Journal: Oncotarget

Article Title: Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

doi: 10.18632/oncotarget.11395

Figure Lengend Snippet: A. Foxp3 expression is examined with western blotting assay in three T24 bladder cancer sublines (T24-P, T24-L, and T24-B). B. Assays for glucose, lactate, and ATP production in three T24 sublines. C. VEGF mRNA and D. GLUT-1~5 mRNA expression is examined in three T24 sublines using qRT-PCR assay. E. Foxp3 mRNA expression is examined in Foxp3-knocking down T24-B transfectants and its control. F. Assays for glucose and lactate production, G. VEGF mRNA expression, and H. GLUT-1~5 as assayed in Foxp3-knocking down transfectant #25 and its control. I. Foxp3 expression is examined with western blotting assay after transient transfection of Foxp3 plasmid into T24-P bladder cancer cells. J. Assays for glucose and lactate production, K. VEGF mRNA expression, and L. GLUT-1~5 as assayed in pooled Foxp3-overexpressing T24-P transfectants and its control. T24-P, T24 parental subline; T24-L, metastatic lung T24 subline; T24-B, metastatic bone T24 subline; ATP, Adenosine triphosphate; GLUT, glucose transporter; EGFR, epidermal growth factor receptor; VEGF, vascular endothelial growth factor; NS, not significant; *, p<0.05; **, p<0.01, ***, p<0.001. Data are represented as mean ± SD.

Article Snippet: The plasmid pCMV3-N-HA-Foxp3 was purchased from Sino Biological Inc. (catalog number: HG11652-NY, Beijing, China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

doi: 10.18632/oncotarget.11395

Figure Lengend Snippet: A. T24-B cell were stained with anti-Foxp3, anti-HIF-1α and DAPI for confocal microscopy studies in normoxic and hypoxic circumstance as indicated times. Scale bars, 10 μm. B. T24-B cell were maintained in normoxic and hypoxic circumstance as indicated times. Cell lysates were immunoprecipitated with anti-Foxp3 antibody and subjected to Foxp3 and HIF-1α western blot.

Article Snippet: The plasmid pCMV3-N-HA-Foxp3 was purchased from Sino Biological Inc. (catalog number: HG11652-NY, Beijing, China).

Techniques: Staining, Confocal Microscopy, Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

doi: 10.18632/oncotarget.11395

Figure Lengend Snippet: A. HIF-1α mRNA expression were examined in T24-B Foxp3 knocking down cells and its control both in normoxic and hypoxic circumstances using qRT-PCR assays. B. Foxp3-knocking down T24B cells and its control were maintained in normoxic and hypoxic circumstance and cell lysates for harvested for western blot. C. After transfection, cells were maintained in normoxic and hypoxic circumstance and treated with MG132 (10μM) for 24 hours before harvesting for western blotting assay. D. Foxp3 and HIF-1α mRNA expression were examined in Foxp3-overexpressing T24-P cells and its control in normoxic circumstance using qRT-PCR assays. E. T24-P cells were transfected with Foxp3 cDNA-containing plasmids or control as indicated conditions with or without MG132 treatment. Cell lysates were immunoprecipitated with anti-Foxp3 or anti-ubiquitin antibody and subjected to Foxp3, HIF-1α or ubiquitin western blot. F. In vivo ubiquitination assay. T24-P cells were transfected with Foxp3 cDNA-carrying plasmid in a dose-dependent manner, as well as histdine-tagged ubiquitin cDNA. Cell lysates were harvested and Histiidine-containing protein complex were pulled down using Ni ++ -NTA magnetic bead for subquentent immunoblotting assay. The expression of β-actin used as a loading control. NS, not significant; *, p<0.05; **, p<0.01, ***, p<0.001. Data are represented as mean ± SD.

Article Snippet: The plasmid pCMV3-N-HA-Foxp3 was purchased from Sino Biological Inc. (catalog number: HG11652-NY, Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Immunoprecipitation, In Vivo, Ubiquitin Assay, Plasmid Preparation

Journal: Oncotarget

Article Title: Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

doi: 10.18632/oncotarget.11395

Figure Lengend Snippet: Female NOD-SCID mice were subcutaneously injected with 1×10 6 Foxp3 knocking-down T24-B in 100 ml serum-free medium or its control. In vivo growth A. and survival curve B. were record and analyzed. C. The growing tumor were harvested at day 58 for immunostaining of von Willebrand factor , Magnification × 200. Scale bars, 50 μ. D. The integrated density were calculated using Imaging-Pro Plus software and compared with unpaired t -test.

