Journal: The Journal of Experimental Medicine

Article Title: Genetic models reveal origin, persistence and non-redundant functions of IL-17–producing γδ T cells

doi: 10.1084/jem.20181439

Figure Lengend Snippet: eGFP, luciferase, and DTx receptor are functionally expressed by γδ T cells. (A) Two-photon in vivo imaging of Tcrd -GDL mouse ear skin and small intestine. Green, γδ T cell eGFP; blue, second harmonics (ear skin) or Hoechst 33342 nuclear staining (small intestine). Bars: 50 µm (ear skin); 15 µm (small intestine; see also Videos 1 and 2). (B) eGFP expression by peripheral lymph node γδ T cells (green, gated as CD45 + Tcrβ – CD3 + TCRγδ + ), αβ T cells (blue, gated as CD45 + Tcrβ + CD3 + ), and B cells (red, gated as CD45 + Tcrβ – CD3 – CD19 + ) of a Tcrd -GDL mouse and CD45 + cells of a C57BL/6-NCrl mouse (WT, filled gray line). Shown is one representative histogram of two experiments with each n = 2–3 mice per group. (C) Bioluminescence by functional luciferase expression was detected by IVIS in at least two independent experiments with n = 1–2 mice each. C57BL/6-NCrl WT and Tcrd -GDL were sacrificed 10 min after injection of d -luciferin and the indicated organs were displayed for imaging. Color bar corresponds to signal intensities measured as photons. c, colon; k, kidney; li, liver; lu, lung; pl: peripheral lymph node; si, small intestine; sp, spleen; st, stomach; t, thymus. (D) Depletion of γδ T cells using DTx was analyzed by flow cytometry. Representative contour plot of pLN T cells (CD3 + cells) of Tcrd -GDL mice treated with PBS (ctrl.) or DTx (top). Cell counts of indicated cell populations in pLNs of DTx- (white bars) and PBS- (black bars) injected Tcrd -GDL and C57BL/6-NCrl WT mice (bottom). Bar graphs show pooled data from four independent experiments with each n = 1–3 mice per group, Kruskal Wallis test with Dunn's Multiple Comparison post-tests. *, P < 0.05; ns, not significant.

Article Snippet: Antibodies directed against CD3 (clone 17A2; APCVio770), TCRβ (clone REA318; APCVio770), CD45.2 (clone 104-2; PerCPVio700 or VioGreen), and CD44 (clone IM7.8.1; VioBlue) were purchased from Miltenyi Biotec.

Techniques: Luciferase, In Vivo Imaging, Staining, Expressing, Functional Assay, Injection, Imaging, Flow Cytometry, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Genetic models reveal origin, persistence and non-redundant functions of IL-17–producing γδ T cells

doi: 10.1084/jem.20181439

Figure Lengend Snippet: Depletion of Tγδ17 cells does not change their repertoire. (A and B) Flow cytometric analysis of indicated cell populations 1 d (d1), 2 wk (2w), and 7 wk (7w) after depletion of γδ T cells in Tcrd -GDL littermate mice. Shown are pooled data from three independent experiments with each n = 2 - 5 mice per group, Student’s t test. (A) Bar graph shows frequencies of γδ T cells (Tcrβ – GFP + ) among T cells (A.dead – CD3 + ) in peripheral lymph nodes, mean ± SD. (B) Scatter plots show frequencies of indicated γδ T cell populations among all γδ T cells in peripheral lymph nodes, one dot represents one mouse, mean. (C) T cell receptor repertoire analysis of γδ T cell nondepleted (ctrl., left) and depleted Tcrd -GDL mice 8–9 wk after depletion (right). Sorted CD44 hi Ly6C – Vγ4 + peripheral lymph node cells were pooled from 7 - 8 mice each and facilitated to Illumina NGS. Amplicons were generated by multiplex PCR. Proportions of Vγ4 (top) and Vδ5 (bottom) top 20 and top 5 amino acid (AA) clonotypes among complete Vγ4 and Vδ5 repertoires. Amino acid sequences of the top 5 clonotypes. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Antibodies directed against CD3 (clone 17A2; APCVio770), TCRβ (clone REA318; APCVio770), CD45.2 (clone 104-2; PerCPVio700 or VioGreen), and CD44 (clone IM7.8.1; VioBlue) were purchased from Miltenyi Biotec.

Techniques: Generated, Multiplex Assay

Journal: The Journal of Experimental Medicine

Article Title: Genetic models reveal origin, persistence and non-redundant functions of IL-17–producing γδ T cells

doi: 10.1084/jem.20181439

Figure Lengend Snippet: Patchy depletion and recovery of ear skin γδ T cells. (A) Frequencies of γδTCR hi epidermal (left) and γδTCR lo dermal (right) γδ T cells in nondepleted (ctrl.) and 7-wk-ago–depleted (7w) Tcrd -GDL mice. Pooled data from two independent experiments, one dot represents one mouse, mean. (B) Longitudinal in vivo two-photon imaging of Tcrd -GDL ear skin. Images were acquired from the same mouse at indicated time points after γδ T cell depletion. As reference, a mouse was injected with PBS (ctrl.). Green: γδ T cell eGFP by Tcrd -GDL mice, shown are representative images of two independent experiments. Bars, 50 µm (see Video 3). (C) Epidermal sheets of DTx- and PBS (ctrl.)–treated Tcrd -GDL mice 4 wk after γδ T cell depletion. Representative fluorescence microscopy images out of two independent experiments with n = 1–2 mice each; blue: Vγ5; red: CD3; white: DAPI nuclear staining. Bars, 50 µm. (D) Epidermal sheets of hetero- and homozygous Tcrd -GDL mice were stained with anti-CD3ε (red) 18 d after depletion (DTx) or PBS injection (ctrl.). Bars, 100 µm. Staining was validated in three experiments with n = 1–2 mice each.

