data_sheet_1.docx

<p>A relatively high affinity/avidity of T cell receptor (TCR) recognition for self-peptide bound to major histocompatibility complex II (self-pMHC) ligands is a distinctive feature of CD4 T regulatory (Treg) cells, including their development in the thymus and maintenance of their suppressive functions in the periphery. Despite such high self-reactivity, however, all thymic-derived peripheral Treg populations are neither homogenous in their phenotype nor uniformly immune-suppressive in their function under steady state condition. We show here that based on the previously defined heterogeneity in the phenotype of peripheral Treg populations, Ly6C expression on Treg marks a lower degree of activation, proliferation, and differentiation status as well as functional incompetence. We also demonstrate that Ly6C expression on Treg in a steady state is either up- or downregulated depending on relative amounts of tonic TCR signals derived from its contacts with self-ligands. Interestingly, peripheral appearance and maintenance of these Ly6C-expressing Treg cells largely differed in an age-dependent manner, with their proportion being continuously increased from perinatal to young adult period but then being gradually declined with age. The reduction of Ly6C⁺ Treg in the aged mice was not due to their augmented cell death but rather resulted from downregulation of Ly6C expression. The Ly6C downregulation was accompanied by proliferation of Ly6C⁺ Treg cells and subsequent change into Ly6C⁻ effector Treg with concomitant restoration of immune-suppressive activity. Importantly, we found that this phenotypic and functional change of Ly6C⁺ Treg is largely driven by conventional effector T cell population. Collectively, these findings suggest a potential cross-talk between peripheral Treg subsets and effector T cells and provides better understanding for Treg homeostasis and function on maintaining self-tolerance.</p

miR-21 contributes to collagen production in fibroblasts MRC-5 cells were transfected with 500nM of anti-miR-21 and then were harvested at indicative times.

<p>These data represent one of the experiments performed four times with duplicate samples (A-C). (A) The level of miR-21 level by taqman miR real-time PCR using U6 snRNA for an internal control (B) The mRNA levels of Col1A2 and Col3A1 by real-time PCR analysis and β-actin was applied for an loading control (C) The protein level of collagen 3 type α1 (Col3A1) at indicative time after miR-21 transfection (30nM) was determined by immunoblotting analysis. β-actin was used for an equal loading control. (D-E) Each sample was loaded as a duplicated and three independent experiments were achieved. The representative results were shown. (D) The level of miR-21 level by taqman miR real-time PCR using U6 snRNA for as internal control (E) The mRNA levels of Col1A2 and Col3A1 by real-time PCR, β-actin for an equal loading control (F) The level of collagen (Col3A1) was determined by immunoblotting analysis. β-actin was applied for an loading control. (G) The mRNA level of Smad7 of mouse lung at 2 weeks after radiation exposure was examined by real-time PCR analysis (n = 3). (H) The levels of α-SMA, Smad2 phosphorylation (pSmad2), Smad7 and collagen (Col3A1) were determined by immunoblotting analysis after ectopic expression of miR-21 mimic. α-tubulin for equal loading control (ns: not significant, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****))</p

Upregulation of miR-21 in IR-damaged lung tissue.

<p>(A) Venn diagram for upregulated miRs from the microRNA array on SBRT tissue (SBRT), Ingenuity pathway analysis (IPA) and Idiopathic pulmonary fibrosis (IPF). (B) miR-21 expression in IR treated lung (left side) compared to control (right side) in 3 week and 4 weeks after IR treatment. The average ct values for miR-21 of 3 week and 4 week are 18.6 and 17.43 respectively. (n = 3, p<0.05 (*), p<0.001 (***)) (C) Level of miR-21 in the left side of lung compared to the control (right side of lung) at indicative time after IR was determined by taqman miR real-time PCR using U6 snRNA for an internal control. (n = 4, p<0.0001 (****)) (D) Heat-map of miR-21 target genes from the IR lung tissue compared with control for indicated time point. (E) In situ hybridization for miR-21 with either scramble probes (miRScr, top panels) or miR-21 (miR-21, middle and bottom panels) in right side of lung (Cont) or left side of lung (IR) (left panel). Pixel density of miR-21 positive area was quantified and shown as a bar graph (right panel).</p

Local IR exposure mimicking SBRT induces lung fibrosis.