Article Snippet: The plasmid pCMV3-N-HA-Foxp3 was purchased from Sino Biological Inc. (catalog number: HG11652-NY, Beijing, China).

Techniques: Injection, In Vivo, Immunostaining, Imaging, Software

Journal: Oncotarget

Article Title: Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

doi: 10.18632/oncotarget.11395

Figure Lengend Snippet: A. Negative Foxp3 expression (× 200; scale bar, 50 μm), B. Positive Foxp3 expression (× 200; scale bar, 50 μm), C. Foxp3+ lymphocytes within the bladder tumor (× 400; scale bar, 50 μm). D. More than 8 CD8+ lymphocyte within bladder tumors, E. 1≤ CD8+ lymphocyte number ≤8, and F. none of CD8+ lymphocytes based on the average number determined from 10 random 0.0328-mm 2 digital images captured under high power field (× 320; scale bar, 20 μm). G. Distribution and mean number of CD8+TILs from 96 bladder cancer specimen according to Foxp3 expression. H. The comparison of average CD8+ TILs number between Foxp3-expressing tumors or not is analyzed using unpaired t -test. Foxp3-expressing lymphocytes (arrow).

Article Snippet: The plasmid pCMV3-N-HA-Foxp3 was purchased from Sino Biological Inc. (catalog number: HG11652-NY, Beijing, China).

Techniques: Expressing

Journal: Oncotarget

Article Title: Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

doi: 10.18632/oncotarget.11395

Figure Lengend Snippet: Clinicopathological correlate of Foxp3 expression in superficial bladder carcinoma tissues

Article Snippet: The plasmid pCMV3-N-HA-Foxp3 was purchased from Sino Biological Inc. (catalog number: HG11652-NY, Beijing, China).

Techniques: Expressing

Journal: Oncotarget

Article Title: Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

doi: 10.18632/oncotarget.11395

Figure Lengend Snippet: A. Proportion of recurrence-free survival according to the Foxp3 expression. B. Proportion of progression-free survival according to the Foxp3 expression. RFS, recurrence-free survival; PFS, progression-free survival.

Article Snippet: The plasmid pCMV3-N-HA-Foxp3 was purchased from Sino Biological Inc. (catalog number: HG11652-NY, Beijing, China).

Techniques: Expressing

Journal: Oncotarget

Article Title: Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

doi: 10.18632/oncotarget.11395

Figure Lengend Snippet: Univariate and multivariate analyses of variables associated with recurrence and progression in 115 superficial bladder cancer patients

Article Snippet: The plasmid pCMV3-N-HA-Foxp3 was purchased from Sino Biological Inc. (catalog number: HG11652-NY, Beijing, China).

Techniques: Expressing

Journal: Oncotarget

Article Title: Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

doi: 10.18632/oncotarget.11395

Figure Lengend Snippet: Correlation of Foxp3 expression with the Glucose transporter family member: result of a meta-analysis of GSE32548 (n=131) and GSE48075 (n=142)

Article Snippet: The plasmid pCMV3-N-HA-Foxp3 was purchased from Sino Biological Inc. (catalog number: HG11652-NY, Beijing, China).

