Journal:

Article Title: Massive T-Lymphocyte Infiltration into the Host Stroma Is Essential for Fibroblast Growth Factor-2-Promoted Growth and Metastasis of Mammary Tumors via Neovascular Stability

doi: 10.2353/ajpath.2009.080471

Figure Lengend Snippet: FGF-2 induces intensive T-lymphocyte and macrophage infiltration into the host stroma of normal mice. Normal and nude mice were inoculated with 4T1 cells into the mammary fat pad, and then FGF-2 was administered from days 1 to 2 after inoculation using the same procedure described in Figure 1. A: Representative photographs show the immunostaining of CD3-positive T lymphocytes in the host stroma on days 5 and 7. B: The number of T lymphocytes was counted in the host stroma on days 5 and 7. C: Representative photographs show the immunostaining of F4/80-positive macrophages in the host stroma on day 7. D: The number of macrophages was counted in the host stroma on day 7. Five mice were examined in each group. Each value represents the mean number of cells per area (mm2) ± SEM. Significant differences from each control group are shown as *P < 0.05; **P < 0.01; and N.S, not significant. T and S in the photographs represent the tumor tissue and host stroma, respectively.

Article Snippet: For the activation of primary T lymphocytes in the culture, mouse IL-2 and hamster monoclonal antibody against mouse CD3ε (145-2C11) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Immunostaining

Journal: Nature Communications

Article Title: CD4 expression in effector T cells depends on DNA demethylation over a developmentally established stimulus-responsive element

doi: 10.1038/s41467-022-28914-4

Figure Lengend Snippet: a Flow-cytometry plot of CD4 expression in control and Cd4 E4mflox/flox T cells, transduced with Cre recombinase and labeled with a cell-proliferation dye prior to activation. Transduced cells were gated on GFP. Data are representative to 3 independent experiments. b CD4 MFI of cells from ( a ) measured at two different time-points post transduction ( n = 3 independent samples) and data shown are representative of three independent experiments. c Integrative Genome View (IGV) browser shot of ATAC-Seq data depicting chromatin accessibility within and upstream of the Cd4 locus. Tracks shown are set at the same scale. Arrow indicate the annotated Cd4 TSS and red box denotes the location of E4a. d FACS plot showing CD4 expression in proliferating T cells that were CFSE-labeled prior to in vitro activation with anti-CD3/CD28. Dot plots at 72hrs (blue dots) and 96hrs (red dots) post activation were overlaid. Data are representative of >3 independent experiments. e Percent of cells with low CD4 expression at indicated proliferation cycles assessed by CFSE labeling ( n = 3 independent samples for Cd4 E4mΔ/Δ and Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are representative >3 experiments. *** p = 0.0003, **** p < 0.0001 (two-way ANOVA with Sidak Multiple Comparisons test). f CD4 mRNA expression (exon 3-4) in activated CD4 T cells. RNA was isolated 96hrs post activation with anti-CD3/CD28 ( n = 4 for group 1 and 3; n = 5 for group 2). Data is expressed as mean ± SEM. *** p = 0.004, p = 0.0002 (one-way ANOVA and Dunnett’s test). g Schematic illustration of experimental design. Naive WT CD45.1 T cells or CD45.2 T cells from Cd4 E4mΔ/Δ/ E4aΔ/Δ mice were FACS-sorted, CFSE labeled and mixed at a 1:1 ratio before i.v. transfer into Rag −/− recipients. Seven days later, LNs and spleen were isolated and analyzed by flow cytometry. h FACS plot showing CD4 expression in CD4 T cells from Rag −/− mice at day 7 post transfer. Cells were gated on congenic markers to distinguish WT and mutant T cells. Data are representative of two independent experiments. i , CD4 geometric MFI in cells gated on CSFE staining profiles ( n = 3 independent samples for Cd4 +/+ and n = 5 independent samples for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data are expressed as mean ± SEM and is representative of two independent experiments. * p = 0.0174, **** p < 0.0001 (two-way ANOVA with Bonferroni multiple comparison test).

