Articles in PresS. Am J Physiol Cell Physiol (December 3, 2014). doi:10.1152/ajpcell.00262.2014
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Krüppel-like factor KLF10 regulates Transforming growth factor receptor II expression
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and TGF-β signaling in CD8+ T lymphocytes
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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
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Hepatology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Tel.: 507-284-2468;
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Fax: 507-255-6318; E-mail:
[email protected].
To whom correspondence should be addressed: Dept. of Gastroenterology and
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The abbreviations used are: KLF; krüppel-like factor, TGF-βRII; TGF-β receptor II
1 Copyright © 2014 by the American Physiological Society.
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ABSTRACT
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KLF10 has recently elicited significant attention as a transcriptional regulator of
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TGF-β1 signaling in CD4+ T cells. In the current study, we demonstrate a novel
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role for KLF10 in the regulation of TGF-βRII expression with functional relevance
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in antiviral immune response. Specifically, we show that KLF10-deficient mice
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have an increased number of effector/memory CD8+ T cells, display higher levels
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of the T helper type 1 cell-associated transcription factor T-bet, and produce
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more IFN-γ following in vitro stimulation. In addition, KLF10-/- CD8+ T cells show
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enhanced proliferation in vitro and homeostatic proliferation in vivo. Freshly
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isolated CD8+ T cells from the spleen of adult mice express lower levels of
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surface TGF-βRII (TβRII). Congruently, in vitro activation of KLF10-deficient
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CD8+ T cells up-regulate TGF-βRII to a lesser extent compared to wt CD8+ T
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cells which results in attenuated Smad-2 phosphorylation following TGF-β1
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stimulation compared to wt CD8+ T cells. Moreover, we demonstrate that KLF10
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directly binds to the TGF-βRII promoter in T cells leading to enhanced gene
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expression. In vivo viral infection with Daniel's strain TMEV also led to lower
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expression of TGF-βRII among viral-specific KLF10-/- CD8+ T cells and a higher
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percentage of IFN-γ-producing CD8+ T cells in the spleen. Collectively, our data
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reveal a critical role for KLF10 in the transcriptional activation of TGF-βRII in
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CD8+ T cells. Thus, KLF10 regulation of TGF-βRII in this cell subset may likely
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play a critical role in viral and tumor immune responses for which the integrity of
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the TGF-β1/TGF- βRII signaling pathway is crucial.
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INTRODUCTION
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Krüppel-like factors (KLFs) have emerged as important regulators of immune
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function (2, 7, 8, 15, 18, 23, 24, 29). KLF proteins constitute a family of transcription
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factors that regulate the expression of a large number of genes with established
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relevance to cell proliferation, apoptosis, differentiation, and transformation (2, 12, 23,
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29). One of these proteins, KLF10 (formerly TGF-β inducible early gene 1; TIEG1) was
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identified as a transcript that is rapidly induced after TGFβ treatment in human
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osteoblasts (17). Under normal conditions, KLF10 regulates the growth and
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differentiation of many epithelial cell types and its disruption is associated to human
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diseases (12). KLF10 is also critical for the homeostasis of mesenchymal cells,
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including the cellular components of tendons, bone, and muscle (12). More relevant to
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the current study, recent reports have identified a role for KLF10 in T lymphocytes and
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innate immune cells (21, 28). We have previously shown that the genetic inactivation of
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KLF10, epigenetically silences FOXP3 via EZH2 (Enhancer of Zeste 2)-mediated
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trimethylation of Histone 3 K27 resulting in an impaired induction of this gene with a
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concomitant inappropriate adaptive T regulatory cell differentiation in vitro and in vivo
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(23).