Techniques: Expressing

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A Naive CD3 + CD4 + CD44 − CD62L + Foxp3 − naive T cells were isolated from the spleens of WT Foxp3 GFP and cultured for 72 h in the presence of CD3/CD28 beads, IL-2 and TGF-β. Culture plates were coated with 5 µg/mL PD-L2Fc or control IgG1Fc for 30 min prior culture. B Representative flow cytometry plots of Foxp3 induction in CD3 + CD4 + CD24 + Foxp3 GFP+ iTregs after 72 h of culture. C Absolute numbers of iTregs after 72 h of culture, presented as mean ± SEM. n = 3 biologically independent samples. D Foxp3 mean fluorescence intensity (MFI) of iTregs after 72 h of culture, presented as mean ± SEM. n = 3 biologically independent samples. E IL-10 secretion by iTregs after 72 h of culture, presented as the mean ± SEM. n = 8 biologically independent samples. F Following culture, cells were transferred to PDL-coated HS mini plates and a Mito Stress test was performed to measure oxygen levels in the culture medium. Oxygen consumption rate (OCR) in response to Oligomycin (oligo.), FCCP and Rotenone (Rot/AA) sequential injections. n = 3 biologically independent samples. G Basal respiration in PD-L2Fc or control IgG1Fc groups. H Spare respiratory capacity in PD-L2Fc or control IgG1Fc groups. I ATP production in PD-L2Fc or control IgG1Fc groups. J Schematic of the Foxp3 promoter region, showing the intron 1 TSDR region containing CpG#19, CpG#20, CpG#21, and CpG#22 within the CNS2. K Average methylation of CpG#19, CpG#20, CpG#21, and CpG#22 in TSDR region. L Methylation of CpG#19, CpG#20, CpG#21, and CpG#22 in TSDR region. n = 4 biologically independent samples. Data are representative of 2 independent experiments and are presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Isolation, Cell Culture, Control, Flow Cytometry, Fluorescence, Methylation, Two Tailed Test

