Journal: Frontiers in Immunology

Article Title: The Reversal of Immune Exclusion Mediated by Tadalafil and an Anti-tumor Vaccine Also Induces PDL1 Upregulation in Recurrent Head and Neck Squamous Cell Carcinoma: Interim Analysis of a Phase I Clinical Trial

doi: 10.3389/fimmu.2019.01206

Figure Lengend Snippet: Tumors from patients treated with Tadalafil and MUC1/polyICLC vaccine show a lower infiltration of MDSCs and Treg and a higher infiltration of activated CD8 in the tumor bed. Computer based image cytometry was performed to enumerate the number of (A) MDSC, (B) IL4Rα − myeloid cells, (C) CD4 + T cell subsets, or (D) CD8 + T cells. (E) CD69 expression within the CD8 is reported normalized on the CD69 expression on all the cells evaluated. Depending on the region of interest evaluated, at least 10 5 -10 6 cells were analyzed. (F) The expression of CD69 in CD8 + T cells was plotted against MUC1 IHC score of the corresponding tumor. Two ways T -test and relevant pearson correlation parameters are reported.

Article Snippet: Rabbit polyclonal anti-human CD8 antibody (dilution 1/30, Abcam) and goat polyclonal anti-human CD69 antibody (dilution 1/25, R&D System).

Techniques: Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: The Reversal of Immune Exclusion Mediated by Tadalafil and an Anti-tumor Vaccine Also Induces PDL1 Upregulation in Recurrent Head and Neck Squamous Cell Carcinoma: Interim Analysis of a Phase I Clinical Trial

doi: 10.3389/fimmu.2019.01206

Figure Lengend Snippet: Tadalafil and MUC1/polyICLC vaccine treatments modulate the expression of PDL1 in the tumor microenvironment. The expression of PDL1 within the CD163+ (A) or the CD163 − cells (B) was quantified by image cytometry in the tumor, at the tumor edge, or in “normal” surrounding tissue in the tumor specimen from the control (black filled circle) or Tadalafil and MUC1/polyICLC vaccine treated (gray filled circle) patients. Two way T -test p -value are reported. (C) Correlation between the expression of CD69 in the CD8 + T cells and PDL1 expression on the CD163 − cells. (D) Summary of the one way RM ANOVA analysis.

Article Snippet: Rabbit polyclonal anti-human CD8 antibody (dilution 1/30, Abcam) and goat polyclonal anti-human CD69 antibody (dilution 1/25, R&D System).

Techniques: Expressing, Cytometry, Control

Journal: Frontiers in Immunology

Article Title: The Reversal of Immune Exclusion Mediated by Tadalafil and an Anti-tumor Vaccine Also Induces PDL1 Upregulation in Recurrent Head and Neck Squamous Cell Carcinoma: Interim Analysis of a Phase I Clinical Trial

doi: 10.3389/fimmu.2019.01206

Figure Lengend Snippet: PDL1 expression limits the beneficial prognostic value of CD8a and CD69 in the tumor. KM plotter analysis (kmplot.com) was performed on tumor RNAseq data from patients with HNSCC ( n = 499, all tumor stages) using the mean expression of the indicated genes with weight =1 and auto select best cutoff selected.

Article Snippet: Rabbit polyclonal anti-human CD8 antibody (dilution 1/30, Abcam) and goat polyclonal anti-human CD69 antibody (dilution 1/25, R&D System).

Techniques: Expressing

Journal: Mediators of Inflammation

Article Title: Dietary Fiber-Derived Microbial Butyrate Suppresses ILC2-Dependent Airway Inflammation in COPD

doi: 10.1155/2024/6263447

Figure Lengend Snippet: SCFAs alleviate inflammatory cell activities in gut ILC2 cells. (a) t-SNE feature plots revealing profiles of eight biomarkers. (b) Arg1, ST2, Thy1, KLRG1, IL-17RB, CCR9, and CD69 protein contents, as evidenced by Western blot analysis. (c) S1P, S1P2, S1P3, S1P4, and S1P5 transcript expressions, as evidenced by qRT-PCR. (d) Lung ILC2s mRNA expression in murine COPD model treated with butyrate. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.01 (relative to control mice).

