Journal: Bioengineering

Article Title: RNU ( Foxn1 RNU -Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats

doi: 10.3390/bioengineering8010012

Figure Lengend Snippet: CD68+ staining is increased in RNU DMM-treated sites compared to Sprague Dawley. CD68 staining was quantified and showed increased levels in RNU rats ( A ). Yet, CD163 ( B ) and FoxP3 ( C ) were unaltered. CD4 staining was increased in RNU rats ( D ) while CD8 staining was lower ( E ). In addition, CD4, CD8, and FoxP3 levels were low. DRAQ5 showed no change in DNA staining ( F ). Data shown are means ± SEM of 8 animals. Letters not shared indicated a significant difference ( p < 0.05, Tukey).

Article Snippet: Primary antibodies used in this experiment were: mouse anti-Pax7 (ab55494, Abcam, Cambridge, UK); rabbit anti-nicotinic acetylcholine receptor-epsilon (AChR-ε, ab65180, Abcam); mouse anti-nicotinic acetylcholine receptor-gamma (AChR-γ, MA3-043, Thermo Fisher Scientific, Waltham, MA, USA); mouse anti-myosin heavy chain-fetal (fMyHC, SC-53097, Santa Cruz Biotechnology); CD68 (ab125212, Abcam); CD163 (ab87099, Abcam); CD8 (MAB116, R&D Systems), CD4 (MAB554, R&D Systems), FoxP3 (MAB8214, R&D Systems) and were diluted in PBS with 1% BSA and 0.3% Tween-20.

Techniques: Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Serine protease inhibitor from the muscle larval Trichinella spiralis ameliorates non-alcoholic fatty liver disease in mice via anti-inflammatory properties and gut-liver crosstalk.

doi: 10.1016/j.biopha.2024.116223

Figure Lengend Snippet: Fig. 4. rTs-SPI attenuated macrophage infiltration and promoted CD4þCD25þFoxp3þ Tregs population in NAFLD mice. (A) Macrophage infiltration and (B) CD4+CD25+Foxp3+ Tregs in spleen using Flow cytometry analysis; Quantitative analysis of (C) F4/80+ macrophages and (D) CD4+CD25+Foxp3+ Tregs in spleen; (E) Microphotographs show total macrophages staining with F4/80 (red) and DAPI (blue) in liver (600x); (F) Quantitative analysis of F4/80+ macrophages in liver; The hepatic mRNA expression levels of (G) F4/80 and (H) Foxp3. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For Treg analysis, cells were stained with Percp/cy5.5 anti-mouse CD4 and APC anti-mouse CD25 antibodies (Elabscience, China), followed by fixing and permeabilizing using a Foxp3/transcription factor staining kit (Elabscience, China) and staining with PE anti-mouse Foxp3 antibody (Elabscience, China).

Techniques: Flow Cytometry, Staining, Expressing

Journal: The Indonesian Biomedical Journal

Article Title: 2-(3-(chloromethyl)benzoyloxy)benzoic Acid Increases CD4+ Regulatory T-Cell Population and FoxP3 Expression in Lipopolysaccharide-induced Mice

doi: 10.18585/inabj.v15i4.2429

Figure Lengend Snippet: Figure 3. Foxp3-expression in splenocytes of LPS- induced mice. Mice were induced with/without LPS and treated orally with/without ASA or 3-CH2Cl. Spleen of the mice were isolated, processed, and the splenic FoxP3 expression was measured with flow cytometry as described in Methods. A: Representative histogram with MFI value obtained from each group. B: MFI value of each group. **0.01≥p>0.001; ***0.001≥p>0.0001, tested with upaired t-test.

Article Snippet: Ten μg of APC Anti-Mouse Foxp3 Antibody (Elabscience) was added to the splenocytes.