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A Thymus of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were collected and analyzed by flow cytometry. Representative plots of CD25 + (CD25 + TregP cells) and Foxp3 lo Treg cell progenitors (Foxp3 lo TregP cells) analyzed based on CD25 and Foxp3 GFP expression. B Corresponding quantitation presented as mean ± SEM. n = 3 biologically independent mice. C Spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were collected and analyzed by flow cytometry. Representative plots of CD3 + CD4 + CD25 + Foxp3 GFP+ total Tregs. D Numbers of total Tregs in WT and PD-L2 KO mice presented as mean ± SEM. n = 5 biologically independent mice. E BALB/c (WT) BMDC were differentiated in vitro for 7–10 days, plated and further cultured with or without 50 ng/mL IL-4 for 24 h to measure PD-L2 expression by flow cytometry. F Representative plots of CD11c + BMDC, expression of PD-L2. G Numbers of PD-L2-expressing CD11c + BMDC presented as mean ± SEM. n = 4 biologically independent samples. H PD-L2 low and PD-L2 high BMDCs were intravenously injected or not to WT or PD-L2 KO Foxp3 GFP mice on day 0, and on day 3 the spleens of recipient mice were analyzed for CD3 + CD4 + CD25 + Foxp3 + total Tregs by flow cytometry. I Numbers of CD3 + CD4 + CD25 + Foxp3 GFP+ total Tregs per spleen at day 3 following BMDC transfer. n = 3 biologically independent mice. J – M PD-L2 high or PD-L2 null BMDCs were i.v. injected or not to WT or PD-L2 KO Foxp3 GFP mice on day 0, and on day 3 the spleens of recipient mice were analyzed for Tregs by flow cytometry. J Numbers of CD3 + CD4 + CD25 + Foxp3 GFP+ total Tregs. K Numbers of CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − pTregs. L Numbers of CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 + tTregs. M Thymus of recipient mice were collected and analyzed by flow cytometry, and the frequencies of CD25 + TregP cells and Foxp3 lo TregP cells was analyzed based on CD25 and Foxp3 GFP expression, presented as mean ± SEM. n = 3 biologically independent mice. Data are representative of three independent experiments and are presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Mouse image provided with permission from Servier Medical Art.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Flow Cytometry, Expressing, Quantitation Assay, In Vitro, Cell Culture, Injection, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A Spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were collected and analyzed by flow cytometry. Representative plots of CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − pTregs and CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 + tTregs. B Quantitation of pTreg and tTreg numbers in the spleen presented as means ± SEM. n = 5 biologically independent mice. C – D pTregs and tTregs—gated as in ( A )—were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and cultured for 24 h with CD3/CD28 beads, IL-2 and TGF-β. IL-10 was measured by ELISA in the culture supernatants. C Levels of IL-10 in pTreg cultures. n = 3 biologically independent samples. D Levels of IL-10 in tTreg cultures. E CD3 + CD4 + CD44 − CD62L + naive T cells were isolated from the spleens of BALB/c mice and co-cultured with FACS-sorted CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTregs isolated from Foxp3 GFP or PD-L2 KO Foxp3 GFP mice for 72 h. Cell proliferation was assessed by measuring thymidine incorporation. F 3 H thymidine incorporation after 72 h of culture. n = 4 biologically independent samples. G CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTregs cells were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and incubated for 24 h with CD3/CD28 beads, IL-2 and TGF-β. Genomic DNA was extracted, followed by bisulfite conversion, purification, cloning, and the degree of methylation of CpG#19, CpG#20, CpG#21, and CpG#22 within the TSDR region was determined by bisulfite sequencing. H Foxp3 GFP expression within WT and PD-L2 KO pTregs. n = 3 biologically independent samples. I Average methylation of CpG#19, CpG#20, CpG#21, and CpG#22 in TSDR region. n = 3 biologically independent samples. J Methylation of CpG#19, CpG#20, CpG#21, and CpG#22 in TSDR region. K 10 6 WT Foxp3 GFP iTregs were adoptively transferred to WT BALB/c or PD-L2 KO hosts on day 0 and on day 3, the frequencies of GFP + Tregs were analyzed in the spleen of recipients. L Representative plots of CD3 + CD4 + CD25 + Foxp3 GFP+ Tregs. M Numbers of transferred Tregs in the spleen presented as mean ± SEM. n = 3 biologically independent mice. Data are representative of two independent experiments and are presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Mouse image provided with permission from Servier Medical Art.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Flow Cytometry, Quantitation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Incubation, Purification, Cloning, Methylation, Methylation Sequencing, Expressing, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTregs cells were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and incubated for 24 h with CD3/CD28 beads, IL-2 and TGF-β. Cells were then lysed and mRNA was isolated to perform RNA-sequencing (RNA-seq). Volcano plot representation of differentially regulated genes in WT and PD-L2 KO pTregs. Dotted lines represent 2FC ( x axis) and p < 0.05 ( y axis) cutoffs. B – G Differentially regulated genes in selected pathways. B Treg function pathway. C Fatty acid (FA) degradation pathway. D Amino acid (AA) degradation pathway. E TCA cycle pathway. F Glycolysis pathway. G Pentose Phosphate Pathway (PPP) pathway. All red dots represent genes whose differences are p < 0.05. H – K CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTreg cells were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and incubated for 24 h with CD3/CD28 beads, IL-2 and TGF-β. Following culture, cells were transferred to PDL-coated HS mini plates and a Mito Stress test was performed to measure oxygen levels in the culture medium. H Oxygen consumption rate (OCR) in response to Oligomycin (oligo.), FCCP, and Rotenone (Rot/AA) sequential injections. I Basal respiration in WT and PD-L2 KO pTregs. n = 3 biologically independent samples. J Spare respiratory capacity in WT and PD-L2 KO pTregs. K Respiration-coupled ATP production. L Representative flow cytometry plots of MitoTracker DR expression within WT and PD-L2 KO pTregs after 24 h of culture. M Quantitation of MitoTracker DR expression within WT and PD-L2 KO pTregs after 24 h of culture, presented as mean ± SEM. n = 3 biologically independent samples. N CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTregs cells were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and incubated for 24 h with CD3/CD28 beads, IL-2 and TGF-β with or without 2 mM methyl pyruvate. O Levels of IL-10 production in the culture supernatants. n = 3 biologically independent samples. H – O Data are representative of two independent experiments and are presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Incubation, Isolation, RNA Sequencing, Flow Cytometry, Expressing, Quantitation Assay, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were i.n. challenged on days −10, −9 and −8 with 100 µg ovalbumin (OVA). On day 0, mice were intraperitoneally sensitized with 50 µg OVA emulsified in alum (1:1) and CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − pTreg numbers were analyzed by flow cytometry on day 7. B Representative plots of pTregs in the lungs of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice. C Numbers of pTregs in the lungs of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice presented as mean ± SEM. n = 4 biologically independent mice. D Representative plots of PD-1 expression in lung pTregs from Foxp3 GFP and PD-L2 KO Foxp3 GFP . E Representative plots of Foxp3 expression in lung pTregs from Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and corresponding quantitation presented as mean ± SEM. n = 3 biologically independent mice. F BALB/c (WT) and PD-L2 KO BMDC were differentiated in vitro for 7–10 days and cultured with FACS-sorted CD4 + CD44 − CD62L + KJ1-26 + naive T cells in the presence of IL-2, TGF-β, 100ng/mL OVA peptide and CD3/CD28 stimulation to induce Treg differentiation for 72 h at 1:20 ratio. G Representative plot of Foxp3 expression. H Foxp3 expression in T cells incubated with either WT or PD-L2 KO BMDC, presented as mean ± SEM. n = 3 biologically independent mice. I Levels of IL-10 in the supernatants, presented as mean ± SEM. n = 4 biologically independent samples. J Representative plots of BODIPY 493/503 expression in T cells incubated with either WT or PD-L2 KO BMDC and corresponding quantitation presented as mean ± SEM. n = 3 biologically independent samples. K Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were challenged as in A . On days 7, 8, and 9 mice were further challenged or not with 100 mg/kg ethyl pyruvate intraperitoneally. L Lung resistance in response to 40 mg/mL of methacholine. M Numbers of CD11b + Ly6G + neutrophils (PMN), CD11c − SiglecF + eosinophils (eos.), CD11c + SiglecF + CD64 + macrophages (mac.) and CD3 + T cells in the BAL fluid. n = 3 biologically independent mice. Data are representative of two independent experiments, presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Mouse image provided with permission from Servier Medical Art.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Flow Cytometry, Expressing, Quantitation Assay, In Vitro, Cell Culture, Incubation, Two Tailed Test, Comparison

