cd4 cd25 treg cells  (Miltenyi Biotec)

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: Tregs produce TNF in several organs in naïve mice. (A) Representative flow cytograms showing percentages of TNF + cells, and geometric mean fluorescence intensity (gMFI) of TNF in gated TNF + cells, in Treg and Tconv from the inguinal lymph node (IngLN), mesenteric lymph node (MesLN), spleen (SPL), lung, liver, and bone marrow (BM), of naïve C57BL/6 mice. (B,C) Cumulative analysis showing percentages of TNF + (B), and gMFI of TNF in gated TNF + cells (C), in Treg and Tconv of the indicated organs. Data are from one representative of two independent experiments, each performed with 3–5 mice. Bars represent means and SD. * p < 0.05, by multiple Mann–Whitney test with Holm–Sidak correction for multiple comparisons.

Article Snippet: CD4 + CD25 + (Treg) and CD4 + CD25 − (Tconv) were immunomagnetically isolated from splenocytes using the CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse (Miltenyi Biotec).

Techniques: Fluorescence, MANN-WHITNEY

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: TNF and TNFR2 are differentially distributed in Tregs. (A) Tregs and Tconvs were immunomagnetically enriched as CD4 + CD25 + and CD25 − , respectively, from the spleens of C57BL/6 mice and restimulated in vitro 4 h before analysis of surface TNFR2 and intracellular TNF. (B, C) TNFR2 + and TNFR2 − Tregs and Tconvs were sorted as YFP + and YFP − CD4 T cells, respectively, from the spleens of Foxp3 CreYFP mice and restimulated in vitro 4 h before analysis of surface TNFR2 and intracellular TNF. (B) Representative cytograms showing the purity of sorted cells, and TNF/TNFR2 expression in unstimulated and stimulated cells. (C) Cumulative analysis showing percentages of TNF + , and gMFI of TNF in gated TNF + cells, in the indicated subpopulation (each tested in 2–3 replicates). Data are from one representative of two independent experiments, each performed with CD4 T cells from the pooled splenocytes of three mice. Bars represent means and SD. * p < 0.05, ** p < 0.01, *** p < 0.001, by one‐way ANOVA.

Article Snippet: CD4 + CD25 + (Treg) and CD4 + CD25 − (Tconv) were immunomagnetically isolated from splenocytes using the CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse (Miltenyi Biotec).

Techniques: In Vitro, Expressing

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: Both TNF + and TNFR2 + Tregs are expanded in tumors. (A) Representative cytograms showing Treg and Tconv frequencies and TNF/TNFR2 expression in spleen (SPL) and tumor (TUM) samples, from mice bearing MC38 subcutaneous tumors. (B–D) Cumulative analysis showing percentages of Tregs among CD4 (B), TNFR2 + TNF − and TNFR2 − TNF + Treg and Tconv among CD4 T cells (C), and the gMFI of the indicated markers (D), in SPL and TUM samples of MC38 tumor‐bearing mice ( n = 9). Bars represent means and SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by multiple Mann–Whitney test with Holm–Sidak correction for multiple comparisons.

Article Snippet: CD4 + CD25 + (Treg) and CD4 + CD25 − (Tconv) were immunomagnetically isolated from splenocytes using the CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse (Miltenyi Biotec).

Techniques: Expressing, MANN-WHITNEY

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: Treg recovery is associated with TNF production in chronic CIA. (A) C57BL/6 females ( n = 16) were immunized with chicken collagen type II as described in the Methods section. Mice were sacrificed after 2 weeks (acute CIA, n = 8) or 10 weeks (chronic CIA, n = 8), after the second immunization, and two draining lymph nodes per mouse were collected. Arrows indicate the two time points of analysis. As control, inguinal lymph nodes were harvested from nonimmunized sex‐ and age‐matched mice at the two time points (ctrl, n = 10). (B, C) Representative plots showing Treg and Tconv frequencies in gated CD4 T cells (B), and the percentage of CD44 Teff in gated Tconv (C). (D) Analysis of Treg and Teff percentages in lymph nodes of mice with acute or chronic CIA, compared with naïve controls (ctrl). (E) Representative plots of intracellular TNF and IFNγ in Treg and Teff (identified as CD44 + Tconv) in the indicated samples. (F) Analysis of the percentages of TNF + IFNγ + / − cells in gated Treg or Teff. In all plots, bars represent means and SD. Each dot is a single lymph node. Statistically significant outliers were identified with the Rout method and removed from the analysis. ** p < 0.01, **** p < 0.0001, by Kruskal–Wallis test with Dunn's multiple comparison.

Article Snippet: CD4 + CD25 + (Treg) and CD4 + CD25 − (Tconv) were immunomagnetically isolated from splenocytes using the CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse (Miltenyi Biotec).

Techniques: Control, Comparison

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: TNFR2 + and TNFR2 − Treg maintain their diversity, while TNFR2 − are more activated when cocultured with TNFR2 + . (A–D) TNFR2 + and TNFR2 − Tregs were sorted from the spleens of Foxp3 CreYFP mice, respectively labeled with eF670 (blue) or CTV (purple) proliferation dyes, cultured 3–4 days with coated aCD3/aCD28 in the presence of IL‐2, and then analyzed by flow cytometry (after restimulation in vitro for 4 h for the analysis of intracellular TNF). (A) TNFR2 expression against each proliferation dye in each cell type. (B) Percentages of TNFR2 + cells, and geometric mean fluorescence intensity (gMFI) of TNFR2 in gated TNFR2 + cells, in the indicated cell types. (C) TNF expression against each proliferation dye in each cell type. (D) Percentages of TNF+ cells, and geometric mean fluorescence intensity (gMFI) of TNF in gated TNF + cells, in the indicated cell types. * p < 0.05, ** p < 0.01, **** p < 0.0001, by Student's t ‐test. (E–I) TNFR2 + and TNFR2 − Tregs were seeded alone or cocultured at 1:1 ratio. (E, F) Representative cytograms and percentages of each cell type in the indicated conditions. (G) Contour plot overlays with adjunct histograms showing the fluorescence of the two proliferation dyes in TNFR2 + and TNFR2 − Tregs, either alone or cocultured. (H, I) Representative histogram overlays and analysis of the gMFI of the indicated dyes and markers in TNFR2 + and TNFR2 − Tregs, either alone or cocultured. ** p < 0.01, *** p < 0.001, **** p < 0.0001, by two‐way ANOVA. Data are from one experiment representative of three. Each condition was tested in triplicate.

