Journal: Nature Communications

Article Title: Topical tacrolimus for the treatment of secondary lymphedema

doi: 10.1038/ncomms14345

Figure Lengend Snippet: ( a ) Representative photographs of mouse tails after surgical excision of superficial/deep collecting lymphatics and treatment with or without topical tacrolimus beginning either 2 weeks (early treatment) or 6 weeks (late treatment) after surgery. Images represent 4 weeks of treatment in the early group and 3 weeks of treatment in the late group. ( b ) Graphical representation of tail volume changes after early (* P =0.021) or late (* P =0.018) treatment with tacrolimus, as compared with controls (red arrows indicate timing of treatment) ( n =8/group). ( c ) Representative cross-sectional histological images (upper panel) of control and early tacrolimus-treated mouse tails harvested 6 weeks after lymphatic ablation. Brackets show soft tissue thickness. Quantification of soft tissue changes (lower panel) after early or late treatment with tacrolimus (* P ≤0.001) ( n =8/group). ( d ) Quantification of whole-blood tacrolimus levels demonstrating immunosuppressive concentrations in systemically treated animals (4 mg kg −1 intraperitoneally daily) and non-immunosuppressive levels in the topically treated group (* P =0.007; n =6/group). ( e ) Representative flow cytometry plots displaying side scatter area (SSA) on the y axis and CD3 + cells (T cells) on the x axis for animals retreated with vehicle control, topical tacrolimus or systemic tacrolimus. ( f ) Quantification of blood T cells for each group. Note significant reduction in T cells only in systemically treated animals (* P =0.012; n =6/group). Experiments were repeated two to three times. All data represent mean±s.d. with P ≤0.05 considered as significant. Two-tailed Student's t -test ( c , d ) and analysis of variance (ANOVA) with post hoc tests ( b , f ).

Article Snippet: Rabbit anti-human/mouse CD3 (A0452; 1:300; Dako, Carpinteria, CA) and Cy3-conjugated mouse anti-αSMA (C6-198; 1:1,000; Sigma-Aldrich).

Techniques: Flow Cytometry, Two Tailed Test

Journal: PLoS ONE

Article Title: 3D investigation shows walls and wall-like structures around human germinal centres, probably regulating T- and B-cell entry and exit

doi: 10.1371/journal.pone.0242177

Figure Lengend Snippet: (A) Alpha-actin + (red) and CD3 + cells (green) relate to each other. The number of T-cells decreases abruptly towards the B-zone where the FRC wall is located. (B) Alpha-actin positive (green) and BCL6 positive (red) cells show proportion of walls towards the germinal centre. BCL6 positive germinal centre cells are situated within the physical boundary. Additionally, the BCL6 and alpha-actin negative surrounding between germinal centre and FRC wall indicates that the follicular mantle is within the compartmentalised area of the walls. (C) Alpha-actin-positive (red) and IgD positive (green) structures reveal orientation of FRC walls. The follicular mantle shows a polar orientation without or with only fragmented FRC walls towards the mantle zone. On the opposite pole, the germinal centre is compartmentalised by smaller FRC walls and is more permeable. The remaining germinal centre is enclosed from both sides by the FRC walls in form of a sufficient physical boundary. This staining combination shows properly that the orientation of the FRC walls shows a coherence with the follicular mantle. The maximum resolution of (A), (B) and (C) was set to 0.13 μm per pixel in the native microscopy dataset.

Article Snippet: We used anti-alpha smooth muscle actin antibody (SP171/Abcam), anti-human BCL6 monoclonal mouse antibody (M7211;Agilent/Dako), anti-human polyclonal rabbit IgD antibody (IR517;Agilent/Dako), anti-human CD3 monoclonal mouse antibody (A0452; Agilent/Dako) and anti-human CD23 monoclonal mouse antibody (M7312; Agilent/Dako).