Techniques: Expressing, Isolation, Flow Cytometry

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Intracellular and intranuclear staining can be improved with extended incubation times. (A) Representative staining obtained in 30 min versus 16 hr. (B) Stain indices for Foxp3 and IL‐2. Mouse splenocytes were stimulated and stained for intracellular cytokines as described in the Methods section. Data were acquired on a Cytek Aurora Spectral Cytometer.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Staining, Incubation, Cytometry

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Increasing incubation time reduces batch effects. (A) Representative staining and MFI of SIGLEC‐8 on CD45 + SSC hi CD16 ‐ cells from the same donor over 3 independent experiments. (B) Representative staining and MFI of CD123 on CD45 + SSC lo CD3 − CD19 − CD14 − CD16 − cells from the same donor over 3 independent experiments. (C) Human whole blood immunophenotyping data from the same donor over three independent experiments, stained for 30 min or 16 hr. tSNE plots were generated using the parameters CD45, SSC‐A, CD4, CD8, CD127, CD16, CD19, CD3, CD123, CD20, CD25, Fcer1a, CD11c, SIGLEC‐8, CD56, CD14, and HLA‐DR. Data were acquired on a BD LSRFortessa cytometer. FlowSOM clusters are shown in a colored overlay. (D) Cross entropy distances between samples stained for 30 min or 16 hr. (E) Mouse data from four experiments over the course of two years. Data were acquired on a BD FACSymphony A5 cytometer. tSNE plots were generated using the parameters CD4, CD8, Foxp3, CD103, Neuropilin, CD44, CD62L, Ki67, ICOS, PD‐1, CTLA‐4, CD25, KLRG1, CD69, ST2, and Helios on CD3 + T cells. FlowSOM clusters are shown in a colored overlay. (F) Cross entropy distances between mouse samples (intra‐batch variation) or batches (inter‐batch variation). Significance was tested by unpaired t‐test.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Incubation, Staining, Generated, Cytometry

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Titration is essential to maximize sensitivity. (A) Titration of CD3‐Spark Blue 550. (B) Intracellular overnight staining for PD‐1 on viable CD4 + CD3 + T cells at the indicated dilutions on cells fixed and permeabilized with the eBioscience Foxp3 Fix/Perm kit.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Titration, Staining

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Controls confirm specificity is maintained with overnight staining. (A) IL‐2 staining on WT or IL‐2‐deficient mouse CD4 + T cells. (B) pSTAT5 and Foxp3 staining on mouse CD4 + T cells with or without IL‐2 stimulation.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Staining

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Reagents Used for This Study

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Concentration Assay

Journal: Nature Communications

Article Title: GPR4 promotes immune exclusion in colon cancer through LOXL2-mediated extracellular matrix remodeling

doi: 10.1038/s41467-025-67967-z

Figure Lengend Snippet: A GPR4 regulates JAK2 and STAT3 phosphorylation via western blotting. n = 3. Scale bar: 100 µm. B Representative IF images of STAT3-Y705 phosphorylation regulated by GPR4. C Quantitative analysis of STAT3-Y705 phosphorylation in GPR4 - OE SW480 cell line and ( D ) GPR4 - KD HCT116 cell lline. Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. E Static treatment reduces both pSTAT3-Y705 and the expression of LOXL2, COL1A1, and TGF-β in GPR4 - OE SW480 cells. F RT-PCR analysis for LOXL2 in SW480 cell lines after receiving Stattic treatment. Two-tailed t test, n = 3 biologically independent samples. G RT-PCR analysis for TGF-β in SW480 cell lines after receiving Stattic treatment. Data are presented as mean ± SD.Two-tailed t test, n = 3 biologically independent samples. H STAT3 transcription factor binding site. I In SW480 cells, STAT3 binds to the LOXL2 promoter region confirmed via ChIP assays. J STAT3 binds to the TGF-β promoter region confirmed via ChIP assays. K RT-PCR assesses the transcriptional activity of LOXL2 promoter regions and TGF-β promoter region L . Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. ns > 0.05.

Article Snippet: Subsequently, Transcription Factor Staining Kit (Elabscience, E-CK-A108) was applied for cells fixation and Permeabilization as recommended method.