Article Snippet: FACS-sorted CD4 + CD8 − CD25 − CD62L + CD44 lo naive T cells were seeded in T-cell medium [RPMI 1640 (Gibco), 10% heat-inactivated FBS (Atlanta), 2 mM L-glutamine, 50 µg/ml gentamicin, 1% Penn/Strep, and 50 uM 2-mercaptoethanol (Gibco)] on plate-bound hamster IgG along with anti-CD3 (BioXcell, clone 145-2C11, 0.25 μg/ml or Tonbo, clone 17A2, 0.25ug/mL) and anti-CD28 (BioXcell, clone 37.5.1, 1 μg/ml or Tonbo, clone 37.51, 1ug/mL) antibodies.

Techniques: Flow Cytometry, Expressing, Transduction, Labeling, Activation Assay, In Vitro, Isolation, Mutagenesis, Staining

Journal: Nature Communications

Article Title: CD4 expression in effector T cells depends on DNA demethylation over a developmentally established stimulus-responsive element

doi: 10.1038/s41467-022-28914-4

Figure Lengend Snippet: a Heatmap depicting percent CpG methylation in control CD4 + (Tet1/3 flox/flox ), Tet1/3 cDKO CD4+ mature thymocytes and Tet1/3 cDKO CD4+ naive peripheral T cells for CpGs −9270bp to −15869bp relative to the Cd4 TSS (Chr6:124847307–124853906; mm9). The blue line underlines CpGs flanking the E4a region. CATCH-seq was performed on genomic DNA from sorted populations of TCRβ hi CD24 lo CD69 − CD4 + CD8 − thymocytes or CD4 + TCRβ + CD62L hi CD44 − T cells from LN/spleen. Replicates are from two independent mice. b H3K4me3 modifications assessed by ChIP-qPCR in sorted naive CD4 T cells. n = 2 or 3 mice/genotype. Data shown are a summary of two independent experiments and expressed as mean ± SEM. * p = 0.0257, ns not significant, p = 0.9797, p = 0.187, and p > 0.9999 (two-way ANOVA and Bonferroni test). c H3K9me3 modifications assessed by ChIP-qPCR in sorted naive CD4 T cells. n = 2 mice/genotype and representative of two independent experiments. d Heatmap depicting percent CpG methylation in activated (WT) Cd4 +/+ , Cd4 E4pΔ/Δ , Cd4 E4mΔ/Δ , Cd4 E4aΔ/Δ , and Cd4 E4mΔ/Δ/ E4aΔ/Δ T cells for CpGs -9270 bp to -15869 bp relative to the Cd4 TSS (Chr6:124847307-124853906; mm9). The blue line underlines CpGs flanking the E4a region. CATCH-seq was performed on genomic DNA from naive T cells activated in vitro with anti-CD3/CD28 for 120 hrs. Note that data for Cd4 E4pΔ/Δ and Cd4 E4mΔ/Δ conditions were from previously published experiments with similar experimental conditions , . e Heatmap depicting percent CpG methylation in WT Cd4 +/+ , Cd4 E4pΔ/Δ , Cd4 E4mΔ/Δ , Cd4 E4aΔ/Δ , and Cd4 E4mΔ/Δ/ E4aΔ/Δ T cells for CpGs from +6200 to −669 relative to the Cd4 TSS (Chr6:124832027–124838896; mm9). A red line underlines CpGs in E4m (indicated by the gap in the mutant mice) and a black arrow indicates the Cd4 TSS. Red box indicates the Cd4 promoter. Note that data for Cd4 E4pΔ/Δ and Cd4 E4mΔ/Δ conditions were from previously published experiments with similar experimental conditions , .

Article Snippet: FACS-sorted CD4 + CD8 − CD25 − CD62L + CD44 lo naive T cells were seeded in T-cell medium [RPMI 1640 (Gibco), 10% heat-inactivated FBS (Atlanta), 2 mM L-glutamine, 50 µg/ml gentamicin, 1% Penn/Strep, and 50 uM 2-mercaptoethanol (Gibco)] on plate-bound hamster IgG along with anti-CD3 (BioXcell, clone 145-2C11, 0.25 μg/ml or Tonbo, clone 17A2, 0.25ug/mL) and anti-CD28 (BioXcell, clone 37.5.1, 1 μg/ml or Tonbo, clone 37.51, 1ug/mL) antibodies.