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 1. The FoxP3 Reporter LV Shows Expression Selective for the Treg Cell Lineage (A) Endogenous human FOXP3 gene shows location of regulatory regions (promoter, CNS1, CNS2, CNS3, and 30 UTR) included in vector. Vector maps show design of CNS123p-mStrawberry and CNS123p-FoxP3-mStrawberry constructs within the pCCL vector backbone. (B) Analysis of human hematopoietic cell lines transduced with CNS123p-mStrawberry. Histograms show endogenous FoxP3 status of each cell line (left) and mStrawberry expression (right) in each cell line transduced with CNS123p-mStrawberry. Plots show mStrawberry expression in human hematopoietic cell lines over a range of vector copy numbers (n = 12 per cell line). (C) Activated human CD4 cells transduced with different doses of CNS123p-mStrawberry. Histograms show mStrawberry expression in viable CD4+ cells analyzed 4 days after activation. (see also Figure S2B). (D) Experimental design to evaluate in vivo lineage-specific expression of CNS123p-mStrawberry. Lin HSPCs were isolated from CD45.2 FoxP3-prom-GFP mice and transduced with CNS123p-mStrawberry. Transduced lin HSPCs were transplanted into lethally irradiated congenic CD45.1 recipients. CD45.2 donor cells within each hematopoietic lineage were analyzed at 20 weeks post-transplant for mStrawberry reporter LV expression (see also Figure S2A). (E) Histograms depict mStrawberry reporter expression in each hematopoietic lineage in bone marrow, thymus, and spleen of engrafted mice. Each individual histogram line represents mStrawberry expression for an individual mouse (n = 9 mice; see also Figure S2C). (F) y axis represents the percentage of mStrawberry+ cells within each hematopoietic lineage in the BM, spleen, and thymus (n = 9 mice). Data in (E) represent mean ± SD.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Expressing, Plasmid Preparation, Construct, Transduction, Activation Assay, In Vivo, Isolation, Irradiation

