Journal: PLoS ONE

Article Title: A Study of T Cell Tolerance to the Tumor-Associated Antigen MDM2: Cytokines Can Restore Antigen Responsiveness, but Not High Avidity T Cell Function

doi: 10.1371/journal.pone.0000353

Figure Lengend Snippet: (A) Lymph nodes cells from 6–8 week old Ag pos and Ag neg mice were stained with antibodies against CD4, CD8 and Vβ7 and analysed by flow cytometry. Numbers in quadrants are percentage of live-gated lymphocytes. (B) Lymph node cells were stained with antibodies against CD8, Vβ7 and the activation markers CD44, CD62L, CD69 and CD25. Histograms display gated viable CD8 + Vβ7 + cells. Numbers in histograms represent the specific MFI of the activation marker staining for the gated population. Data are representative of at least three independent experiments.

Article Snippet: The following mAbs were used for flow cytometric staining: rat-anti-mouse Vβ7 FITC (Serotec, UK), rat-anti-mouse CD4 FITC, rat-anti-mouse CD8α APC, rat-anti-mouse CD8α Cy-Chrome 5 (CY-5), rat-anti-mouse Vβ7 PE, hamster-anti-mouse CD69 PE, rat-anti-mouse CD25 PE, rat-anti-mouse CD62L-FITC, rat-anti-mouse CD44 PE, rat-anti-mouse CD43 activation-associated glycoform-PE (all BD Biosciences).

Techniques: Staining, Flow Cytometry, Activation Assay, Marker

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Schematic of experimental design. (B) Flow cytometry contour plots demonstrating the loss of the endogenous splenic CD4 + Foxp3 -GFP + Treg population in Foxp3 GFP-DTR mice following administration of diphtheria toxin (DTx). (C) Lung and spleen Foxp3 -GFP + Treg cell quantification in Foxp3 GFP-DTR mice that received influenza A virus but no DTx (No DTx + flu, n = 3) or influenza and DTx (DTx + flu, n = 4) at 13 DPI compared with mice that received DTx but no influenza (DTx No flu, n = 3). (D) Mass over time of Foxp3 GFP-DTR mice receiving no DTx or DTx until pre-specified timepoints (DTx withdrawal) following intra-tracheal inoculation of 5 plaque-forming units (PFU) of influenza A/WSN/33 H1N1 virus (n = 5 per group). (E) Representative lung histopathology (H&E staining) at 60 DPI of control (No DTx) and DTx withdrawal mice following influenza A virus infection. Original magnification x10, scale bar = 1 mm. (F) Metagene analysis of DNA methylation across Treg-SE of Tregs recovered from the mice described in 1D compared with splenic nTregs and iTregs from culture. Data presented as mean and SD. * q < 0.05 according to one-way ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (C).* q < 0.05 according to mixed-effects model (REML) with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (D).

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Flow Cytometry, Virus, Histopathology, Staining, Control, Infection, DNA Methylation Assay

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Flow cytometry contour plots for phenotyping of the CD4 + Foxp3 -GFP + tdTomato + iTreg population following culture in the presence of αCD3/αCD28, IL-2, TGF-β, and tamoxifen. (B) Mass over time of Foxp3 GFP-DTR mice receiving adoptive transfer of nTreg (n = 27), PBS (n = 21), Tconv (n = 25), or iTreg (n = 18) cells 5 days following intra-tracheal inoculation of 6.5 PFU of influenza A/WSN/33 H1N1 virus. Negative controls included mice that received influenza but no DTx (no DTx + flu, n = 9) and DTx but no influenza (DTx no flu, n = 3). (C) Total number of lung CD45 + cells at 24 DPI in mice that received No DTx (n = 3), nTreg (n = 15), PBS (n = 11), or Tconv (n = 10). (D-H) Total number of lung CD326 + CD31 − CD45 − cells (D), CD326 + MHCII + T1A − cells (E), KRT5 + CD326 + cells (F), Ki-67 + CD326 + MHCII + cells (G), and CD326 − CD31 + cells (H) at 24 DPI in mice that received No DTx (n = 3), nTreg (n = 15), PBS (n = 11), or Tconv (n = 10) cells. * q < 0.05 according to mixed-effects model (REML) with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (B). Data presented as mean and SD (C, F-H) with * q < 0.05 according to multiple Mann-Whitney tests and correcting for multiple comparisons using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (C). UHRF1 is dispensable for iTreg FOXP3 induction and stability but is required to maintain transcriptional and epigenetic programs in vitro .

