Journal: Nature immunology

Article Title: T-bet controls regulatory T cell homeostasis and function during type-1 inflammation

doi: 10.1038/ni.1731

Figure Lengend Snippet: (a,b) Representative flow cytometric analysis of CXCR3 and Foxp3 expression by splenocytes isolated from wild-type (WT) mice, ( a ) or age-matched WT and Tbx21 –/- mice ( b ). Plots are gated on CD4 + splenocytes. rIgG2a, isotype control. Numbers display the frequency of cells expressing the indicated markers. Data are representative of greater than six mice analyzed in this fashion. ( c) Migration of CD4 + Foxp3 + splenocytes isolated from the indicated mice in response to media alone, 100nM CXCL10 or 100nM CCL21 in a transwell chemotaxis assay. Data are mean and s.d. of triplicate measurements. ( d) CD45.2 and CXCR3 expression on CD4 + Foxp3 + cells recovered from the indicated tissues of recipients of a mixture of CD45.1 + WT and CD45.2 + Tbx21 –/– BM. Numbers depict the percent of cells positive for the indicated markers. Data are representative of three independent experiments with four mice analyzed per experiment. ( e) T-bet expression (open histograms) in CD4 + Foxp3 + CXCR3 + or CD4 + Foxp3 + CXCR3 - splenocytes. Histograms (right) correspond to indicated gates (left). Data are representative of greater than ten mice analyzed in this fashion.

Article Snippet: Tbx21 -/- and Foxp3 gfp mice (on C57BL/6 background) were generously provided by A. Weinmann and A. Rudensky, respectively (University of Washington, Seattle, WA).

Techniques: Expressing, Isolation, Control, Migration, Chemotaxis Assay

Journal: Nature immunology

Article Title: T-bet controls regulatory T cell homeostasis and function during type-1 inflammation

doi: 10.1038/ni.1731

Figure Lengend Snippet: (a) Experimental design showing cell transfer and treatment schedule. Briefly, a mixture of CD45.1 + WT and CD45.2 + Tbx21 –/– T reg cells were injected into TCRβδ-KO mice, followed by treatment with the indicated antibodies. BrdU was added to the drinking water when indicated. (b) CD45.1 expression on splenocytes of recipient mice was analyzed by flow cytometry. Histograms are gated on Foxp3 + CD4 + TCRβ + B220 - cells, and numbers indicate the percent of cells positive and negative for CD45.1. Graphs show absolute numbers of WT- and Tbx21 –/– -derived T reg cells recovered from the spleens of recipient mice. Each point represents an individual treated mouse. (c) BrdU incorporation by splenocytes of recipient mice was analyzed by flow cytometry. Dot plots are gated on Foxp3 + CD4 + TCRβ + B220 - splenocytes. Numbers in plots indicate percentage of BrdU + cells among total WT- (CD45.1 + ) or Tbx21 –/– -derived (CD45.1 - ) T reg cells. Graphs depict the frequency of BrdU + cells among WT and Tbx21 –/– -derived T reg cell populations. For b and c , statistical significance was determined using a two-way repeated measures ANOVA. Bonferroni post-tests were used to obtain the P -values for the indicated pairwise comparisons. Data are representative of three independent experiments with three or greater mice per group

Article Snippet: Tbx21 -/- and Foxp3 gfp mice (on C57BL/6 background) were generously provided by A. Weinmann and A. Rudensky, respectively (University of Washington, Seattle, WA).

Techniques: Injection, Expressing, Flow Cytometry, Derivative Assay, BrdU Incorporation Assay

Journal: Nature immunology

Article Title: T-bet controls regulatory T cell homeostasis and function during type-1 inflammation

doi: 10.1038/ni.1731

Figure Lengend Snippet: (a) T-bet and Foxp3 expression on cells isolated from the indicated tissues of an Mtb -infected mouse (left) or an uninfected age-matched control (right). Plots are gated on CD4 + T cells. Numbers display the percent of cells in each of the indicated quadrants. Data are representative of five independent experiments. (b) (Left) Recipients of a mixture of WT (CD45.1 + ) and Tbx21 –/– (CD45.2 + ) BM were infected with Mtb for 105 days. Cells isolated from the indicated organs were analyzed by flow cytometry. Contour plots depict gating strategy, with numbers indicating the percent of cells positive for the indicated markers. Graphs depict the ratio of WT and Tbx21 –/– -derived cells among gated CD4 - CD8 - DN, CD4 + Foxp3 + T reg and CD4 + Foxp3 - CD44 hi T eff populations. Each point represents a value from an individual infected BM chimera. Data are representative of two independent experiments (n=3 per experiment).

Article Snippet: Tbx21 -/- and Foxp3 gfp mice (on C57BL/6 background) were generously provided by A. Weinmann and A. Rudensky, respectively (University of Washington, Seattle, WA).

Techniques: Expressing, Isolation, Infection, Control, Flow Cytometry, Derivative Assay

Journal: Nature immunology

Article Title: T-bet controls regulatory T cell homeostasis and function during type-1 inflammation

doi: 10.1038/ni.1731

Figure Lengend Snippet: (a) CD44 and T-bet expression (top) or IFN-γ production (bottom) by splenocytes isolated from age-matched WT or scurfy ( sf ) mice, as measured by flow cytometry. Plots are gated on CD4 + Foxp3 - cells. Numbers in plots indicate the percent of T-bet + (top) or IFN-γ + (bottom) cells among total CD44 hi CD4 + Foxp3 - cells. Data are representative of three independent experiments. (b) T-bet and Foxp3 expression by splenocytes isolated from age-matched WT mice or sf mice given WT T reg cells. Plots are gated on CD4 + CD8 - lymphocytes. Numbers in plots indicate the percentage of CD4 + Foxp3 + cells expressing T-bet. Data are representative of six independent experiments. (c) Cells isolated from the spleen and peripheral lymph nodes (pLN) of sf neonate recipients of WT or Tbx21 -/- T reg cells were analyzed by flow cytometry. Numbers in plots indicate fraction of Foxp3 + T cells among total CD4 + reg splenocytes. (d, e) Flow cytometric and quantitative analysis of splenocytes isolated from sf mice given WT or Tbx21 –/– T reg cells, or from age-matched WT mice. Splenocytes in e were stimulated with PMA and ionomycin prior to analysis. Plots are gated on CD4 + Foxp3 - cells. Numbers in plots display percentage of T-bet + ( d ) and IFN-γ + ( e ) cells among total CD4 + Foxp3 - CD44 hi cells. (f) Photograph of representative spleen and peripheral LNs isolated from 6 week old sf mice given WT (left) or Tbx21 –/– (right) T reg cells as neonates. Representative of six independent experiments. For c , d and e , each point represents an individual mouse, and significance was measured using two-tailed, unpaired student’s t tests.

Article Snippet: Tbx21 -/- and Foxp3 gfp mice (on C57BL/6 background) were generously provided by A. Weinmann and A. Rudensky, respectively (University of Washington, Seattle, WA).

Techniques: Expressing, Isolation, Flow Cytometry, Two Tailed Test