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TGF-β acting through transforming growth factor-β (TGF-β) receptor I (TGF-βRI)
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and II (TGF-βRII) plays a critical role also in the control of CD8+ T cell differentiation in
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lymphoid and peripheral organs (26, 27). Indeed, recent studies have shown that TGF-
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β-signaling promotes IL7Rα expression and CD8+ T cell differentiation (14). Moreover,
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TGF-β-signaling inhibits the migration of effector CD8+ T cells from the spleen to the gut
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by dampening the expression of the integrin α4β7 (26). T cell-specific deletion of TGF-
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βRII receptor early in development (Tgfbr2f/f CD4-cre) leads to an early onset lethal
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autoimmune disease (9, 11). Notably, however, the signals that control the expression
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and regulation of TGF-βR and hence TGF-β1 signaling in T cells remain largely
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unidentified (27). Our laboratory has focused on better understanding the functional role
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of the transcription factor KLF10 in regulating TGF-β signaling in CD4+ T cells. Both our
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group and Cao et. al., have previously shown that KLF10 constitutes an important
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component of T regulatory cell suppressive function and CD4+CD25- T cell activation
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through distinct mechanisms involving transforming growth factor (TGF)-beta1 and
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Foxp3 (1, 23). Interestingly, KLF10-/- T reg cells have reduced suppressor function,
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independent of Foxp3 expression, with decreased expression and elaboration of TGF-
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beta1 (1). In response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and
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Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-
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intrinsic control of T cell activation (1, 23).
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Thus, given the established importance of KLF10 in TGF-β signaling in CD4+ T
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cells, in the current study, we hypothesize that this protein controls CD8+ T cell
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responses by transcriptionally regulating genes encoding key signaling proteins within
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this pathway. We hypothesized that the TGF βRII promoter is a good candidate for a
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KLF10 target in T cells. We were guided by previous studies, performed in pancreatic
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epithelial cells, which revealed the existence of several functional KLF cis-regulatory
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sites within the TGF-βRII promoter (20). Indeed, our results show that KLF10 is critically
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involved in the regulation of CD8+ T cell phenotype by transcriptionally regulating TGF-
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βRII expression. Thus, insights into the KLF10-mediated mechanisms of TGF-βR
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regulation are critical for understanding CD8+ T cell differentiation and function and thus
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provide important therapeutic applications for vaccine development and the treatment of
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certain autoimmune diseases and cancer.
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MATERIALS AND METHODS
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Mouse Strains
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C57BL/6 mice were purchased from the Jackson Laboratory. KLF10−/− mice were
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kindly provided by Thomas C. Spelsberg (Mayo Clinic, Rochester, MN)(16). The
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CAR transgenic mouse was obtained through the NIAID Exchange Program of
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the National Institutes of Health: Balb/cJ[Tg]CARdelta1-[Tg]DO11.10 mouse line
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4285 (22).All of the mice used in experiments were of 6–8 weeks in age and have
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been maintained under SPF conditions. The mice were age-matched in
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experiments comparing wild type with KLF10−/−. All animal experiments were
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performed per the recommendations outlined in the Guide for Care and Use of
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Laboratory Animals from the National Institutes of Health as required by Mayo
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Clinic. These guidelines were incorporated into the current study protocol
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(IACUC No. A13313), which was reviewed and approved by the Institutional
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Animal Care and Use Committee (IACUC), at Mayo Clinic, Rochester, MN.
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Isolation of primary murine CD8+ T Cells and T cell Stimulation
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Murine CD8+ splenocytes were isolated using a CD8+ T cell isolation kit (Miltenyi
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Biotec, San Diego, CA). In vitro activation of murine T cells was done by plate-
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bound anti-CD3, (clone 145-2C11, BD Biosciences) at 2 μg/ml. 100 units/ml of
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IL-2 was added to the cultures throughout the incubation period. Recombinant
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human TGFβ-1 (AUSTRAL Biologicals, San Ramon, CA ) at a concentration of 5
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ng/ml was used to induce CD103 expression and SMAD2 phosphorylation.
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Flow cytometry
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Fluorescent dye–labeled Abs against murine CD8α, CD4, CD3, CD45.1, CD45.2,
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CD62L, CD44, CD103 (integrin αE) and T-bet were purchased from BioLegend
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(San Diego, CA). Anti-IFN-γ and anti-IL-17 Abs were from BioLegend.
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Fluorescent dye–labeled antibody to TGF-βRII was from R & D Systems
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(Minneapolis, MN). For intracellular cytokine staining, CD8+ T cells from wt or
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KLF10-/- mice were stimulated with plate-bound anti-CD3 145-2C11
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Biosciences, Franklin Lakes, NJ) in the presence of Golgi-stop (BD Biosciences)
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for 4 h, followed by fixing and permeabilization according to the manufacturer’s
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instructions (BD Biosciences). For cell division assay, purified CD8+ T cells were
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stained with CFSE (Life Technologies, Grand Island, NY) and cultured in the
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presence of anti-CD3 or IL-15 (30 ng/ml; R & D Systems) for 2–4 d. The cells
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were analyzed on an LSRII or FACSCalibur (Becton Dickinson, Franklin Lakes,
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NJ), and data were analyzed with FlowJo software (Tree Star, Ashland, OR).