Techniques: Staining, Microscopy

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Maintenance of WT1 expression in tumor cells is associated with a good prognosis in malignant glioma patients treated with WT1 peptide vaccine immunotherapy

doi: 10.1007/s00262-021-02954-z

Figure Lengend Snippet: Summary of IHC staining results of both pre- and post- WT1 vaccination specimens

Article Snippet: The sections were then incubated overnight at 4 °C with anti-human WT1 mouse monoclonal antibody 6F-H2 (DAKO, Carpinteria, CA, USA; diluted 1:50), anti-human Transforming growth factor-β (TGF-β) rabbit polyclonal antibody (Abcam, Cambridge, MA, USA; diluted 1:200), anti–human Ki-67 mouse monoclonal antibody MIB-1 (DAKO; diluted 1:40), anti-human HLA class I-ABC mouse monoclonal antibody EMR8-5 (Hokudo, Sapporo, Japan; diluted 1:100), anti-human programmed death-ligand 1 (PD-L1) rabbit monoclonal antibody ab205921 clone 28–8, (Abcam; diluted 1:100), anti-human CD3 mouse monoclonal antibody F7.2.38 (DAKO; diluted 1:5), anti–human CD4 mouse monoclonal antibody 4B12 (DAKO; diluted 1:80), anti–human CD8 mouse monoclonal antibody C8/14B (Nichirei Biosciences, Tokyo, Japan; undiluted), anti-human CD79α mouse monoclonal antibody JCB117 (DAKO; diluted 1:200), anti-human PD-1 mouse monoclonal antibody clone NAT105 (Abcam; diluted 1:100), anti-human Foxp3 mouse monoclonal antibody 236A/E7 (Abcam; diluted 1:100), or anti IDH1­R132H mouse monoclonal Antibody (FUJIFILM Wako Chemicals, Osaka, Japan; diluted 1:100).

Techniques: Immunohistochemistry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The role of Ly49E receptor expression on murine intraepithelial lymphocytes in intestinal cancer development and progression

doi: 10.1007/s00262-016-1894-6

Figure Lengend Snippet: Tumor-infiltrating T cells and tumor uPA expression in ApcMin/+ Ly49E WT versus ApcMin/+ Ly49E KO mice. a Hematoxylin/eosin- (upper), and CD3-(lower) stained paraffin tumor sections from ApcMin/+ Ly49E WT and ApcMin/+ Ly49E KO mice. Scale bar: 250 µm, ×100 magnification. A graph showing CD3 mean gray value/mm2 according to tumor surface area (mm2) is shown for ApcMin/+ Ly49E WT and ApcMin/+ Ly49E KO mice (n = 6). b Small intestinal tumor-infiltrating IEL subpopulation frequencies in ApcMin/+ Ly49E WT and ApcMin/+ Ly49E KO mice, and small intestinal IEL subpopulation frequencies from control littermates, at 14 weeks of age (mean ± SEM; n = 6). The percentage of TCRαβ and TCRγδ IEL is shown as a fraction of the total numbers of T cells. TCRαβ CD4, TCRαβ CD8αβ and TCRαβ CD8αα IEL subpopulation frequencies are shown as a percentage of the total TCRαβ IELs. TCRγδ DN and TCRγδ CD8αα IEL subpopulation frequencies are shown as a percentage of total TCRγδ IEL. c Tumor uPA expression in tumors from ApcMin/+ Ly49E WT and ApcMin/+ Ly49E KO mice at 14 weeks of age. n.s. not significant

Article Snippet: Antibodies used for immunofluorescent staining were polyclonal rabbit anti-human/mouse CD3 (Dako) and Dylight649 donkey anti-rabbit (BioLegend).

Techniques: Expressing, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The role of Ly49E receptor expression on murine intraepithelial lymphocytes in intestinal cancer development and progression

doi: 10.1007/s00262-016-1894-6

Figure Lengend Snippet: Tumor-infiltrating T cells and tumor uPA expression in AOM-treated Ly49E WT versus Ly49E KO mice. a Hematoxylin/eosin-(H&E)- (upper), and CD3-(lower) stained paraffin tumor sections from AOM-treated Ly49E WT and Ly49E KO mice. Scale bar 250 µm, ×100 magnification. A graph showing CD3 mean gray value/mm2 according to tumor surface area (mm2) is shown for AOM-treated Ly49E WT and Ly49E KO mice (n = 6). b Colon tumor-infiltrating IEL subpopulation frequencies in AOM-treated Ly49E WT and Ly49E KO mice 14–22 weeks following the start of treatment, and colon IEL subpopulation frequencies from untreated Ly49E WT and Ly49E KO mice (mean ± SEM; n = 5 for AOM-treated mice; n = 3 for untreated mice). The percentage of TCRαβ and TCRγδ IEL is shown as a fraction of the total numbers of T cells. TCRαβ CD4, TCRαβ CD8αβ and TCRαβ CD8αα IEL subpopulation frequencies are shown as a percentage of the total TCRαβ IELs. TCRγδ DN and TCRγδ CD8αα IEL subpopulation frequencies are shown as a percentage of the total TCRγδ IEL. c Dot plots are shown for CD4/CD8β versus CD8α expression in colon tumor-infiltrating IEL in AOM-treated Ly49E WT and Ly49E KO mice, and colon IEL from untreated Ly49E WT and Ly49E KO mice. Numbers indicate the percentage of cells in each quadrant. Dot plots are representative for n = 5 AOM-treated mice and n = 3 untreated mice. d Tumor uPA expression in tumors of varying size from AOM-treated Ly49E WT and Ly49E KO mice at 14–22 weeks following the start of treatment. n.s. not significant. Data were analyzed using the nonparametric two-tailed Mann–Whitney U-test or the Kruskall–Wallis test. A p value ≤0.05, (*), a p value ≤0.01 (**) and p value ≤0.001 (***), were considered statistically significant

