Journal: Cancer Immunology, Immunotherapy : CII

Article Title: CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

doi: 10.1007/s00262-013-1392-z

Figure Lengend Snippet: Phenotypes of CD8+ Treg (a) and CD4+CD25hi Treg (b) from a healthy subject (donor # 1). CD8+ Treg were generated in vitro from purified CD8+ T lymphocytes; CD4+CD25+ Treg were directly purified from the peripheral blood. Shown data are representative of the CD8+ and CD4+CD25hi Treg phenotypic characterization performed with cells from all the other donors. In the dotplots Treg populations are in black. The percentages of CD39+ Treg are indicated in the histograms

Article Snippet: The following mAbs were used: phycoerythrin (PE) or fluorescein isothiocynate (FITC)-conjugated anti-CD127, allophycocianin (APC)-cyanin (Cy) 7-conjugated anti-CD3 (e-Biosciences, San Diego, CA), FITC-conjugated anti-CD25 (Miltenyi, Bergisch Gladbach, Germany), PE-Cy7 or eFluor 450-conjugated anti-CD8 (Biolegend, San Diego, CA or e-Biosciences, San Diego, CA, respectively), Peridinin Chlorophyll Protein Complex-cyanin 5.5 (PerCP-Cy5.5)-conjugated anti-CD28 (eBioscience), APC-conjugated anti-CD39 (Serotec, MorphoSys US Inc), FITC-conjugated anti-CD103 (Becton–Dickinson, BD Biosciences, San Josè CA), PE-conjugated anti-NKG2A (IL, Milan, Italy), FITC-conjugated anti-HLA-G (Exbio Praha a.s., Vestec, CZ), PE-conjugated anti-LAG3 (Bioss Inc., Vobum, MA), PE-conjugated anti-CD122 (BD Biosciences, San Josè CA), FITC-conjugated anti-PD1 (e-Biosciences, San Diego, CA), PE-conjugated anti-TGFβ (BD Biosciences, San Josè CA).

Techniques: Generated, In Vitro, Purification

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

doi: 10.1007/s00262-013-1392-z

Figure Lengend Snippet: Tumor details and relative percentage expression of CD39 on intratumoral CD8+ and CD4+CD25 hi Treg

Article Snippet: The following mAbs were used: phycoerythrin (PE) or fluorescein isothiocynate (FITC)-conjugated anti-CD127, allophycocianin (APC)-cyanin (Cy) 7-conjugated anti-CD3 (e-Biosciences, San Diego, CA), FITC-conjugated anti-CD25 (Miltenyi, Bergisch Gladbach, Germany), PE-Cy7 or eFluor 450-conjugated anti-CD8 (Biolegend, San Diego, CA or e-Biosciences, San Diego, CA, respectively), Peridinin Chlorophyll Protein Complex-cyanin 5.5 (PerCP-Cy5.5)-conjugated anti-CD28 (eBioscience), APC-conjugated anti-CD39 (Serotec, MorphoSys US Inc), FITC-conjugated anti-CD103 (Becton–Dickinson, BD Biosciences, San Josè CA), PE-conjugated anti-NKG2A (IL, Milan, Italy), FITC-conjugated anti-HLA-G (Exbio Praha a.s., Vestec, CZ), PE-conjugated anti-LAG3 (Bioss Inc., Vobum, MA), PE-conjugated anti-CD122 (BD Biosciences, San Josè CA), FITC-conjugated anti-PD1 (e-Biosciences, San Diego, CA), PE-conjugated anti-TGFβ (BD Biosciences, San Josè CA).

Techniques: Expressing, In Situ

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

doi: 10.1007/s00262-013-1392-z

Figure Lengend Snippet: Suppression activity of intratumoral CD8+ T cells from different tumor patients in the presence or not of CD39 and/or IL10 inhibitors

Article Snippet: The following mAbs were used: phycoerythrin (PE) or fluorescein isothiocynate (FITC)-conjugated anti-CD127, allophycocianin (APC)-cyanin (Cy) 7-conjugated anti-CD3 (e-Biosciences, San Diego, CA), FITC-conjugated anti-CD25 (Miltenyi, Bergisch Gladbach, Germany), PE-Cy7 or eFluor 450-conjugated anti-CD8 (Biolegend, San Diego, CA or e-Biosciences, San Diego, CA, respectively), Peridinin Chlorophyll Protein Complex-cyanin 5.5 (PerCP-Cy5.5)-conjugated anti-CD28 (eBioscience), APC-conjugated anti-CD39 (Serotec, MorphoSys US Inc), FITC-conjugated anti-CD103 (Becton–Dickinson, BD Biosciences, San Josè CA), PE-conjugated anti-NKG2A (IL, Milan, Italy), FITC-conjugated anti-HLA-G (Exbio Praha a.s., Vestec, CZ), PE-conjugated anti-LAG3 (Bioss Inc., Vobum, MA), PE-conjugated anti-CD122 (BD Biosciences, San Josè CA), FITC-conjugated anti-PD1 (e-Biosciences, San Diego, CA), PE-conjugated anti-TGFβ (BD Biosciences, San Josè CA).