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 2. Lineage-Specific FoxP3 Expression Restores Treg Cell Development from Scurfy (FoxP3 Mutant) HSCs (A) Transplant setup to evaluate Treg cell development. Scurfy (FoxP3mut) mice were rescued with WT CD45.1 splenocytes at birth to allow survival into adulthood to serve as bone marrow donors. Lin HSPCs were isolated from rescued scurfy (FoxP3mut) or wild-type (FoxP3-prom-GFP) donor mice and transduced with CNS123p-mStrawberry or CNS123p-FoxP3-mStrawberry. Transduced lin HSPCs were transplanted into lethally irradiated WT CD45.1 congenic recipients. After 12 weeks, donor cells from each transplant cohort were evaluated for thymic and splenic reconstitution of Treg cells. Treg cell populations from each group were identified as CD4+mStrawberry+ cells (uncorrected scurfy Treg cells [Sf-Treg cells]; corrected scurfy Treg cells [cSf-Treg cells]) or CD4+GFP+ cells (wild- type Treg cells [WT-Treg cells]). (B) Lineage distribution of total donor thymocytes in mice reconstituted with Sf, cSf, or WT BM lin cells (n = 3–5 mice/arm). (C) Thymic Treg cell reconstitution. FACS plots show donor CD45.2+CD4SP cells in the thymus of transplant recipients. Gates delineate thymic Sf-Treg cells, cSf-Treg cells, and WT-Treg cells. Bottom panel shows expression of the Treg cell surface markers CD25, GITR, and CTLA4 within each putative Treg cell population (surface marker expression for Sf-Treg cells and cSf-Treg cells is shown for mStrawberryhigh cells or cells expressing the top 50% of mStrawberry expression). Histograms depict one representative experiment of 3 (see also Figure S2D). (D) FACS sort for splenic Treg and Tconv cells. Splenic Treg cell populations (mStrawberryhigh or GFP+ gates) were FACS sorted for a Treg cell suppression assay. Tconv cell populations (mStrawberry or GFP gates) were sorted for an iTreg cell induction assay. (E) In vitro Treg cell suppression assay. Sorted Treg cells (shown in D) were co-cultured with responder T cells (Tresp cells) (congenic WT CD4+ cells labeled with a fluorescent proliferation dye) at a 1:1 ratio in the presence of bead-bound CD3 and CD28 antibodies. Histograms depict Tresp cell proliferation in one of three representative experiments. Bar graph shows proliferation index for each Treg cell culture condition (n = 6–9 Tresp cell cultures per arm from 3 different Treg cell sources per arm; data normalized to internal ‘‘no Treg cell’’ control for each experiment). (F) Sorted splenic Tconv cells (shown in D) were activated with CD3 and CD28 antibodies in the presence of 20 ng/mL interleukin-2 (IL-2) and 20 ng/mL TGF-b for 4 days to induce iTreg cells. FACS plots show mStrawberry or GFP expression from each group (n = 3 mice/group). Data in (B), (C), (E), and (F) are presented as mean ± SD. Data in (B) were analyzed by Kruskal-Wallis test for each lineage. Data in (E) are analyzed by Kruskal- Wallis test for overall comparison for all groups and Mann-Whitney U test for pairwise comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Expressing, Mutagenesis, Isolation, Transduction, Irradiation, Marker, Suppression Assay, In Vitro, Cell Culture, Labeling, Control, Comparison, MANN-WHITNEY