Article Snippet: The membrane was blocked with 5% nonfat milk at room temperature for 2 hr and further incubated overnight with rabbit anti-arginase1 (anti-Arg1) (Sangon Biotech, Shanghai, China), Thy.1 monoclonal antibody (Proteintech Group, Inc, Wuhan, China), ST2 monoclonal antibody (Proteintech Group, Inc., Wuhan, China), rabbit antikiller cell lectin-like receptor G1 (KLRG1) (Abcam, Cambridge, MA, USA), rabbit anti-IL17RB antibody (bioss, Bejing, China), anti-CCR9 antibody (BosterBio, USA), rabbit anti-CD69 polyclonal antibody (absin, Shanghai, China), NFIL3 polyclonal antibody (Proteintech Group, Inc., Wuhan, China), rabbit anti-GATA3 polyclonal antibody (absin, Shanghai, China), histone-H3 polyclonal antibody (Proteintech Group, Inc., Wuhan, China), rabbit anti-acetyl-histone H3 monoclonal antibody (absin, Shanghai, China), or β -actin polyclonal antibody (Proteintech Group, Inc., Wuhan, China) at 4°C.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Control

Journal: Nature immunology

Article Title: Lymph Node Conduits Transport Virions for Rapid T Cell Activation

doi: 10.1038/s41590-019-0342-0

Figure Lengend Snippet: a ) Blended projections of T cells (red) clustered around a conduit (white)-associated VACV-infected cell (green) near a high endothelial venule (HEV, indicated in picture). Arrows point to conduits. Scalebar = 20 μm. Area in circle is magnified on the right. Scalebar = 5 μm. ERTR7= white, VACV-infected cells = green, OT-I CD8 + T cells = red, B220 = blue. Results are representative of 30 LN sections taken from 5 experiments. b ) OT-I CD8 + T cell activation as determined by flow cytometry of single-cell suspensions of popliteal LNs harvested 8 h p.i. 10 6 OT-I cells were transferred 12–24 h prior to infection. NP-eGFP (no SIINFEKL) was given at 10 7 pfu; all other infections used NP-S-eGFP (containing SIINFEKL) at the indicated dose. Activation was determined by CD69 expression. MFI is shown on the right. Dots = individual LNs. n = 6. Results were repeated 3 times with 3–6 mice/group. Bars = mean. Error bars = SEM. c ) OT-I CD8 + T cell activation in (b) but 24 h p.i. Dots = individual LNs. n = 6. Results were repeated 3 times with 3–6 mice/group. Bars = mean. Error bars = SEM. d ) Quantitation of the percentage of VACV- or MVA-infected cells in either the SCS and IFA region or T cell zone contacted by OT-I CD8 + T cells (n = 37 VACV or 32 MVA; dots indicate individual sections). Bars = mean. Error bars = SEM. e ) Percentage of activated OT-I CD8 + T cells (those with CD69 MFI > 50) in each region of 10 LN sections harvested 8 h after infection with 10 8 pfu VACV. Percentages shown are activated cells/total cells in each region. Dots = individual LN sections. Bars = mean. Error bars = SEM. f ) Percentage of only the activated OT-I CD8 + T cells (as opposed to total cells in (e)) found in each LN region. g ) MIP LN section 8 h p.i. showing OT-I CD8 + T cell activation. B = B cell follicle, SCS = subcapsular sinus. Arrow indicates activated T cell cluster in the T cell zone, magnified on the right. Scalebars = 50 μm, left and 5 μm, right. OT-I CD8 + T cells = red, CD69 = white, VACV-infected cells = green, B220 = blue. h ) MFI of CD69 on all activated cells in each region of the popliteal LN shown in (g). n =197 T cells. Bars = mean. Error bars = SEM. i ) MIP of popliteal LN harvested 8 h post infection with 10 8 pfu VACV (green) without OT-I transfer. Endogenous, polyclonal CD8 + T cells = red. Colocalization of polyclonal CD8 + T cell signal (red) and CD69 (white) signal is shown in purple. B220 = blue, VACV-infected cells = green. The boxed area is magnified in (j). scalebars = 100 μm j ) Higher magnification MIP image of (i) showing both the SCS&IFA (labeled SCS) and T cell zone (T). Colocalization of polyclonal CD8 + T cell signal (red) and CD69 (white) signal is shown in purple. scalebars = 50 μm. f,h,i,j) 6 LNs from 3 experiments were analyzed. g) 10 LNs from 5 experiments were analyzed. Statistics = unpaired two-tailed t test.

Article Snippet: Cells were stained with the following antibodies: CD45 (clone 30-F11, eBioscience, Lot#E10032–1635); CD8α (clone 53–6.7, eBioscience, many different lots); CD69 (clone H1.2F3, eBioscience, Lot#B207773); and IFN-γ (clone XMG1.2, eBioscience, Lot#E024667).