Article Snippet: Antibodies used for immunofluorescent staining were polyclonal rabbit anti-human/mouse CD3 (Dako) and Dylight649 donkey anti-rabbit (BioLegend).

Techniques: Expressing, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Preventive effect of tertiary lymphoid structures on lymph node metastasis of lung adenocarcinoma

doi: 10.1007/s00262-022-03353-8

Figure Lengend Snippet: Characterization of tertiary lymphoid structures and tumor-infiltrating lymphocytes. Characterization of tertiary lymphoid structures (TLSs) in lung adenocarcinoma by staining of formalin-fixed, paraffin-embedded tissues (A–D). TLS in lung adenocarcinoma stained by hematoxylin and eosin (A), CD20 (B), CD3 (C) and DC-Lamp (D). Tumor-infiltrating lymphocytes stained by CD3 (E), CD8 (F), CD4 (G) and Foxp3 (H). Histogram of the number of TLSs within low-power field (LPF) (I). Histogram of the number of DC-Lamp+ mature dendritic cells in TLSs (/0.04 mm2) (J)

Article Snippet: The primary antibodies were: mouse monoclonal anti-human CD20cy antibody (clone #L26, dilution 1:200; Dako); rat monoclonal anti-mouse DC-Lamp antibody (clone #1010E1.01, dilution 1:100; eurobio SCIENTIFIC); mouse monoclonal anti-human CD3 antibody (clone #F7.2.38, dilution 1:50; DAKO); mouse monoclonal anti-human CD8 antibody (clone #C8/144B, dilution 1:100; Dako); rabbit monoclonal anti-human CD4 antibody (clone #sp35, dilution 1:100; abcam); mouse monoclonal anti-human Foxp3 antibody (clone #236A/E7, dilution 1:100; eBioscience); rabbit polyclonal anti-granzyme B antibody (dilution 1:200; abcam); mouse monoclonal anti-human NKp46/NCR1 antibody (clone #195,314, dilution 1:200; R&D systems); rabbit polyclonal anti-MIP-3 beta/CCL19 antibody (dilution 1:500; abcam); rabbit polyclonal anti-CCL21 antibody (dilution 1:200; abcam); and rabbit polyclonal anti-CCR7 antibody (dilution 1:200; abcam).

Techniques: Staining, Formalin-fixed Paraffin-Embedded

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Preventive effect of tertiary lymphoid structures on lymph node metastasis of lung adenocarcinoma

doi: 10.1007/s00262-022-03353-8

Figure Lengend Snippet: Clinicopathological characteristics of the patients according to mature/immature TLS

Article Snippet: The primary antibodies were: mouse monoclonal anti-human CD20cy antibody (clone #L26, dilution 1:200; Dako); rat monoclonal anti-mouse DC-Lamp antibody (clone #1010E1.01, dilution 1:100; eurobio SCIENTIFIC); mouse monoclonal anti-human CD3 antibody (clone #F7.2.38, dilution 1:50; DAKO); mouse monoclonal anti-human CD8 antibody (clone #C8/144B, dilution 1:100; Dako); rabbit monoclonal anti-human CD4 antibody (clone #sp35, dilution 1:100; abcam); mouse monoclonal anti-human Foxp3 antibody (clone #236A/E7, dilution 1:100; eBioscience); rabbit polyclonal anti-granzyme B antibody (dilution 1:200; abcam); mouse monoclonal anti-human NKp46/NCR1 antibody (clone #195,314, dilution 1:200; R&D systems); rabbit polyclonal anti-MIP-3 beta/CCL19 antibody (dilution 1:500; abcam); rabbit polyclonal anti-CCL21 antibody (dilution 1:200; abcam); and rabbit polyclonal anti-CCR7 antibody (dilution 1:200; abcam).

