approved final version of manuscript; J

approved final version of manuscript; J.K., J.R., P.S., Y.X., O.F.S., A.H., and A.J.J. strain Theiler’s PRKAA2 murine encephalomyelitis computer virus (TMEV) also led to lower expression of TGF-RII among viral-specific KLF10?/? CD8+ T cells and a higher percentage of IFN–producing CD8+ T cells in the spleen. Collectively, our data reveal a critical role for KLF10 in the Ophiopogonin D’ transcriptional activation of TGF-RII in CD8+ T cells. Thus, KLF10 regulation of TGF-RII in this cell subset may likely play a critical role in viral and tumor immune responses for which the integrity of the TGF-1/TGF-RII signaling pathway is crucial. via EZH2 (enhancer of zeste 2)-mediated trimethylation of histone 3 K27, resulting in an impaired induction of this gene with a concomitant inappropriate adaptive T regulatory (Treg) cell differentiation in vitro and in vivo (23). TGF- acting through TGF- receptor I (TGF-RI) and II (TGF-RII) plays a critical role also in the control of CD8+ T cell differentiation in lymphoid and peripheral organs (26, 27). Indeed, recent studies have shown that TGF- signaling promotes IL-7R expression and CD8+ T cell differentiation (14). Moreover, TGF- signaling inhibits the migration of effector CD8+ T cells from the spleen to the gut by dampening the expression of the integrin 47 (26). T cell-specific deletion of TGF-RII receptor early in development (Tgfbr2f/f CD4-cre) leads to an early onset lethal autoimmune disease (9, 11). Notably, however, the signals that control the expression and regulation of TGF-R and hence TGF-1 signaling in T cells remain largely unidentified (27). Our laboratory has focused on better understanding the functional role of the transcription factor KLF10 in regulating TGF- signaling in CD4+ T cells. Both our group (23) and Cao et al. (1) have previously shown that KLF10 constitutes an important component of T regulatory cell-suppressive function and CD4+CD25? T cell activation through distinct mechanisms involving TGF-1 and Foxp3. Interestingly, KLF10?/? Treg cells have reduced suppressor function, impartial of Foxp3 expression, with decreased expression and elaboration of TGF-1 (1). In response to TGF-1, KLF10 can transactivate both TGF- and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation (1, 23). Thus, given the established importance of KLF10 in TGF- signaling in CD4+ T cells, in the current study, we hypothesize that this protein controls CD8+ T cell responses by transcriptionally regulating genes Ophiopogonin D’ encoding key signaling proteins within this pathway.1 We hypothesized that this TGF-RII promoter is a good candidate for a KLF10 target in T cells. We were guided by previous studies, performed in pancreatic epithelial cells, which revealed the presence of several functional KLF from the National Institutes of Health as required by Mayo Clinic. These guidelines were incorporated into the current study protocol (IACUC no. A13313), which was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic (Rochester, MN). Isolation of primary murine CD8+ T cells and T cell stimulation. Murine CD8+ splenocytes were isolated using a CD8+ T cell isolation kit (Miltenyi Biotec, San Diego, CA). In vitro activation of murine T cells was done by plate-bound anti-CD3, (clone 145-2C11, BD Biosciences) at 2 g/ml. IL-2 (100 U/ml) was added to the cultures throughout the incubation period. Recombinant human TGF-1 (Austral Biologicals, San Ramon, CA) at a concentration of 5 ng/ml was used to induce CD103 expression and SMAD2 phosphorylation. Flow cytometry. Fluorescent dye-labeled Abs against murine CD8, CD4, CD3, CD45.1, CD45.2, CD62L, CD44, CD103 (integrin E), and T-bet were purchased from BioLegend (San Diego, CA). Anti-IFN- and anti-IL-17 Abs were from BioLegend. Fluorescent dye-labeled antibody to TGF-RII was from R&D Systems (Minneapolis, MN). For intracellular cytokine staining, CD8+ T cells from WT or KLF10?/? mice were stimulated with plate-bound anti-CD3 145C2C11 (BD Biosciences, Franklin Lakes, NJ) in the presence of Golgi-stop (BD Biosciences) for 4 h, followed by fixing and permeabilization according to the manufacturer’s instructions (BD Ophiopogonin D’ Biosciences). For cell division assay, purified CD8+ T cells were stained with CFSE (Life Technologies, Grand Island, NY) Ophiopogonin D’ and cultured in the presence of anti-CD3 or IL-15 (30 ng/ml; R&D Systems) for 2C4 days. The cells were analyzed on an LSRII or FACSCalibur (Becton Dickinson, Franklin Lakes, NJ), and data were analyzed with FlowJo software (Tree Star, Ashland, OR). Western blot analysis. CD8+ T cells were activated with plate-bound anti-CD3 antibodies for 72 h and.