Techniques: Infection, Activation Assay, Flow Cytometry, Expressing, Quantitation Assay, Labeling, Two Tailed Test

Journal: Nature immunology

Article Title: Lymph Node Conduits Transport Virions for Rapid T Cell Activation

doi: 10.1038/s41590-019-0342-0

Figure Lengend Snippet: a ) Maximum intensity projection (MIPs) of sections from two different popliteal LNs (left and right) harvested 8 h after footpad injection of 10 6 pfu of VACV. ERTR7 = red, Lyve-1 = white, VACV-infected cells = green, B220 = blue. Arrows indicate the location of VACV-infected paracortical cell. scalebars = 100 μm. Higher magnification images are shown on the right; lower panels lack the blue channel for clarity. Scalebars = 50 μm. Results are representative of 10 LNs from 3 experiments. b ) MIP of a section of a popliteal LN harvested 8 h after footpad injection of 10 6 pfu of VACV. Prior to infection, 10 6 OT-I CD8 + T cells were transferred (pink). ERTR7 = red, B220 = blue, CD69 = white, VACV-infected cells = green. Arrows indicate two areas of T cell activation that are shown in higher magnification insets on the left. Scalebar = 200 μm. Top panels of insets show merge; bottom panels do not show OT-I signal (pink) in order to reveal CD69 staining more clearly. Scalebars = 20 μm. Results are representative of 10 LNs from 3 experiments. c ) MIP of a section of a popliteal LN harvested 8 h after footpad infection with 10 6 pfu of VACV showing an infected paracortical CD205 + DC. Scalebar = 200 μm. Higher magnification images are shown in the insets showing: top left) merge; top right) conduits + VACV-infected cells; lower left) conduits + VACV-infected cells + CD11c + CD11b; lower right) conduits + VACV-infected cells + CD205. Scalebar = 20 μm. ERTR7 = red, Lyve-1 = white, VACV-infected cells = green, B220 = blue, CD11c = yellow, CD205 = purple, and CD11b = brown. Results are representative of 10 LNs from 3 experiments.

Article Snippet: Cells were stained with the following antibodies: CD45 (clone 30-F11, eBioscience, Lot#E10032–1635); CD8α (clone 53–6.7, eBioscience, many different lots); CD69 (clone H1.2F3, eBioscience, Lot#B207773); and IFN-γ (clone XMG1.2, eBioscience, Lot#E024667).

Techniques: Injection, Infection, Activation Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: The pro‐inflammatory signature of lipopolysaccharide in spontaneous contracting embryoid bodies differentiated from mouse embryonic stem cells

doi: 10.1111/jcmm.17805

Figure Lengend Snippet: Expression of CD68 and CD69 in embryoid bodies treated with LPS. (A) Western blot analysis of CD68 expression in embryoid bodies treated from Day 9 to Day 15 of cell culture with 0.1, 1 and 5 μg/mL LPS. Upper panel, representative western blot, lower panel, bar chart of the means ± SD of n = 3 experiments. (B) Immunohistochemical analysis of CD 68 expression in plated embryoid bodies treated with different concentrations of LPS. Upper panel, representative images of CD68 + cells (left, green), DRAQ5 nuclear staining (middle, blue) and overlay (right). Lower panel, bar chart of the means ± n = 3 experiments. (C) Western blot analysis CD69 expression in embryoid bodies treated from Day 9 to Day 15 of cell culture with 0.1, 1 and 5 μg/mL LPS. Upper panel, representative western blot, lower panel, bar chart of the means ± SD of n = 3 experiments. (D) Immunohistochemical analysis of CD 69 expression in plated embryoid bodies treated with different concentrations of LPS. Upper panel, representative images of CD69 + cells (left, green), DRAQ5 nuclear staining (middle, blue) and overlay (right). Lower panel, bar chart of the means ± n = 3 experiments. The scale bars in immunohistochemical images represent 30 μm. (E) Flow cytometry analysis of CD68 + and CD69 + cells/total cells (%) ( n = 3). Cells were enzymatically dissociated from embryoid bodies treated from Day 9 to Day 15 of cell culture with 5 μg/mL LPS. * p < 0.05, significantly different as compared to 2nd antibody, # p < 0.05, significantly different as compared to the untreated control.

Article Snippet: Blocking against unspecific binding was performed in 10% BSA for 1 h. Cells were resuspended in FACS buffer [0.1% sodium azide, 5% foetal calf serum (FCS), 0.05% in PBS], and stained with either rabbit polyclonal anti CD68 (Bioss, cat. no. Bs – 0649R) or rabbit polyclonal CD69 (Invitrogen, Thermo Fisher Scientific, cat. no. PA5‐114989) antibodies.

Techniques: Expressing, Western Blot, Cell Culture, Immunohistochemical staining, Staining, Flow Cytometry, Control