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 3. The Lineage-Specific FoxP3 cDNA Vector Generates Functional Treg Cells Capable of Rescuing the Scurfy Mouse (A) Assay for in vivo Treg cell function: three groups of CD4 cells containing putative Treg cells were generated by congenic bone marrow transplants: uncorrected scurfy CD4 (Sf-CD4; no. 1); corrected scurfy CD4 (cSf-CD4; no. 2); and wild-type CD4 (WT-CD4; no. 3). To obtain Sf-CD4 and cSf-CD4, CD45.2 Lin HSPCs were isolated from scurfy donors (rescued at birth by CD45.1 splenocytes) and transduced with either CNS123p-mStrawberry (Sf-CD4; no. 1) or CNS123p-FoxP3- mStrawberry (cSf-CD4; no. 2). To obtain WT-CD4, CD45.2 Lin HSPCs were isolated from FoxP3-prom-GFP donors and transduced with CNS123p- mStrawberry (no. 3). Transduced cells were transplanted into lethally irradiated congenic (CD45.1) recipients. 8 weeks post-transplant, donor CD45.2+CD4+ cells were purified with magnetic beads from the spleens of transplant recipients and injected intraperitoneally into scurfy neonates. Scurfy neonates and age-matched WT receiving PBS injection were also analyzed as control conditions no. 4 and no. 5. The autoimmune phenotype of all groups was evaluated at 21 days of life (see also Figure S3). (B) Photographs of scurfy mice or WT controls at 21 days. White arrows highlight ear skin phenotype in mice from each group. Ear skin inflammation (char- acterized by small, thickened, scaly ears) is seen in untreated scurfy mice (Sf + PBS) and scurfy mice receiving uncorrected scurfy CD4 cells (‘‘Sf + Sf-CD4’’). Normal ear skin without inflammation is seen in WT controls (‘‘WT + PBS’’) and scurfy mice receiving wild-type or corrected scurfy CD4 cells (‘‘Sf + WT-CD4’’ and ‘‘Sf + cSf-CD4’’). (C) Spleen-to-body-weight ratio for rescued scurfy mice or WT littermate controls. (D) Activated (CD44+CD62L) CD4 T cells (expressed as a percentage of total CD4 splenocytes) in the spleens of rescued scurfy mice or WT littermate controls. (E) Serum cytokine levels in rescued scurfy mice or WT littermate controls. Data in (C)–(E) are presented as mean ± SD. Data on (C)–(E) represent n = 3 independent experiments pooled for analysis with a total of 3–6 mice/arm. Data in (C)–(E) were analyzed by Kruskal-Wallis test for overall comparison for all groups, and Mann-Whitney U test was performed for pairwise comparisons. *p < 0.05; **p < 0.01.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Plasmid Preparation, Functional Assay, In Vivo, Cell Function Assay, Generated, Isolation, Transduction, Irradiation, Magnetic Beads, Injection, Control, Comparison, MANN-WHITNEY

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 4. The FoxP3 Reporter Vector CNS123p-mStrawberry Shows Treg Cell Lineage-Selective Expression in a Humanized Mouse Model (A) Experimental setup for humanized mouse models. Cord blood CD34+ HSPCs were transduced with CNS123p-mStrawberry and transplanted into neonatal NSG mice (B, C, E, and F) or NSG-SGM3 mice (D). 12–16 weeks post-transplant, engrafted hCD45+ cells were analyzed for mStrawberry expression. (B) mStrawberry reporter expression in each hematopoietic lineage. Each overlaid histogram represents mStrawberry expression in an individual mouse (n = 10–14 mice humanized with 2 different cord blood CD34+ donors; see also Figures S4A–S4C). (C) Percentage of mStrawberry+ cells in each lineage shown in (B). (D) Co-expression of FoxP3 and mStrawberry in humanized mice. Splenic human CD4+ cells were FACS sorted into mStrawberry+ and mStrawberry pop- ulations followed by intracellular staining for FoxP3 expression. Left panel shows sorting of human CD4+ cells by mStrawberry expression, and right panel shows FoxP3 expression in sorted populations. Results are representative of 2 independent experiments. (E) CNS2 methylation analysis of T cell populations from humanized mice. Figure shows the locations of CNS2 within the endogenous FOXP3 gene and CNS2 within the viral genome. Red arrows indicate differential primer binding sites for amplification of endogenous or viral CNS2. FACS plot shows sorting gates used to define Treg cell (CD4+CD25+) and Tconv cell (CD4+CD25) populations in CD4-enriched cells isolated from the pooled spleens of 3–5 humanized mice. Heatmap represents the percentage of methylated reads detected at each of the 9 CpG sites within endogenous and viral CNS2. Results are representative of 2 independent experiments using pooled NSG cohorts humanized from 2 different CB CD34+ donors (see also Figures S4D–S4F). (F) Experimental setup for NSG competitive repopulation assay. ‘‘Test’’ CB CD34+ cells were transduced with either CNS123p-mStrawberry or CNS123p-FoxP3- mStrawberry, and ‘‘competitor’’ CD34+ cells were transduced with a UBC-mCitrine vector. Test and competitor cells were co-transplanted at a 1:1 ratio into NSG neonates, and the percentage of competitor (mCitrine+) CD45+ cells engrafted in the BM at 12 weeks was determined for each group (n = 6 mice per group; humanized from 2 different CB CD34+ donors). Data in (F) represent mean ± SD. Data in (F) were analyzed by Mann-Whitney U test.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Plasmid Preparation, Expressing, Transduction, Staining, Methylation, Binding Assay, Isolation, MANN-WHITNEY