipients. The CD8+ T cells were positively selected on the MACS
184
column. Day 3 recipient mice were anesthetized with isoflurane and intracranially
185
injected with 2 X 106 PFUs of Daniels strains TMEV. Animals were euthanized 7
186
days after TMEV infection and spleen was harvested according to the Mayo
187
Clinic Institutional Animal Care and Use Committee standards. Splenocytes were
188
stained for the expression of cell surface TGF-βRII and viral Ag-tetramers and
189
analyzed by flow cytometry. Db:VP2121-130 and Db:GARC tetramers were made as
9
190
previously described (19). In additional experiments, splenocytes were stimulated
191
in vitro with viral peptides or control antigen and analyzed for the expression of
192
IFN-γ by intracellular staining among viral-specific CD8+ T cells.
193
Statistical Methodology
194
Statistical analyses were performed using GraphPad Software version 4
195
(GraphPad Software, Inc. La Jolla, CA, USA). Descriptive analyses including
196
means and standard deviations were performed in normally distributed
197
data. t tests were used to compare means between two groups. Paired t test was
198
used to compare means between paired samples. A p value of
1
Krüppel-like factor KLF10 regulates Transforming growth factor receptor II expression
2
and TGF-β signaling in CD8+ T lymphocytes
3 4 5 6 7 8 9 10 11 12 13
Konstantinos A. Papadakis1, James Krempski1, Jesse Reiter1, Phyllis Svingen1, Yuning Xiong1, Olga F. Sarmento1, April Huseby2, Aaron J. Johnson2, Gwen A. Lomberk3,Raul A. Urrutia3,and William A. Faubion1. 1
Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA Division of Immunology and Neurology, Mayo Clinic, Rochester, Minnesota, USA 3 Epigenetics and Chromatin Dynamics Laboratory, Departments of Medicine and Biochemistry and Molecular Biology. Epigenetic Translational Program, Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota, USA 2
14 15 16 17
2
18
Hepatology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Tel.: 507-284-2468;
19
Fax: 507-255-6318; E-mail: [email protected].
To whom correspondence should be addressed: Dept. of Gastroenterology and
20 21 22
The abbreviations used are: KLF; krüppel-like factor, TGF-βRII; TGF-β receptor II
1 Copyright © 2014 by the American Physiological Society.
23
ABSTRACT
24
KLF10 has recently elicited significant attention as a transcriptional regulator of
25
TGF-β1 signaling in CD4+ T cells. In the current study, we demonstrate a novel
26
role for KLF10 in the regulation of TGF-βRII expression with functional relevance
27
in antiviral immune response. Specifically, we show that KLF10-deficient mice
28
have an increased number of effector/memory CD8+ T cells, display higher levels
29
of the T helper type 1 cell-associated transcription factor T-bet, and produce
30
more IFN-γ following in vitro stimulation. In addition, KLF10-/- CD8+ T cells show
31
enhanced proliferation in vitro and homeostatic proliferation in vivo. Freshly
32
isolated CD8+ T cells from the spleen of adult mice express lower levels of
33
surface TGF-βRII (TβRII). Congruently, in vitro activation of KLF10-deficient
34
CD8+ T cells up-regulate TGF-βRII to a lesser extent compared to wt CD8+ T
35
cells which results in attenuated Smad-2 phosphorylation following TGF-β1
36
stimulation compared to wt CD8+ T cells. Moreover, we demonstrate that KLF10
37
directly binds to the TGF-βRII promoter in T cells leading to enhanced gene
38
expression. In vivo viral infection with Daniel's strain TMEV also led to lower
39
expression of TGF-βRII among viral-specific KLF10-/- CD8+ T cells and a higher
40
percentage of IFN-γ-producing CD8+ T cells in the spleen. Collectively, our data
41
reveal a critical role for KLF10 in the transcriptional activation of TGF-βRII in
42
CD8+ T cells. Thus, KLF10 regulation of TGF-βRII in this cell subset may likely
43
play a critical role in viral and tumor immune responses for which the integrity of
44
the TGF-β1/TGF- βRII signaling pathway is crucial.
45
2
46
INTRODUCTION
47 48
Krüppel-like factors (KLFs) have emerged as important regulators of immune
49
function (2, 7, 8, 15, 18, 23, 24, 29). KLF proteins constitute a family of transcription
50
factors that regulate the expression of a large number of genes with established
51
relevance to cell proliferation, apoptosis, differentiation, and transformation (2, 12, 23,
52
29). One of these proteins, KLF10 (formerly TGF-β inducible early gene 1; TIEG1) was
53
identified as a transcript that is rapidly induced after TGFβ treatment in human
54
osteoblasts (17). Under normal conditions, KLF10 regulates the growth and
55
differentiation of many epithelial cell types and its disruption is associated to human
56
diseases (12). KLF10 is also critical for the homeostasis of mesenchymal cells,
57
including the cellular components of tendons, bone, and muscle (12). More relevant to
58
the current study, recent reports have identified a role for KLF10 in T lymphocytes and
59
innate immune cells (21, 28). We have previously shown that the genetic inactivation of
60
KLF10, epigenetically silences FOXP3 via EZH2 (Enhancer of Zeste 2)-mediated
61
trimethylation of Histone 3 K27 resulting in an impaired induction of this gene with a
62
concomitant inappropriate adaptive T regulatory cell differentiation in vitro and in vivo
63
(23).