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Flow Cytometry, Adoptive Transfer Assay, Virus, MANN-WHITNEY, In Vitro

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Schematic of experimental design. (B) Frequency of Foxp3 -GFP + and Foxp3 -GFP − tdTomato + (ex-FOXP3) cells in culture of iTregs with UHRF1 deleted concurrent with (early) or 5 days after (delayed) FOXP3 induction compared with Uhrf1 +/+ controls on days 5 and 12 of culture (n = 3 per group). (C) Division index of CD4 + CTV + Foxp3 -GFP − splenic responder T cells co-cultured for 72 hours with varying ratios of Uhrf1 +/+ CD4 + Foxp3 -GFP + iTregs, Uhrf1 fl/fl CD4 + Foxp3 -GFP + iTregs, or Uhrf1 +/+ nTregs (n = 3 per group). (D) Principal component analysis of 6,978 differentially expressed genes from sorted cells described in A, identified from ANOVA-like testing with FDR q < 0.05. Ellipses represent normal contour lines with 1 standard deviation probability. (E) K-means clustering of 127 genes with an FDR q < 0.05 comparing the Uhrf1 fl/fl and Uhrf1 +/+ iTreg populations on day 12 in which UHRF1 was deleted after FOXP3 induction (delayed) with k = 2. (F) MA plot comparing gene expression of delayed Uhrf1 +/+ and Uhrf1 fl/fl iTregs on day 12 of culture. Genes of interest are annotated. (G) Enrichment plot of the GSE14415_INDUCED_TREG_VS_TCONV_UP, GSE14415_INDUCED_TREG_VS_TCONV_DN, GSE14415_INDUCED_TREG_VS_FAILED_INDUCED_TREG_UP, GSE37605_TREG_VS_TCONV_C57BL6_FOXP3_FUSION_GFP_UP, GSE13306_TREG_VS_TCONV_UP gene sets ( p < 0.05, FDR q < 0.25) generated through GSEA preranked testing of the expressed genes of delayed Uhrf1 +/+ iTregs (control) and delayed Uhrf1 fl/fl iTregs on day 12 of culture. (H) PCA of 81,179 differentially methylated cytosines identified from ANOVA-like testing with FDR q < 0.05. Ellipses represent normal contour lines with 1 standard deviation probability. (I) Cumulative distribution function plot of differentially methylated cytosines expressed as β scores, with 0 representing unmethylated and 1 representing fully methylated; a shift in the cumulative distribution function up and to the left represents relative hypomethylation. * q < 0.05 according to two-way ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Cell Culture, Standard Deviation, Gene Expression, Generated, Control, Methylation

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) MA plot comparing gene expression of Uhrf1 +/+ iTregs (control) with CD4 + Foxp3 -GFP + nTregs on day 5 of culture. Genes of interest are annotated. (B) Enrichment plots of the HALLMARK_MYC_TARGETS_V1, HALLMARK_E2F_TARGETS, HALLMARK_TGF_BETA_SIGNALING, HALLMARK_WNT_BETA_CATENIN_SIGNALING, HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_MTORC1_SIGNALING, HALLMARK_IL2_STAT5_SIGNALING, and HALLMARK_KRAS_SIGNALING_UP gene sets generated through GSEA preranked testing of the expressed genes of delayed Uhrf1 +/+ iTregs (control) and delayed Uhrf1 fl/fl iTregs on day 12 of culture.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Gene Expression, Control, Generated

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A-F) Total numbers of CD45 + (A), CD3 + (B), CD4 + CD8 − (C), CD4 − CD8 + (D), CD11b − CD64 + CD11c + SiglecF + (E), and CD11b + Ly6G + (F) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 8), Uhrf1 +/+ iTregs (n = 16), or U hrf1 fl/fl iTregs. (n = 16) (G) Total number of CD326 + CD31 − CD45 − (epithelial) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 7), Uhrf1 +/+ iTregs (n = 15), or U hrf1 fl/fl iTregs (n = 16). (H) Total number of KRT5 + CD326 + epithelial cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 7), Tconv (n = 7), PBS (n = 7), Uhrf1 +/+ iTregs (n = 13), or U hrf1 fl/fl iTregs (n = 14). (I) Total number of CD326 − CD31 + (endothelial) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 8), Uhrf1 +/+ iTregs (n = 14), or U hrf1 fl/fl iTregs (n = 15). Data presented as mean and SD. ns = not significant by multiple Mann-Whitney tests and correcting for multiple comparisons using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% test.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: MANN-WHITNEY

