Journal: Journal of Alzheimer's Disease Reports

Article Title: Utility of DNA Methylation as a Biomarker in Aging and Alzheimer’s Disease

doi: 10.3233/ADR-220109

Figure Lengend Snippet: Summary of DNA methylation clocks

Article Snippet: Cho Model 2 , First , 5 , 20-74 , Pyrosequencing , Bisulphite Conversion (EZ DNA Methylation - Lightning ™ Kit; Zymo) , Blood , 100 , Multivariate linear model , Association with chronological age , [ ] .

Techniques: DNA Methylation Assay, Cell Culture, Marker, Modification, Sequencing, Methylation, Methylation Sequencing, Biomarker Assay

Journal: Redox Biology

Article Title: Folic acid modulates VPO1 DNA methylation levels and alleviates oxidative stress-induced apoptosis in vivo and in vitro

doi: 10.1016/j.redox.2018.08.005

Figure Lengend Snippet: Folic acid modulates the vascular peroxidase 1 (VPO1) promoter methylation levels of the CpG sites and the expression in HUVCEs. HUVCE were treated with 0–500 nmol/L folic acid for 48 h and exposed to 120 μg/mL ox-LDL for the first 24 h. (A) Schematic diagram of mice VPO1 promoter (1897–2233 bp). Pyrosequencing assay data were evaluated by dividing the VPO1 promoters into 26 CpG sites. The locations of CpG sites were indicated with red font numbers. (B) Mean methylation levels of CpG sites in VPO1 in HUVCEs. (n = 3) *, p < 0.05 was considered the significance of any differences between test groups. (C) Gene expression levels of VPO1 (×10 3 ) in HUVECs. (D) Representative western blot bands of VPO1 and β-actin proteins. (E) Semiquantitative levels of VPO1 proteins calculated by band density analysis normalized to β-actin expression. The plotted values are mean ± SE values for 3 independent experiments. a, p < 0.05 compared with the ox-LDL + FA20. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Article Snippet: Subsequently, the membranes were blocked with 5% (w/v) milk in Tris-buffered saline and 0.1% Tween 20 for 2 h at room temperature, followed by incubation with rabbit polyclonal anti-VPO1 (1:500; Merck Millipore Corporation), mouse anti-DNMT1 (1:500; Abcam, Cambridge, UK), rabbit polyclonal anti-DNMT3a (1:1000; Abcam), rabbit polyclonal anti-DNMT3b (1:500; Abcam), and rabbit polyclonal anti-β-actin antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C on a rocking table and washing with PBST.

Techniques: Methylation, Expressing, Pyrosequencing Assay, Gene Expression, Western Blot

Journal: Redox Biology

Article Title: Folic acid modulates VPO1 DNA methylation levels and alleviates oxidative stress-induced apoptosis in vivo and in vitro

doi: 10.1016/j.redox.2018.08.005

Figure Lengend Snippet: Responses of DNA methyltransferase (DNMT) activity and expression in HUVCEs. HUVCE were treated with 0–1000 nmol/L folic acid for 48 h and exposed to 120 μg/mL ox-LDL for the first 24 h. (A) The total DNMTs activity in HUVECs. (B) Global methylation levels in HUVECs. The plotted values are mean ± SE values for 3 independent experiments. a, p < 0.05 compared with the ox-LDL + FA20. (C) Gene expression levels of DNMT1 (×10 5 ), DNMT3A (×10 3 ), and DNMT3B (×10 5 ) in HUVECs. (D) Representative western blot bands of DNMT1, DNMT3A, DNMT3B and β-actin proteins. (E) Semiquantitative levels of DNMT1, DNMT3A, DNMT3B proteins calculated by band density analysis normalized to β-actin expression. The plotted values are mean ± SE values for 3 independent experiments. a, p < 0.05 compared with the ox-LDL + FA20. b, p < 0.05 compared with the ox-LDL + FA0.

Article Snippet: Subsequently, the membranes were blocked with 5% (w/v) milk in Tris-buffered saline and 0.1% Tween 20 for 2 h at room temperature, followed by incubation with rabbit polyclonal anti-VPO1 (1:500; Merck Millipore Corporation), mouse anti-DNMT1 (1:500; Abcam, Cambridge, UK), rabbit polyclonal anti-DNMT3a (1:1000; Abcam), rabbit polyclonal anti-DNMT3b (1:500; Abcam), and rabbit polyclonal anti-β-actin antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C on a rocking table and washing with PBST.

Techniques: Activity Assay, Expressing, Methylation, Gene Expression, Western Blot

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

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: Virchows Archiv

Article Title: EPM2 AIP1 immunohistochemistry as a surrogate of promoter methylation analysis in endometrial carcinoma

doi: 10.1007/s00428-025-04132-3

Figure Lengend Snippet: Representative images of an EC with MLH1 deficiency, absence of MLH1 promoter methylation, and positive EPM2 AIP1 immunostaining. The tumor exhibited biallelic somatic alteration of MLH1 ; one pathogenic variant in MLH1 ( NM_000249.4 ): p.Glu633 Ter (c.1897G > T), classified as a stop-gained mutation, truncating the MLH1 protein, as well as loss of heterozygosity (LOH) detected in chromosomal region 3p22.2-q27.1

Article Snippet: In summary, results show concordance between MLH1 promoter methylation analysis by pyrosequencing and EPM2 AIP1 IHC, with 100% EPM2 AIP1 negativity in the 70 cases that exhibited MLH1 promoter methylation by pyrosequencing.

Techniques: Methylation, Immunostaining, Variant Assay, Mutagenesis

Journal: Virchows Archiv

Article Title: EPM2 AIP1 immunohistochemistry as a surrogate of promoter methylation analysis in endometrial carcinoma

doi: 10.1007/s00428-025-04132-3

Figure Lengend Snippet: Representative images of an EC with MLH1 with a methylation score close to the cut-off. The initial biopsy A showed negative EPM2 AIP1 immunostaining and a methylation score of 9.8. In the surgical specimen B , EPM2 AIP1 immunostaining was negative, but the methylation score increased to 60% after tumor microdissection

Article Snippet: In summary, results show concordance between MLH1 promoter methylation analysis by pyrosequencing and EPM2 AIP1 IHC, with 100% EPM2 AIP1 negativity in the 70 cases that exhibited MLH1 promoter methylation by pyrosequencing.

Techniques: Methylation, Immunostaining, Laser Capture Microdissection