64
TGF-β acting through transforming growth factor-β (TGF-β) receptor I (TGF-βRI)
65
and II (TGF-βRII) plays a critical role also in the control of CD8+ T cell differentiation in
66
lymphoid and peripheral organs (26, 27). Indeed, recent studies have shown that TGF-
67
β-signaling promotes IL7Rα expression and CD8+ T cell differentiation (14). Moreover,
68
TGF-β-signaling inhibits the migration of effector CD8+ T cells from the spleen to the gut
3
69
by dampening the expression of the integrin α4β7 (26). T cell-specific deletion of TGF-
70
βRII receptor early in development (Tgfbr2f/f CD4-cre) leads to an early onset lethal
71
autoimmune disease (9, 11). Notably, however, the signals that control the expression
72
and regulation of TGF-βR and hence TGF-β1 signaling in T cells remain largely
73
unidentified (27). Our laboratory has focused on better understanding the functional role
74
of the transcription factor KLF10 in regulating TGF-β signaling in CD4+ T cells. Both our
75
group and Cao et. al., have previously shown that KLF10 constitutes an important
76
component of T regulatory cell suppressive function and CD4+CD25- T cell activation
77
through distinct mechanisms involving transforming growth factor (TGF)-beta1 and
78
Foxp3 (1, 23). Interestingly, KLF10-/- T reg cells have reduced suppressor function,
79
independent of Foxp3 expression, with decreased expression and elaboration of TGF-
80
beta1 (1). In response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and
81
Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-
82
intrinsic control of T cell activation (1, 23).
83
Thus, given the established importance of KLF10 in TGF-β signaling in CD4+ T
84
cells, in the current study, we hypothesize that this protein controls CD8+ T cell
85
responses by transcriptionally regulating genes encoding key signaling proteins within
86
this pathway. We hypothesized that the TGF βRII promoter is a good candidate for a
87
KLF10 target in T cells. We were guided by previous studies, performed in pancreatic
88
epithelial cells, which revealed the existence of several functional KLF cis-regulatory
89
sites within the TGF-βRII promoter (20). Indeed, our results show that KLF10 is critically
90
involved in the regulation of CD8+ T cell phenotype by transcriptionally regulating TGF-
91
βRII expression. Thus, insights into the KLF10-mediated mechanisms of TGF-βR
4
92
regulation are critical for understanding CD8+ T cell differentiation and function and thus
93
provide important therapeutic applications for vaccine development and the treatment of
94
certain autoimmune diseases and cancer.
95 96 97 98
5
99
MATERIALS AND METHODS
100
Mouse Strains
101
C57BL/6 mice were purchased from the Jackson Laboratory. KLF10−/− mice were
102
kindly provided by Thomas C. Spelsberg (Mayo Clinic, Rochester, MN)(16). The
103
CAR transgenic mouse was obtained through the NIAID Exchange Program of
104
the National Institutes of Health: Balb/cJ[Tg]CARdelta1-[Tg]DO11.10 mouse line
105
4285 (22).All of the mice used in experiments were of 6–8 weeks in age and have
106
been maintained under SPF conditions. The mice were age-matched in
107
experiments comparing wild type with KLF10−/−. All animal experiments were
108
performed per the recommendations outlined in the Guide for Care and Use of
109
Laboratory Animals from the National Institutes of Health as required by Mayo
110
Clinic. These guidelines were incorporated into the current study protocol
111
(IACUC No. A13313), which was reviewed and approved by the Institutional
112
Animal Care and Use Committee (IACUC), at Mayo Clinic, Rochester, MN.
113 114
Isolation of primary murine CD8+ T Cells and T cell Stimulation
115
Murine CD8+ splenocytes were isolated using a CD8+ T cell isolation kit (Miltenyi
116
Biotec, San Diego, CA). In vitro activation of murine T cells was done by plate-
117
bound anti-CD3, (clone 145-2C11, BD Biosciences) at 2 μg/ml. 100 units/ml of
118
IL-2 was added to the cultures throughout the incubation period. Recombinant
119
human TGFβ-1 (AUSTRAL Biologicals, San Ramon, CA ) at a concentration of 5
120
ng/ml was used to induce CD103 expression and SMAD2 phosphorylation.
121
6
122
Flow cytometry
123
Fluorescent dye–labeled Abs against murine CD8α, CD4, CD3, CD45.1, CD45.2,
124
CD62L, CD44, CD103 (integrin αE) and T-bet were purchased from BioLegend
125
(San Diego, CA). Anti-IFN-γ and anti-IL-17 Abs were from BioLegend.