<p>(A) Schematic diagram of IR exposure on mouse lung (left panel). Lung tissue isolated from the left lung was stained with Masson’s Trichrome at indicative time after 90Gy exposure (right panel). The representative images were shown (right panel) (n = 4). (B) Heat-map of collagen subtypes from the IR lung tissue (left side lung) compared to the control (right side of lung) (n = 4) (red dotted line and asterisk for significantly upregulated collagen subtypes after IR) (C) The mRNA level of Col1A2 was determined by real-time PCR at indicative time after 90Gy exposure (n = 4, * p<0.05).</p

miR-21 expression is dispensible for EndMT HPECs were transfected with scramble miRs (miRN.C inh) or miR-21 inhibitor (anti-miR-21) (500nM).

<p>24 hours after, cells were exposed to 5 Gy of X-ray (IR) and were harvested after 3 days. (A-D) Each sample was loaded as a duplicated and four independent experiments were achieved. The representative results were shown. (A) The level of miR-21 after either scramble miRs (miRN.C) or miR-21 inhibitor (anti-miR-21) transfection was determined by taqman miR realtime-PCR. U6 snRNA for equal loading control (B) The mRNA levels of ICAM-1 by real-time PCR analysis 3 days after 5 Gy of X-ray (C) The mRNA levels of α-SMA (top panel), FN (bottom panel) by real-time PCR (D) The mRNA levels of collagen subtypes (Col1A2: top panel and Col3A1: bottom panel) by real-time PCR, β-actin for a equal loading control. (E) HPECs were immunostained with phalloidin (green) and α-VE-cadherin antibody (red). DAPI was used for nuclear counterstaining (blue). Corresponding bright-field microscopic images were displayed in the bottom panel. (F) Protein level of CD31 and p21 were determined by immunoblotting analysis. β-actin was used for an equal loading control. (G-H) HPECs were transfected with 30nM of miR-21 mimics (hsa-mir-21-3p and -5p) and then were harvested at 48hr. Each sample was loaded as a duplicated and three independent experiments were accomplished. The representative results were presented. (G) The level of miR-21 level by taqman miR real-time PCR with U6 snRNA for an internal control (H) The mRNA levels of α-SMA and FN by real-time PCR, β-actin was used for an equal loading control. (ns: not significant, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****))</p

IR promotes EndMT in lung ECs concurrent with miR-21 induction HPECs were exposed with either 5 or 20 Gy of X-ray and were harvested after 3 days.

<p>(A) The mRNA levels of ICAM-1 (left panel), α-SMA (middle panel), FN (right panel) were determined by real-time PCR analysis. β-actin for an equal loading control. Each data represents one of the experiments conducted three times with duplicate samples. (B) The protein level of CD31, ICAM-1, α-SMA and p21 were determined by immunoblotting analysis. α-tubulin was used for loading control (C) HPECs was immunostained with phalloidin (green) and α-VE-cadherin antibody (red). DAPI was used for nuclear counterstaining (blue). Corresponding bright-field microscopic images were displayed in the bottom panel. (D) The mRNA levels of collagen genes (Col1A2 and Col3A1) were determined by real-time PCR analysis. β-actin for a loading control (E) The level of miR-21 of HPECs was determined via taqman miR real-time PCR using U6 snRNA for an internal control. (D-E) Each result reproduced four times and the representative results were shown. ((p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****)).</p

Data_Sheet_6.PDF

<p>γδ T cells, known to be an important source of innate IL-17 in mice, provide critical contributions to host immune responses. Development and function of γδ T cells are directed by networks of diverse transcription factors (TFs). Here, we examine the role of the zinc finger TFs, Kruppel-like factor 10 (KLF10), in the regulation of IL-17-committed CD27⁻ γδ T (γδ²⁷⁻-17) cells. We found selective augmentation of Vγ4⁺ γδ²⁷⁻ cells with higher IL-17 production in KLF10-deficient mice. Surprisingly, KLF10-deficient CD127^hi Vγ4⁺ γδ²⁷⁻-17 cells expressed higher levels of CD5 than their wild-type counterparts, with hyper-responsiveness to cytokine, but not T-cell receptor, stimuli. Thymic maturation of Vγ4⁺ γδ²⁷⁻ cells was enhanced in newborn mice deficient in KLF10. Finally, a mixed bone marrow chimera study indicates that intrinsic KLF10 signaling is requisite to limit Vγ4⁺ γδ²⁷⁻-17 cells. Collectively, these findings demonstrate that KLF10 regulates thymic development of Vγ4⁺ γδ²⁷⁻ cells and their peripheral homeostasis at steady state.</p