Article Snippet: CD4 + CD25 + (Treg) and CD4 + CD25 − (Tconv) were immunomagnetically isolated from splenocytes using the CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse (Miltenyi Biotec).

Techniques: Labeling, Cell Culture, Flow Cytometry, In Vitro, Expressing, Fluorescence

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: TNFR2 + Tregs display superior suppressive function and survival in vitro. (A, B) TNFR2 + and TNFR2 − Tregs were sorted from the spleens of Foxp3 eGFP‐Cre‐ERT2 mice, and their phenotype was freshly analyzed through flow cytometry. The plots display histogram overlays (A) and a heatmap of the z‐score (B), calculated from means and SD of gMFI of each marker in Tconvs, TNFR2 + , and TNFR2 − Tregs ( n = 4). Data are from one experiment representative of two. * p < 0.05, by Mann–Whitney test. (C–F) Tconv were sorted as YFP − cells, labeled with the CTV proliferation dye, and cultured in vitro either alone or at scaled ratios with eF670‐labeled TNFR2 + or TNFR2 − Tregs. (C) Histogram overlays showing CTV dilution and respective gMFI in gated Tconv cultured alone (black) or cocultured with TNFR2 + (red) or TNFR2 − (blue) Tregs in the indicated conditions. (D) Analysis of the gMFI of CTV and of the percentage of proliferating cells in gated Tconv in the indicated conditions. (E, F) Representative cytograms and cumulative analysis showing the percentages and the counts of Treg in each coculture condition. Data are from one representative of two independent experiments. Each condition was tested in 2–6 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by unpaired t ‐test with Holm–Sidak correction for multiple comparisons.

Article Snippet: CD4 + CD25 + (Treg) and CD4 + CD25 − (Tconv) were immunomagnetically isolated from splenocytes using the CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse (Miltenyi Biotec).

Techniques: In Vitro, Flow Cytometry, Marker, MANN-WHITNEY, Labeling, Cell Culture

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: The molecular program of TNFR2 + Tregs supports survival and resistance to oxidative stress. (A) Volcano plot showing the fold change in gene expression by RNAseq of TNFR2 + versus TNFR2 − Treg sorted from spleens of Foxp3 CreYFP mice ( n = 4). Genes with FDR <0.05 and with fold change >2 (red) or <−2 (blue) are highlighted. (B) Statistically significant (FDR <0.05) results from the gene set enrichment analysis of the transcriptome of TNFR2 + versus TNFR2 − Treg in the hallmarks gene sets. (C) Gene set enrichment analysis of the transcriptome of TNFR2 + versus TNFR2 − Treg. A gene set of oxidative stress was obtained from a published signature . Normalized enrichment scores (NES) and FDR q values are shown under the plot. (D) Expression of Foxp3 was estimated as the gMFI of GFP in TNFR2 + versus TNFR2 − Treg sorted from spleens of Foxp3 eGFP‐Cre‐ERT2 mice and treated for 1 h with the indicated concentrations of menadione. **** p < 0.0001, by two‐way ANOVA. (E) Suppression assay was performed with GFP + TNFR2 + versus TNFR2 − Treg, either untreated or pretreated for 1 h with menadione, against CTV‐labeled Tconv. Treg percentage (as a measure of survival) and gMFI of CTV (as a measure of suppression) are shown at different Treg:Tconv ratios. Right plots represent the percentage of change between untreated and menadione‐treated cells. Each condition was tested in triplicate. Data are from one experiment representative of two. * p < 0.05, *** p < 0.001, **** p < 0.0001, by two‐way ANOVA. (F) Representative cytograms and cumulative analysis of CellROX staining in gated TNFR2 + or TNFR2 − Treg from spleens of Foxp3 CreYFP mice, either unstimulated or stimulated with aCD3 for 18 h. Numbers indicate the geometric mean fluorescence intensity (gMFI). The fmo of CellROX as a negative control is shown. Each condition was tested in quadruplicate. Bars represent means and SD. * p < 0.05, by unpaired t ‐test with Holm–Sidak correction for multiple comparisons. (G) Representative cytograms and cumulative analysis of CellROX staining in gated TNFR2 + or TNFR2 − Treg in unstimulated lymphocytes from spleens or tumors of Foxp3 eGFP‐Cre‐ERT2 mice ( n = 5), previously injected with MC38 tumor cells. Numbers indicate the geometric mean fluorescence intensity (gMFI). The fmo of CellROX as a negative control is shown. Data are from one experiment representative of two. Bars represent means and SD. * p < 0.05, *** p < 0.001, by two‐way ANOVA with Geisser–Greenhouse correction and Sidak multiple comparisons test.

Article Snippet: CD4 + CD25 + (Treg) and CD4 + CD25 − (Tconv) were immunomagnetically isolated from splenocytes using the CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse (Miltenyi Biotec).

Techniques: Gene Expression, Expressing, Suppression Assay, Labeling, Staining, Fluorescence, Negative Control, Injection