Journal: Clinical and Translational Medicine

Article Title: LRH‐1/NR5A2 targets mitochondrial dynamics to reprogram type 1 diabetes macrophages and dendritic cells into an immune tolerance phenotype

doi: 10.1002/ctm2.70134

Figure Lengend Snippet: Reagents and resources used in this study.

Article Snippet: CD194 , Miltenyi Biotec , Cat#130‐117‐376.

Techniques: Recombinant, Cell Isolation, TUNEL Assay, Extraction, Modification, Multiplex Assay, Software, Selection, Control

Journal: Clinical and Translational Medicine

Article Title: LRH‐1/NR5A2 targets mitochondrial dynamics to reprogram type 1 diabetes macrophages and dendritic cells into an immune tolerance phenotype

doi: 10.1002/ctm2.70134

Figure Lengend Snippet: LRH‐1/NR5A2 agonism alters CD4 + and CD8 + T‐cell subpopulations in T1D individuals. PBMCs were purified from individuals with T1D and exposed to 10 µM BL001 every 24 h for a total duration of 48 h with a final dose given 30 min prior to analysis. Flow cytometry immunophenotyping of (A) CD4 + CD25 + and (B) Tregs; CD4 + CD25 + FoxP3 + cell subpopulations. The initial gating defining the CD4 population was always set at 10 000 cells. n = 8 independent individuals with T1D. Relative transcript levels of (C) FoxP3, (D) CTLA4 and (E) GATA3. Data were normalised to the housekeeping gene RSP9. n = 5 T1D independent donors. Flow cytometry immunophenotyping of (F) Th1; CD4 + CD196 − CD183 + CD194 − and (G) Th2; CD4 + CD196 − CD183 − CD194 + . The initial gating defining the CD4 population was always set at 10000 cells. n = 8 independent individuals with T1D. Relative (H) transcript and (I) secreted levels of IFNγ as well as of (J) IL‐4. Transcript levels of IFNγ were normalised to the housekeeping gene RSP9. n = 4–6 T1D independent donors. (K) Th17/22; CD4 + CD196 + CD183 − CD194 + immunophenotyping and (L) IL17 transcript levels normalised to the housekeeping gene RSP9. n = 6–8 T1D independent donors. (M) CD8 + immunophenotyping. n = 8 independent individuals with T1D. (O) CD4 + cells were isolated from PBMCs and treated with BL001 as described above. Cells were then analysed by flow cytometry for the cell surface markers CD25 + FoxP3 + . Relative (P) LRH‐1/NR5A2 and (Q) FoxP3 transcript levels in either siScrambled (siSc) or siNR5A2‐treated PBMCs. Data were normalised to the housekeeping gene RSP9. n = 3 T1D independent donors. CD14 − PBMCs were labelled with CFSE and stimulated/expanded using antiCD3/CD28 before the addition of CD4 + T‐cells (at a 1:2 ratio, respectively, from the same donor). Proliferation of (R) CD4 + /CFSE + and (S) CD8 + /CFSE + subpopulations was assessed by flow cytometry 4 days post co‐culturing. n = 6 T1D independent donors. Each donor is colour coded. Data are presented as means ± SEM. Paired Student t ‐test * p < 0.05 and ** p < 0.01 as compared with untreated cells.

Article Snippet: CD194 , Miltenyi Biotec , Cat#130‐117‐376.

Techniques: Purification, Flow Cytometry, Isolation

Journal: Haematologica

Article Title: Histone deacetylase inhibitors downregulate CCR4 expression and decrease mogamulizumab efficacy in CCR4-positive mature T-cell lymphomas

doi: 10.3324/haematol.2017.177279

Figure Lengend Snippet: The pan-histone deacetylase inhibitor, vorinostat reduces CCR4 expression in various T-cell and natural killer-cell lymphoma cell lines. Bars indicate the mean ± standard error of the mean (SEM) of three independent experiments. Asterisks (*) indicate statistical significance: *0.01 ≤ P < 0.05, **0.001 ≤ P < 0.01, *** P < 0.001, n.s: not siginificant. (A) Quantitative RT-PCR analysis of CCR4 in vorinostat-treated T- and NK-cell lymphoma cell lines (5 μM for 24 h). The Student t -test was used to examine statistical significance. x-axis: cell lines. y-axis: 2 −ΔCt values for microRNA expression. (B) Flow cytometry analysis of CCR4 in indicated T- and NK-cell lymphoma cells treated with vorinostat. Cells were stained with CCR4-FITC 24 h after vorinostat treatment (5 μM). Representative flow cytometry histograms are shown. ΔMFI (mean fluorescence intensity) values were obtained after subtraction of the isotype control, MFI from CCR4 MFI. SAHA: suberoylanilide hydroxamic acid. (C) ΔMFI of CCR4 in vorinostat-treated T- and NK-cell lymphoma cell lines (5 μM for 24 h). x-axis: cell lines. y-axis: ΔMFI of CCR4 in dimethylsulfoxide (DMSO) or vorinostat-treated T- and NK-cell lymphoma cell lines. (D) Migration assay of lymphoma cells treated with 5 μM vorinostat for 24 h. A schematic illustration of the migration assay is also shown. RFU: relative fluorescence units.