Article Snippet: Antibodies directed against CD3 (clone 17A2; APCVio770), TCRβ (clone REA318; APCVio770), CD45.2 (clone 104-2; PerCPVio700 or VioGreen), and CD44 (clone IM7.8.1; VioBlue) were purchased from Miltenyi Biotec.

Techniques: In Vivo, Imaging, Injection, Fluorescence, Microscopy, Staining

Journal: The Journal of Experimental Medicine

Article Title: Genetic models reveal origin, persistence and non-redundant functions of IL-17–producing γδ T cells

doi: 10.1084/jem.20181439

Figure Lengend Snippet: Tγδ17 cells are long lived and self-renewing. (A) Inducible γδ T cell fate-mapping model. RFP expression in γδ T cells was induced by oral administration of tamoxifen (left). Representative gating for RFP + peripheral lymph node γδ T cells 7 wk after tamoxifen administration (right). (B and C) Analysis of RFP + cells from indicated tissues and time points after RFP induction. One dot equals one mouse, pooled data from three independent experiments with each three to six littermate mice per time point, mean. (B) Frequencies of RFP + γδ T cells. (C) Frequencies of RFP + cells among IFN-γ (left) or IL-17 (right) phenotype γδ T cell subsets, defined as CD3 + Tcrβ – γδTCR + CD44 hi CD27 – and CD3 + Tcrβ – γδTCR + CD44 – CD27 + , respectively. (D) Mean frequency of RFP + cells among skin-derived γδTCR hi DETCs and γδTCR lo dermal γδ T cells. One dot equals one mouse, pooled data from two experiments with each three to five littermate mice per time point, mean.

Article Snippet: Antibodies directed against CD3 (clone 17A2; APCVio770), TCRβ (clone REA318; APCVio770), CD45.2 (clone 104-2; PerCPVio700 or VioGreen), and CD44 (clone IM7.8.1; VioBlue) were purchased from Miltenyi Biotec.

Techniques: Expressing, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Genetic models reveal origin, persistence and non-redundant functions of IL-17–producing γδ T cells

doi: 10.1084/jem.20181439

Figure Lengend Snippet: Freshly γδ T cell-depleted mice develop less severe IMQ-induced psoriasis. (A–C) IMQ-induced psoriasis. Ears of indicated genotypes were treated with IMQ or Vaseline (Vas.), starting at day 3 after γδ T cell depletion (DTx). (A) Ear skin immunofluorescence histology at day 8 of IMQ treatment of mice shown in B, CD3 + T cells (blue), and CD11b + Ly6G + neutrophils (yellow). Bars, 100 µm. Representative images from of two independent experiments with n = 1–4 mice each. (B) Change of ear thickness given as percent diameter of untreated ears (day 0; left) and disease score (right) over time. Graphs show pooled data from three experiments, each one to four mice per group (total numbers of mice: seven Tcrd -GDL DTx IMQ; eight Tcrd -GDL ctrl. IMQ; seven Tcrd −/− IMQ; six Tcrd -GDL DTx Vas; five Tcrd -GDL ctrl. Vas; six Tcrd −/− Vas); two-way ANOVA with Bonferroni posttests, mean ± SD. (C) Epidermal hyperplasia measured on H&E-stained ear skin sections of IMQ- and Vaseline-treated ears of the indicated genotypes, respectively. Scatter plot shows quantification of the epidermal thickening measured on the sections; each dot represents one measurement acquired on 36 slides of two mice per group (left). Representative images out of two mice per group. Bars, 50 µm (right). Data were validated in two independent experiments. (D) Steady state mean frequency of IL-17A–producing CD45 + lymphocytes in ear skin of control (ctrl.) and γδ T cell–depleted Tcrd -GDL mice, day 3 (d3) after depletion, compared with Tcrd −/− mice by intracellular flow cytometry. Pooled data from three to five experiments with each n = 2–4 mice per group; one dot equals one mouse, mean. One-way ANOVA with Bonferroni posttests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Antibodies directed against CD3 (clone 17A2; APCVio770), TCRβ (clone REA318; APCVio770), CD45.2 (clone 104-2; PerCPVio700 or VioGreen), and CD44 (clone IM7.8.1; VioBlue) were purchased from Miltenyi Biotec.

Techniques: Immunofluorescence, Staining, Control, Flow Cytometry

Journal: Cancer Cell

Article Title: Retinoic acid receptor activation reprograms senescence response and enhances anti-tumor activity of natural killer cells

doi: 10.1016/j.ccell.2024.02.004

Figure Lengend Snippet:

Article Snippet: CD3 Antibody, anti-mouse, APC-Vio770, REAfinity , Miltenyi Biotec , Cat# 130-119-793; RRID: AB_2751847.

Techniques: Control, Purification, Recombinant, Staining, Reverse Transcription, Cell Isolation, Isolation, cDNA Synthesis, Gene Expression, shRNA, Plasmid Preparation, CRISPR, Software, Transfection, In Vitro, Imaging