Data_Sheet_3.PDF

<p>γδ T cells, known to be an important source of innate IL-17 in mice, provide critical contributions to host immune responses. Development and function of γδ T cells are directed by networks of diverse transcription factors (TFs). Here, we examine the role of the zinc finger TFs, Kruppel-like factor 10 (KLF10), in the regulation of IL-17-committed CD27⁻ γδ T (γδ²⁷⁻-17) cells. We found selective augmentation of Vγ4⁺ γδ²⁷⁻ cells with higher IL-17 production in KLF10-deficient mice. Surprisingly, KLF10-deficient CD127^hi Vγ4⁺ γδ²⁷⁻-17 cells expressed higher levels of CD5 than their wild-type counterparts, with hyper-responsiveness to cytokine, but not T-cell receptor, stimuli. Thymic maturation of Vγ4⁺ γδ²⁷⁻ cells was enhanced in newborn mice deficient in KLF10. Finally, a mixed bone marrow chimera study indicates that intrinsic KLF10 signaling is requisite to limit Vγ4⁺ γδ²⁷⁻-17 cells. Collectively, these findings demonstrate that KLF10 regulates thymic development of Vγ4⁺ γδ²⁷⁻ cells and their peripheral homeostasis at steady state.</p

Data_Sheet_2.PDF

<p>γδ T cells, known to be an important source of innate IL-17 in mice, provide critical contributions to host immune responses. Development and function of γδ T cells are directed by networks of diverse transcription factors (TFs). Here, we examine the role of the zinc finger TFs, Kruppel-like factor 10 (KLF10), in the regulation of IL-17-committed CD27⁻ γδ T (γδ²⁷⁻-17) cells. We found selective augmentation of Vγ4⁺ γδ²⁷⁻ cells with higher IL-17 production in KLF10-deficient mice. Surprisingly, KLF10-deficient CD127^hi Vγ4⁺ γδ²⁷⁻-17 cells expressed higher levels of CD5 than their wild-type counterparts, with hyper-responsiveness to cytokine, but not T-cell receptor, stimuli. Thymic maturation of Vγ4⁺ γδ²⁷⁻ cells was enhanced in newborn mice deficient in KLF10. Finally, a mixed bone marrow chimera study indicates that intrinsic KLF10 signaling is requisite to limit Vγ4⁺ γδ²⁷⁻-17 cells. Collectively, these findings demonstrate that KLF10 regulates thymic development of Vγ4⁺ γδ²⁷⁻ cells and their peripheral homeostasis at steady state.</p

Data_Sheet_4.PDF

<p>γδ T cells, known to be an important source of innate IL-17 in mice, provide critical contributions to host immune responses. Development and function of γδ T cells are directed by networks of diverse transcription factors (TFs). Here, we examine the role of the zinc finger TFs, Kruppel-like factor 10 (KLF10), in the regulation of IL-17-committed CD27⁻ γδ T (γδ²⁷⁻-17) cells. We found selective augmentation of Vγ4⁺ γδ²⁷⁻ cells with higher IL-17 production in KLF10-deficient mice. Surprisingly, KLF10-deficient CD127^hi Vγ4⁺ γδ²⁷⁻-17 cells expressed higher levels of CD5 than their wild-type counterparts, with hyper-responsiveness to cytokine, but not T-cell receptor, stimuli. Thymic maturation of Vγ4⁺ γδ²⁷⁻ cells was enhanced in newborn mice deficient in KLF10. Finally, a mixed bone marrow chimera study indicates that intrinsic KLF10 signaling is requisite to limit Vγ4⁺ γδ²⁷⁻-17 cells. Collectively, these findings demonstrate that KLF10 regulates thymic development of Vγ4⁺ γδ²⁷⁻ cells and their peripheral homeostasis at steady state.</p