Article Snippet: For flow cytometric analysis, cells were stained at 4°C with fluorescein isothiocyanate-conjugated anti-human CCR4 (R&D Systems, Minneapolis, USA).

Techniques: Histone Deacetylase Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Fluorescence, Control, Migration

Journal: Haematologica

Article Title: Histone deacetylase inhibitors downregulate CCR4 expression and decrease mogamulizumab efficacy in CCR4-positive mature T-cell lymphomas

doi: 10.3324/haematol.2017.177279

Figure Lengend Snippet: Effect of isoform-specific histone deacetylase inhibitors and histone deacetylase knockdown on CCR4 expression. Bars indicate the mean ± standard error of the mean (SEM) of three independent experiments. Asterisks (*) indicate statistical significance: *0.01 ≤ P < 0.05, *** P < 0.001. (A) Flow cytometry analysis of CCR4 in CTCL cells (My-La) treated with various HDAC inhibitors. Cells were stained with CCR4-FITC 24 h after treatment (5 μM vorinostat, 10 nM romidepsin, 50 μM CI994, 20 μM RGFP966, 10 μM ricolinostat, and 75 μM PCI-34051). Left panel: representative flow cytometry histograms are shown. Right panel: ΔMFI of CCR4 in My-La cells treated with HDAC inhibitors. (B) Transient knockdown of HDAC (HDAC1, HDAC2, and HDAC3) in CTCL (My-La and HH) cells. Western blot analysis of HDAC1, HDAC2, and HDAC3 in cells after transient transduction of siHDAC or a scrambled control (scr). Tubulin: protein positive control. (C) Quantitative RT-PCR analysis of CCR4 after transient transfection of siHDAC and a scr in CTCL cells. x-axis: cell lines; y-axis: expression relative to control cells that were assigned a value of 1.0. (D) ΔMFI of CCR4 after transient transfection of siHDAC and scr in CTCL (My-La and HH) and ATLL (MT-1 and MT- 4) cells. x-axis: cell lines; y-axis: ΔMFI of CCR4. MFI: mean fluorescence intensity.

Article Snippet: For flow cytometric analysis, cells were stained at 4°C with fluorescein isothiocyanate-conjugated anti-human CCR4 (R&D Systems, Minneapolis, USA).

Techniques: Histone Deacetylase Assay, Knockdown, Expressing, Flow Cytometry, Staining, Western Blot, Transduction, Control, Positive Control, Quantitative RT-PCR, Transfection, Fluorescence

Journal: Haematologica

Article Title: Histone deacetylase inhibitors downregulate CCR4 expression and decrease mogamulizumab efficacy in CCR4-positive mature T-cell lymphomas

doi: 10.3324/haematol.2017.177279

Figure Lengend Snippet: CCR4 expression of clinical lymphoma samples before and after vorinostat treatment. Bars indicate the mean ± standard error of the mean (SEM) of three independent experiments. Asterisks (*) indicate statistical significance: *0.01 ≤ P < 0.05, **0.001 ≤ P < 0.01, *** P < 0.001, n.s: not siginificant. (A) CCR4 expression determined by immunohistochemical staining before and after vorinostat treatment. Representative results from samples (skin and lymph nodes) from patient with mycosis fungoides (MF) are shown. Specimens were stained with hematoxylin and eosin (HE), CD4, and CCR4 for a pathological diagnosis and confirmation: 200× magnification. LN: lymph node. (B) Clinical time course in patient (Pt) #1 with MF before and after vorinostat treatment. (C) CCR4-positive lymphocytes (% of total infiltrating lymphocytes) in specimens from six patients (Pt #1 – 6) before and after vorinostat treatment. The Student t -test was used to examine statistical significance. (D) Quantitative RT-PCR analysis of CCR4 in samples of primary T-cell lymphomas (ATLL, CTCL, and PTCL-NOS) (Pt #7 – 9) exposed to 5 μM vorinostat for 24 h. (E) ΔMFI of CCR4 in a sample of primary ATLL (Pt #7) treated with 5 μM vorinostat for 24 h. (F) ADCC assay against a sample of primary ATLL. Cells were treated with 5 μM vorinostat for 24 h or control cells treated with dimethylsulfoxide (DMSO) before mogamulizumab treatment. Cytotoxicity was measured using the lactate dehydrogenase assay in peripheral blood mononuclear cells obtained from healthy volunteers and mogamulizumab (10 mg/mL) or the same volume of solvent (control). The ratio of target: effector cells was fixed at 1:50.