(BD
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Western blot
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CD8+ T cells were activated with plate-bound (pb) anti-CD3 antibodies for 72
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hours and incubated with TGF-β1 for different time points. The cells were lysed in
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RIPA buffer (1X lysis buffer-150 with protease inhibitors) for p-SMAD2
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measurement or Laemmli buffer for total SMAD2 and β-actin measurements.
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Proteins were separated by 10% SDS/PAGE, transferred to membrane, blocked
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with 5% milk and probed overnight with primary antibodies at the following
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dilutions: p-SMAD2 (1:5000; Cell Signaling, Danvers, MA), SMAD2 (1:1000; Cell
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signaling), and β-actin (1:1000). Blots were washed and incubated with HRP-
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conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology Inc., Dallas,
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TX) and developed with chemiluminescence.
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Transfection and Luciferase Assays
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Two million Jurkat cells were transfected using the Amaxa® Cell Line
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Nucleofector® Kit V from Lonza (Köln Germany) according to the optimized
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protocol provided with the kit. Two ug of plasmid DNA for TβRIIluc promoter,
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KLF10, or empty vector (EV) were added to the cells. The nucleofection was
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done using a Nucleofector®II device. Cells were divided into triplicate and
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allowed to sit for 24 hours. Luciferase assays were done following the
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manufacturer's recommendations (Promega, Madison, WI).
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For primary CD8+ T cells transfection experiments, mouse CD8+ T cells form
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spleens were negatively selected using the CD8+ T Cell Isolation kit from Miltenyi
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Biotec. Ten million cells were transfected using the Amaxa Mouse T Cell
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Nucleofector kit. For siRNA nucleofection, 600nM total scrambled or KLF10
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targeted siRNA was used (ON-TARGET plus siRNA;Thermo Scientific
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Dharmacon, Lafayette, CO). In order to quantify nucleofected cells, 2.5μg of
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pMAXGFP® was added to each sample.
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ChIP Assays
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ChIP assays were performed as previously described (23). Two million murine
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CAR-expressing CD8+ T cells were transfected with KLF10-His adenovirus or 8
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empty vector. After 48 hours cells were treated with 1% formaldehyde to cross-
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link histones to DNA. Fixed cells were sonicated to yield chromatin fragments of
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200–1000 bp. Antibody used in the ChIP assays was Omni-probe (D-8) (catalog
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# sc7270) from Santa Cruz Biotechnology (Santa Cruz, CA). DNA was recovered
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by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation with the
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addition of an inert carrier. Options for critically relevant control samples include
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total IgG or pre-enriched chromatin (input). We chose to control with pre-enriched
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chromatin because nonspecific IgG frequently does not control adequately for
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nonspecific cross-reactivity. Furthermore, the chromatin input generates a more
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accurate estimation of biases introduced through sonication of chromatin and
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subsequent PCR (23). The TGF-βRII ChiP primers #1 to #4 have been
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previously described (27).
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Viral Infection
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Recipient Thy1.1 mice were partially irradiated (400 rads) on day 1. Splenocytes
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from B6-UBC-GFP and KLF10-/- (thy1.2) were isolated on day 2 and 7.2 X 106
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total CD8+ T cells (1/1 mix of GFP and KLF10-/-) were adoptively transferred into
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the thy1.1 recipients. The CD8+ T cells were positively selected on the MACS
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column. Day 3 recipient mice were anesthetized with isoflurane and intracranially
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injected with 2 X 106 PFUs of Daniels strains TMEV. Animals were euthanized 7
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days after TMEV infection and spleen was harvested according to the Mayo
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Clinic Institutional Animal Care and Use Committee standards. Splenocytes were
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stained for the expression of cell surface TGF-βRII and viral Ag-tetramers and
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analyzed by flow cytometry. Db:VP2121-130 and Db:GARC tetramers were made as
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previously described (19). In additional experiments, splenocytes were stimulated
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in vitro with viral peptides or control antigen and analyzed for the expression of
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IFN-γ by intracellular staining among viral-specific CD8+ T cells.
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Statistical Methodology
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Statistical analyses were performed using GraphPad Software version 4
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(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