Techniques: Phospho-proteomics, Western Blot, Two Tailed Test, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Safe and Efficient Sigma1 Ligand: A Potential Drug Candidate for Multiple Sclerosis

doi: 10.3390/ijms231911893

Figure Lengend Snippet: List of monoclonal antibodies used for flow cytometric analysis.

Article Snippet: FoxP3 Antibody, anti-human, APC , Miltenyi Biotec , 130-125-580.

Techniques: Bioprocessing

Journal: Biomedicines

Article Title: Clinicopathological Features of Gastric Cancer with Autoimmune Gastritis

doi: 10.3390/biomedicines10040884

Figure Lengend Snippet: Mucin type, immune cell numbers, PD-L1 expression, and bacteria.

Article Snippet: For immunofluorescence, the paraffin-embedded sections were incubated with primary antibodies against MUC2 (cat. no. ab90007; Abcam, Cambridge, UK), MUC5AC (cat. no. ab3649; Abcam), MUC6 (cat. no. NBP2-44374; Novusbio, Littleton, CO, USA), CD3 (cat. no. NCL-L-CD3-565; Leika, Wetzlar, Germany), CD11b (cat. no. 14011282; Invitrogen, Waltham, MA, USA), Foxp3 (cat. no. 98377; Cell Signaling Technology), PD1 (cat. no. 86163; Cell Signaling Technology), Bacillus cereus (cat. no. ab20556; Abcam), and Streptococcus Group B (cat. no. ab53584; Abcam).

Techniques: Expressing, Bacteria

Journal: Biomedicines

Article Title: Clinicopathological Features of Gastric Cancer with Autoimmune Gastritis

doi: 10.3390/biomedicines10040884

Figure Lengend Snippet: Immunohistochemical staining for PD-L1, CD3, CD11b, Foxp3, and PD1 in gastric cancer in pure AIG and H. pylori /APCA-negative patients. Elevated PD-L1 expression and infiltration of CD3/Foxp3/PD1-positive cells in gastric cancer is associated with pure AIG. Scale bars, 200 μm.

Article Snippet: For immunofluorescence, the paraffin-embedded sections were incubated with primary antibodies against MUC2 (cat. no. ab90007; Abcam, Cambridge, UK), MUC5AC (cat. no. ab3649; Abcam), MUC6 (cat. no. NBP2-44374; Novusbio, Littleton, CO, USA), CD3 (cat. no. NCL-L-CD3-565; Leika, Wetzlar, Germany), CD11b (cat. no. 14011282; Invitrogen, Waltham, MA, USA), Foxp3 (cat. no. 98377; Cell Signaling Technology), PD1 (cat. no. 86163; Cell Signaling Technology), Bacillus cereus (cat. no. ab20556; Abcam), and Streptococcus Group B (cat. no. ab53584; Abcam).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: PLoS ONE

Article Title: Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function

doi: 10.1371/journal.pone.0173773

Figure Lengend Snippet: There was no difference in GFR of patients with predominantly methylated (>75% Foxp3 TSDR methylation) Treg and patients with a sizable proportion of demethylated (≤75% Foxp3 TSDR methylation) Treg (p>0.01; Mann-Whitney U test). Enriched IFNy+ and IFNy- Treg of male and female patients were tested separately using primer ADS783 (primer P1) and primer ADS3576 (primer P2).

Article Snippet: PCR was carried out in a 21 μl total volume containing: 2.1 μl PCR buffer, 0.27 μl MgCl 2 , 0.42 μl dNTPs, 1.05 μl SYBR Green dye (EpigenDx), 1.26 μl each of primers ADS783 and ADS3576 (Human Foxp3 Methylation Panel, EpigenDx), 0.12 μl Hot Start Taq polymerase (Qiagen), and 1.5 μl of bisulfite-treated genomic DNA (concentration 5 ng/μl).

Techniques: Methylation, 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