Article Snippet: For flow cytometric analysis, cells were stained at 4°C with fluorescein isothiocyanate-conjugated anti-human CCR4 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Biomarker Discovery, Quantitative RT-PCR, ADCC Assay, Control, Lactate Dehydrogenase Assay, Solvent

Journal: Veterinary immunology and immunopathology

Article Title: Porcine Treg depletion with a novel diphtheria toxin-based anti-human CCR4 immunotoxin

doi: 10.1016/j.vetimm.2016.10.014

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: The monovalent, bivalent and single-chain foldback diabody anti-human CCR4 immunotoxins as well as DT390 alone were produced in our lab using unique diphtheria toxin resistant yeast Pichia Pastoris expression system ( Wang et al., 2015 ) and biotinylated as previously described ( Wang et al., 2015 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Name Clone# Source Cat# FITC- mouse anti-pig CD1 76-7-4 In house FITC-mouse anti-pig CD3 898H-2-6-15 In house FITC-mouse anti-pig CD4a 74-12-4 In house FITC-mouse anti-pig CD45RA Fg2F9 In house Biotin-mouse anti-pig CD45RA Fg2F9 In house FITC-mouse anti-pig CD16 G7 In house PerCp-Cy5.5-mouse anti-pig CD4a 74-12-4 BD Bioscience 561474 PE-mouse anti-pig CD8α 76-2-11 BD Bioscience 559584 Alexa Fluor® 647-mouse anti pig CD8α 76-2-11 BD Bioscience 561475 PE-mouse anti-human CD21 B-ly4 BD Bioscience 555422 PE-rat anti-pig γδ T lymphocytes MAC320 BD Bioscience 561486 PE-mouse anti-pig CD172a 74-22-15A BD Bioscience 561499 PE-mouse IgG 2b κ MPC-11 BD Bioscience 559529 Fluorescein-mouse anti-human/rat CCR4 205410 R&D Systems FAB1567F PE-mouse anti-human/rat CCR4 205410 R&D Systems FAB1567P Mouse IgG2B fluorescein isotype control 133303 R&D Systems IC0041F PE-mouse anti-human CD194 (CCR4) L291H4 BioLegend 359412 PE-mouse IgG1, κ isotype control MOPC-21 BioLegend 400139 PE-streptavidin BioLegend 405204 APC-streptavidin Biolegend 405207 FITC-rat anti-mouse Foxp3 FJK-16s eBioscience 11-5773-82 FITC-rat IgG2a, κ isotype control eBR2a eBioscience 11-4321-42 Propidium Iodide Sigma 81845 7-Aminoactinomycin (7-AAD) Sigma A9400 Open in a separate window Antibodies used in this study

Techniques: Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Interactions between LAMP3+ dendritic cells and T-cell subpopulations promote immune evasion in papillary thyroid carcinoma

doi: 10.1136/jitc-2024-008983

Figure Lengend Snippet: Interaction between LAMP3 + DCs and T-cell subpopulations contribute to the formation of an immunosuppressive microenvironment in progressive PTC. (A) Summary of selected ligand-receptor interactions between the LAMP3 + DCs, CD8 + T cells, and Tregs. Circle size indicates the p value (permutation test). The color gradient represents the interaction strength. Heatmaps (B) and circle (C) plots show the comparison of interaction quantity and interaction strength between LAMP3 + DCs cells, CD8 + T cells, and Tregs between non-progressive PTC and progressive PTC. Red indicated that the increase of communication in the latter. (D) Comparison of the differences in specific cell communication intensity between non-progressive PTC and progressive PTC. (E) Representative immunofluorescence images illustrating the interaction between LAMP3 + DCs and CD8 + T cells or LAMP3 + DCs and Treg cells in one progressive PTC sample (B9). The small panels show the magnification of the selected region highlighted in red. Scale bars correspond to 50 µm in the large panel. CCL17, chemokine (C-C motif) ligand 17;CCR4, chemokine (C-C motif) Receptor 4; cDC. conventional DC; DC, dendritic cell; LAMP3, lysosomal associated membrane protein 3; PTC, papillary thyroid cancer; Treg, regulatory T cell.

Article Snippet: The following antibodies were used in the current study: primary antibodies against DC_LAMP/CD208 (1:400, CST, Cat#47778), CD8A (1:100 dilution; Abcam, Cat#ab217344), TIGIT (1:1,000, Cell Signaling Technology, Cat#99567T), NECTIN2 (1:400, Cell Signaling Technology, Cat#95333T), NECTIN3 (1:500, R&D, Cat#AF3064-SP), CCL17 (1:1,000, Abcam, Cat#ab195044), KRT19 (1:2,000, Abcam, Cat#ab76539), FOXP3 (1:2,000, Abcam, Cat#ab215206), or CCR4 (1:2,000, Novus Biologicals, Cat#NBP1-86584).

Techniques: Comparison, Immunofluorescence, Membrane