Techniques: Mutagenesis, Expressing

Journal: bioRxiv

Article Title: A mosaic renal myeloid subtype with T-cell inhibitory and protumoral features is linked to immune escape and survival in clear cell renal cell cancer

doi: 10.1101/2020.01.20.912865

Figure Lengend Snippet: (A) Clustered heatmaps depicting relative gene expression levels of human macrophage and DC core genes comparing ercDC, in vitro- generated M1- and M2-macrophages, and CD1c+ DC from blood. Genes whose expression was validated on protein level (B, C) are set apart. (B), (C) Protein expression by flow cytometry. In vitro -generated M1- and M2-macrophages, PBMCs and ccRCC tissue cell suspensions were stained with marker combinations and gated on ercDCs (CD209+CD14+ cells among CD45+ live single CD3-CD11c+ cells of ccRCC cell suspension), CD1c+DCs (CD1c+cells among live single CD3-CD11c+ of PBMCs) and on M1- and M2-macrophage population among live single cells. Depicted are representative histograms of human macrophage markers CD64A, CD14, MerTK, CD32A (B) as well as DC markers ANPEP/CD13 and FLT3 (C) from at least 6 different patient tissues or PBMCs. Black line histogram: specific staining, gray filled histogram: control staining. Numbers indicate the control-corrected median fluorescence intensity.

Article Snippet: Cryosections were fixed with 4% paraformaldehyde (PFA, Merck) and stained with primary and fluorescent-labeled secondary antibodies as described ( ). human CD209/DC-SIGN (mouse IgG2a, SantaCruz Biotechnology) followed by secondary antibodies donkey anti-goat-A488 (ThermoScientific) and rat anti-mouse-IgG-Cy5 or donkey anti-mouse-IgG-A647 (both Jackson ImmuneResearch); goat-anti-human VSIG4, rabbit-anti-human FOXO1 (Cell Signaling) and mouse-anti-human CD3 (UCHT1, BD) followed by secondary antibodies donkey anti-goat-A488, donkey anti-rabbit-RRX (Jackson ImmuneResearch) and donkey anti-mouse-A647 or rat anti-mouse IgG-Cy5.

Techniques: Expressing, In Vitro, Generated, Flow Cytometry, Staining, Marker, Fluorescence

Journal: bioRxiv

Article Title: A mosaic renal myeloid subtype with T-cell inhibitory and protumoral features is linked to immune escape and survival in clear cell renal cell cancer

doi: 10.1101/2020.01.20.912865

Figure Lengend Snippet: (A) Gene expression of macrophage-associated transcription factors and growth factor receptor CSF1R. Bars: median of each group, symbols correspond to individual array replicates of a cell type. Histogram depicts surface expression of CSF1R on ercDCs of ccRCC tissue cell suspensions by flow cytometry (B) Linear expression of genes for DC-associated transcription factors and growth factor receptor FLT3. (C) Clustered heatmaps depicts relative expression levels of genes associated with M1- and M2-macrophage polarization. Surface protein levels of indicated markers validated by flow cytometry. See table S3 and S4 for M1- and M2-genes. (D) Dot plot of polychromatic flow cytometry demonstrates co-expression of M1-(CD64A) and M2-macrophage markers (CD204/MSR1 and MerTK) on ercDCs in ccRCC tissue cell suspensions (n=4). Overlay of MerTK+ cells on gated CD64A+CD204+ double-positive CD209+CD14+ ercDCs (upper right quadrant) depicted in pink. (E) Linear expression of chemokines associated with host defense activity of macrophages. M1-macrophages are the positive reference. Bars: median of each group; symbols correspond to individual array replicates of a cell type. (F), (G) Clustered heatmap of relative expression levels of genes associated with wound healing and tissue remodeling (F) and immunoregulation (G). References are M2-macrophages and CD11cLO macrophages from decidua (CD11cLO MΦ_decidua). (H) Clustered heatmap depicting relative expression levels of genes associated with angiogenesis and invasion. Positive reference: in vitro -generated M2-macrophages; negative control: CD1c+ DCs from blood. Surface expression of indicated proteins on ercDCs by flow cytometry of ccRCC tissue-cell suspensions and gating on ercDCs (CD209+CD14+ cells among CD45+ live single CD3-CD11c+ cells). Histograms or dot plots are representative of at least n= 6) different tissue suspensions. Black line histogram: specific stainings, filled grey histogram: control stainings. Numbers indicate the control-corrected median fluorescence intensity.