126
Fluorescent dye–labeled antibody to TGF-βRII was from R & D Systems
127
(Minneapolis, MN). For intracellular cytokine staining, CD8+ T cells from wt or
128
KLF10-/- mice were stimulated with plate-bound anti-CD3 145-2C11
129
Biosciences, Franklin Lakes, NJ) in the presence of Golgi-stop (BD Biosciences)
130
for 4 h, followed by fixing and permeabilization according to the manufacturer’s
131
instructions (BD Biosciences). For cell division assay, purified CD8+ T cells were
132
stained with CFSE (Life Technologies, Grand Island, NY) and cultured in the
133
presence of anti-CD3 or IL-15 (30 ng/ml; R & D Systems) for 2–4 d. The cells
134
were analyzed on an LSRII or FACSCalibur (Becton Dickinson, Franklin Lakes,
135
NJ), and data were analyzed with FlowJo software (Tree Star, Ashland, OR).
(BD
136 137
Western blot
138
CD8+ T cells were activated with plate-bound (pb) anti-CD3 antibodies for 72
139
hours and incubated with TGF-β1 for different time points. The cells were lysed in
140
RIPA buffer (1X lysis buffer-150 with protease inhibitors) for p-SMAD2
141
measurement or Laemmli buffer for total SMAD2 and β-actin measurements.
142
Proteins were separated by 10% SDS/PAGE, transferred to membrane, blocked
143
with 5% milk and probed overnight with primary antibodies at the following
144
dilutions: p-SMAD2 (1:5000; Cell Signaling, Danvers, MA), SMAD2 (1:1000; Cell
7
145
signaling), and β-actin (1:1000). Blots were washed and incubated with HRP-
146
conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology Inc., Dallas,
147
TX) and developed with chemiluminescence.
148
Transfection and Luciferase Assays
149
Two million Jurkat cells were transfected using the Amaxa® Cell Line
150
Nucleofector® Kit V from Lonza (Köln Germany) according to the optimized
151
protocol provided with the kit. Two ug of plasmid DNA for TβRIIluc promoter,
152
KLF10, or empty vector (EV) were added to the cells. The nucleofection was
153
done using a Nucleofector®II device. Cells were divided into triplicate and
154
allowed to sit for 24 hours. Luciferase assays were done following the
155
manufacturer's recommendations (Promega, Madison, WI).
156
For primary CD8+ T cells transfection experiments, mouse CD8+ T cells form
157
spleens were negatively selected using the CD8+ T Cell Isolation kit from Miltenyi
158
Biotec. Ten million cells were transfected using the Amaxa Mouse T Cell
159
Nucleofector kit. For siRNA nucleofection, 600nM total scrambled or KLF10
160
targeted siRNA was used (ON-TARGET plus siRNA;Thermo Scientific
161
Dharmacon, Lafayette, CO). In order to quantify nucleofected cells, 2.5μg of
162
pMAXGFP® was added to each sample.
163 164
ChIP Assays
165
ChIP assays were performed as previously described (23). Two million murine
166
CAR-expressing CD8+ T cells were transfected with KLF10-His adenovirus or 8
167
empty vector. After 48 hours cells were treated with 1% formaldehyde to cross-
168
link histones to DNA. Fixed cells were sonicated to yield chromatin fragments of
169
200–1000 bp. Antibody used in the ChIP assays was Omni-probe (D-8) (catalog
170
# sc7270) from Santa Cruz Biotechnology (Santa Cruz, CA). DNA was recovered
171
by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation with the
172
addition of an inert carrier. Options for critically relevant control samples include
173
total IgG or pre-enriched chromatin (input). We chose to control with pre-enriched
174
chromatin because nonspecific IgG frequently does not control adequately for
175
nonspecific cross-reactivity. Furthermore, the chromatin input generates a more
176
accurate estimation of biases introduced through sonication of chromatin and
177
subsequent PCR (23). The TGF-βRII ChiP primers #1 to #4 have been
178
previously described (27).
179
Viral Infection
180
Recipient Thy1.1 mice were partially irradiated (400 rads) on day 1. Splenocytes
181
from B6-UBC-GFP and KLF10-/- (thy1.2) were isolated on day 2 and 7.2 X 106
182
total CD8+ T cells (1/1 mix of GFP and KLF10-/-) were adoptively transferred into
183
the thy1.1 recipients. The CD8+ T cells were positively selected on the MACS
184
column. Day 3 recipient mice were anesthetized with isoflurane and intracranially
185
injected with 2 X 106 PFUs of Daniels strains TMEV. Animals were euthanized 7
186
days after TMEV infection and spleen was harvested according to the Mayo
187
Clinic Institutional Animal Care and Use Committee standards. Splenocytes were
188
stained for the expression of cell surface TGF-βRII and viral Ag-tetramers and
189
analyzed by flow cytometry. Db:VP2121-130 and Db:GARC tetramers were made as
9
190
previously described (19). In additional experiments, splenocytes were stimulated
191
in vitro with viral peptides or control antigen and analyzed for the expression of
192
IFN-γ by intracellular staining among viral-specific CD8+ T cells.
193
Statistical Methodology
194
Statistical analyses were performed using GraphPad Software version 4
195
(GraphPad Software, Inc. La Jolla, CA, USA). Descriptive analyses including
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means and standard deviations were performed in normally distributed
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data. t tests were used to compare means between two groups. Paired t test was
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used to compare means between paired samples. A p value of