Journal: Journal of Parasitology Research

Article Title: Induction of Cellular Immune Response by DNA Vaccine Coexpressing E. acervulina 3-1E Gene and Mature CHIl-15 Gene

doi: 10.1155/2012/654279

Figure Lengend Snippet: Flow cytometric analysis of CD4 + and CD8 α + cells isolated from splenocytes of chickens. Chickens in each group were, respectively, intramuscularly immunized with 100 μ g pcDNA-3-1E-linker-mChIL-15 (group 1), 100 μ g pcDNA3.1 (group 2), and sterile TE buffer (pH 7.6) (group 3) at 14 and 21 days of age. All chickens were inoculated orally with 5 × 10 4 sporulated oocysts of E. acervulina at 28 days of age (day 7 after secondary immunization). The ratio of CD4 + and CD8 α + cells in spleen of randomly selected five chickens was assayed at 28 and 34 days of age (day 6 after challenge). Data represent mean ± standard errors ( n = 5 per group). Highly significant difference ( P < 0.01) between bars not sharing the same capital letters in each time point. Significant difference ( P < 0.05) between bars not sharing the same small letters in each time point.

Article Snippet: The cells were respectively incubated with 5 μ L of fluorescein-isothiocyanate- (FITC-) conjugated mouse monoclonal antibodies (mAbs) against chicken CD4 + (AbD Serotec) and 5 μ L of FITC-conjugated mouse mAbs against chicken CD8 α + (AbD Serotec) for 30 min at room temperature.

Techniques: Isolation, Sterility

Journal: PLoS ONE

Article Title: A Comparative Study of N -glycolylneuraminic Acid (Neu5Gc) and Cytotoxic T Cell (CT) Carbohydrate Expression in Normal and Dystrophin-Deficient Dog and Human Skeletal Muscle

doi: 10.1371/journal.pone.0088226

Figure Lengend Snippet: GRMD muscle (cranial sartorius) was triple stained for Neu5Gc (green), Pax7 (A), CD11b (B), CD4 (C), CD8 (D) (all red), and DAPI (blue). Arrows indicated co-staining of Neu5Gc and Pax7 (in A), CD11b (in B) or CD4 (in C). Bar is 100 µm (A) and 50 µm (B–D) for all panels.

Article Snippet: Co-staining antibodies used in dog were mouse anti-chicken Pax7 (Developmental Studies Hybridoma Bank, clone P3U1), mouse anti-dog CD4 (AbD Serotec, MCA1998S), rat anti-dog CD8 (AbD Serotec, MCA1039GA), mouse anti-dog CD11b (AbD Serotec, MCA1777S), mouse anti-dog CD21 (AbD Serotec, MCA1781R), mouse anti-human β spectrin (Novus, NB300-574) or mouse anti-rat embryonic myosin (NovaCastra, NCL-MHCd).

Techniques: Staining

Journal: PLoS ONE

Article Title: A Comparative Study of N -glycolylneuraminic Acid (Neu5Gc) and Cytotoxic T Cell (CT) Carbohydrate Expression in Normal and Dystrophin-Deficient Dog and Human Skeletal Muscle

doi: 10.1371/journal.pone.0088226

Figure Lengend Snippet: (A) Cranial sartorius muscle sections from GR and GRMD dogs were stained with markers for satellite cells (Pax7), T lymphocytes (CD4 or CD8) or macrophages (CD11b) and quantified for numbers of cells stained per 40X visual field. (B) The percentage of cells co-stained for Neu5Gc and CD4, CD8, CD11b or Pax7 in GRMD muscles was quantified. Errors are SEM. ***P<0.001, for each GR vs. GRMD comparison in A.

Article Snippet: Co-staining antibodies used in dog were mouse anti-chicken Pax7 (Developmental Studies Hybridoma Bank, clone P3U1), mouse anti-dog CD4 (AbD Serotec, MCA1998S), rat anti-dog CD8 (AbD Serotec, MCA1039GA), mouse anti-dog CD11b (AbD Serotec, MCA1777S), mouse anti-dog CD21 (AbD Serotec, MCA1781R), mouse anti-human β spectrin (Novus, NB300-574) or mouse anti-rat embryonic myosin (NovaCastra, NCL-MHCd).

Techniques: Staining, Muscles, Comparison