Techniques: CpG Methylation Assay, In Vitro, Mutagenesis

Journal: Nature Communications

Article Title: CD4 expression in effector T cells depends on DNA demethylation over a developmentally established stimulus-responsive element

doi: 10.1038/s41467-022-28914-4

Figure Lengend Snippet: a Venn diagram showing the number of genes that upregulate gene expression (a fold change >2 in gene expression from DN3 to CD4 + SP) intersected with genes that have 5hmC in either DP or CD4 + SP T cells (intragenic 5hmC log2 CMS-IP/input >2) intersected with genes containing open-chromatin ATAC-Seq peaks and H3K27Ac marks in either DP or CD4 + T cells. b Metascape bar graph showing top nonredundant enrichment clusters among genes that have novel-accessibility ATAC-Seq in activated T cells, undergo DNA demethylation, and upregulate RNA expression during thymic development. The number of genes in each cluster is indicated on the left and cluster IDs are shown on the right. Bar graph was generated using the Metascape gene annotation and analysis online resource. p -values were computed using hypergeometric test and Benjamini–Hochberg p -value correction algorithm as previously described in the publicly available Metascape interface . List of genes used for the enrichment analysis is found in Supplementary Table . c FACS plot showing CD4, ThPOK, CD5, and CD6 expression in CFSE-labeled T cells from control or Rorc(t) CreTg Tet1 fl/fl Tet2 fl/+ Tet3 fl/fl mice 72 hrs post activation. Naive CD4 T cells were FACS-sorted and activated with anti-CD3/CD28. Data is a representative of two experiments with at least two animals/genotype/experiment. d Cd4, Zbtb7b, Cd5 and Cd6 mRNA expression in control or Rorc(t) CreTg Tet1 fl/fl Tet2 fl/+ Tet3 fl/fl T cells activated in vitro for 96 hrs with anti-CD3/CD28. Data shown are mean ± SEM ( n = 3). p -values are indicated on graphs (unpaired two-tailed t -test). e IGV snapshots of the Zbtb7b locus depicting p300 and H3K27Ac signals by ChIP-Seq in activated T (Th0) cells and ATAC-Seq peaks in DP, CD4 + thymocytes, and Th0 CD4 + T cells. Red arrows denote currently undefined CREs. Green arrow shows the location of a previously validated CRE PE in CD4 + T cells , . f IGV snapshot of the Cd5 locus depicting p300 and H3K27Ac signals by ChIP-Seq in activated T (Th0) cells and ATAC Seq peaks in DP, CD4 + thymocytes and Th0 CD4 + T cells. Red arrows denote currently undefined CREs. g IGV snapshot of the Cd6 locus depicting p300 and H3K27Ac signals by ChIP-Seq in activated T (Th0) cells and ATAC-Seq peaks in DP, CD4 + thymocytes and activated CD4 + T cells. Red arrows denote currently undefined CREs.

Article Snippet: FACS-sorted CD4 + CD8 − CD25 − CD62L + CD44 lo naive T cells were seeded in T-cell medium [RPMI 1640 (Gibco), 10% heat-inactivated FBS (Atlanta), 2 mM L-glutamine, 50 µg/ml gentamicin, 1% Penn/Strep, and 50 uM 2-mercaptoethanol (Gibco)] on plate-bound hamster IgG along with anti-CD3 (BioXcell, clone 145-2C11, 0.25 μg/ml or Tonbo, clone 17A2, 0.25ug/mL) and anti-CD28 (BioXcell, clone 37.5.1, 1 μg/ml or Tonbo, clone 37.51, 1ug/mL) antibodies.

Techniques: Expressing, RNA Expression, Generated, Labeling, Activation Assay, In Vitro, Two Tailed Test, ChIP-sequencing