Article Snippet: Cryosections were fixed with 4% paraformaldehyde (PFA, Merck) and stained with primary and fluorescent-labeled secondary antibodies as described ( ). human CD209/DC-SIGN (mouse IgG2a, SantaCruz Biotechnology) followed by secondary antibodies donkey anti-goat-A488 (ThermoScientific) and rat anti-mouse-IgG-Cy5 or donkey anti-mouse-IgG-A647 (both Jackson ImmuneResearch); goat-anti-human VSIG4, rabbit-anti-human FOXO1 (Cell Signaling) and mouse-anti-human CD3 (UCHT1, BD) followed by secondary antibodies donkey anti-goat-A488, donkey anti-rabbit-RRX (Jackson ImmuneResearch) and donkey anti-mouse-A647 or rat anti-mouse IgG-Cy5.

Techniques: Expressing, Flow Cytometry, Activity Assay, In Vitro, Generated, Negative Control, Fluorescence

Journal: bioRxiv

Article Title: A mosaic renal myeloid subtype with T-cell inhibitory and protumoral features is linked to immune escape and survival in clear cell renal cell cancer

doi: 10.1101/2020.01.20.912865

Figure Lengend Snippet: (A) Linear expression of VSIG4 . Bars are the median of each group; symbols correspond to individual array replicates of a cell type. CD1c+DC_blood is the negative reference, M2-MΦ_in vitro the positive reference. (B) Validation of VSIG4 protein on ercDCs in ccRCC tissue cell suspension by flow cytometry. ercDCs are gated as CD209+CD14+ cells among CD45+ live single CD3-CD11c+ cells in ccRCC cell suspension, CD1c+DCs are gated within PBMCs as CD1c+ cells among live single CD3-CD11c+ cells. Control staining: grey filled histogram. Numbers depict the difference in median fluorescence intensity (MFI) between specific antibody and control. Shown is a representative example of n = 4 different ccRCC tissue cell suspensions. (C) Confocal images of ccRCC tissues stained with CD209 (red), VSIG4 (green) and DAPI (blue). Original magnification, x400. Cells coexpressing CD209 and VSIG4 are yellow. Images are merged fluorescent channels and maximal projection of 6-9 z-planes (z-step size = 0.7 µm). Left image: late stage ccRCC (RCC114, T3cG2), right image: early stage ccRCC (RCC90, T1aG2), (D). Confocal image of an ccRCC tissue area (RCC118) depicting a T cell (CD3+, cyan) expressing FOXO1 (red) in contact with VSIG4+ cell (original magnification, x400, zoom 5). Histograms document fluorescence intensities of a single z-plane along indicated line. (E) Scatter plot showing the ratio of the % FOXO+ T cells among T cells that are in contact with VSIG4+ cells to the % of FOXO+ T cells among T cells that are not in contact with VSIG4+ cells. Values above >1 indicate that the majority of CD3+FOXO+ cells is in contact with VSIG4+ cells. **** = p < 0.001, Mann Whitney test comparing across all 4 ccRCC tissues.

Article Snippet: Cryosections were fixed with 4% paraformaldehyde (PFA, Merck) and stained with primary and fluorescent-labeled secondary antibodies as described ( ). human CD209/DC-SIGN (mouse IgG2a, SantaCruz Biotechnology) followed by secondary antibodies donkey anti-goat-A488 (ThermoScientific) and rat anti-mouse-IgG-Cy5 or donkey anti-mouse-IgG-A647 (both Jackson ImmuneResearch); goat-anti-human VSIG4, rabbit-anti-human FOXO1 (Cell Signaling) and mouse-anti-human CD3 (UCHT1, BD) followed by secondary antibodies donkey anti-goat-A488, donkey anti-rabbit-RRX (Jackson ImmuneResearch) and donkey anti-mouse-A647 or rat anti-mouse IgG-Cy5.

Techniques: Expressing, In Vitro, Flow Cytometry, Staining, Fluorescence, MANN-WHITNEY