Techniques: Activity Assay, Inhibition

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

doi: 10.1007/s00262-013-1392-z

Figure Lengend Snippet: CD39 expression on intratumoral Treg subsets and its relationship with their relative function. a Suppression activity of CD8+ Treg treated or not with the SASI_Hs02_00318598 CD39-specific siRNA: the experiment was performed with cells from patient # 12, and it is representative of all the experiments performed with cells from the other patients. The histograms refer to the proliferation activity of a responder PBMC (from one healthy donor) stimulated with an anti-CD3 mAb cultured without CD8+ Treg (left panel), with CD8+ Treg (middle panel), or with CD8+ Treg treated with the inhibitory CD39-specific siRNA for 24 h before the test (right panel). A control sample was also prepared culturing the anti-CD3 mAb-stimulated PBMC with CD8+ Treg treated with the liposomal transfection reagent used to transfect the cells with the siRNA but without the siRNA itself: in all the experiments, the suppression activity of these cells was comparable to that of untreated CD8+ Treg (not shown). The percentages of proliferating cells are shown in all the three panels while the percentages of suppression activity are indicated between parentheses in the middle and right panel, respectively. b Counteraction of CD8+ Treg suppression activity by a CD39-specific siRNA. CD8+ T lymphocytes purified from tumor specimens were transfected or not with the SASI_Hs02_00318598 CD39-specific siRNA 24 h before being tested for their suppression activity. The efficient downmodulation of the CD39 expression is shown in the insert. The results are expressed as mean ± SD of data from 4 independent experiments performed with cells from patients # 8, 9, 10, 12, respectively. c Histogram showing CD39 expression on control CD8+ Treg untreated (dark gray) or treated with the liposomal transfection reagent in the absence of the CD39 specific siRNA (light gray)

Article Snippet: The following mAbs were used: phycoerythrin (PE) or fluorescein isothiocynate (FITC)-conjugated anti-CD127, allophycocianin (APC)-cyanin (Cy) 7-conjugated anti-CD3 (e-Biosciences, San Diego, CA), FITC-conjugated anti-CD25 (Miltenyi, Bergisch Gladbach, Germany), PE-Cy7 or eFluor 450-conjugated anti-CD8 (Biolegend, San Diego, CA or e-Biosciences, San Diego, CA, respectively), Peridinin Chlorophyll Protein Complex-cyanin 5.5 (PerCP-Cy5.5)-conjugated anti-CD28 (eBioscience), APC-conjugated anti-CD39 (Serotec, MorphoSys US Inc), FITC-conjugated anti-CD103 (Becton–Dickinson, BD Biosciences, San Josè CA), PE-conjugated anti-NKG2A (IL, Milan, Italy), FITC-conjugated anti-HLA-G (Exbio Praha a.s., Vestec, CZ), PE-conjugated anti-LAG3 (Bioss Inc., Vobum, MA), PE-conjugated anti-CD122 (BD Biosciences, San Josè CA), FITC-conjugated anti-PD1 (e-Biosciences, San Diego, CA), PE-conjugated anti-TGFβ (BD Biosciences, San Josè CA).

Techniques: Expressing, Activity Assay, Cell Culture, Control, Transfection, Purification

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

doi: 10.1007/s00262-013-1392-z

Figure Lengend Snippet: Relationship between suppression activity and CD39 expression in Treg from healthy subjects. CD8+ Treg were generated in vitro from purified CD8+ T lymphocytes; CD4+CD25+ Treg were directly purified from the peripheral blood. a Correlation between percent suppression activity and percent expression of CD39 on CD8+ Treg from 13 healthy subjects; b Correlation between percent suppressive activity and percent expression of CD39 on CD4+CD25hi Treg from 10 healthy subjects; c Experiment of suppression activity by CD8+ Treg treated or not with the SASI_Hs02_00318598 CD39-specific siRNA performed with cells from donor # 3. The histograms refer to the proliferation activity of a responder PBMC stimulated with an anti-CD3 mAb cultured without CD8+ Treg (left panel), with CD8+ Treg (middle panel), or with CD8+ Treg treated with the inhibitory CD39-specific siRNA for 24 h before the test (right panel). The percentages of proliferating cells are shown in all the three panels while the percentages of suppression activity are indicated between parentheses in the middle and right panel, respectively. The experiment is representative of all the experiments performed with cells from the other donors. d Counteraction of CD8+ Treg suppression activity by a CD39-specific interfering RNA. In vitro generated CD8+ Treg were transfected or not with the SASI_Hs02_00318598 CD39-specific siRNA for 24 h before being tested for their suppression activity (big panel). The percentage of CD8+ CD28-CD127loCD39+ Treg in control cells and in cells treated with the CD39-specific siRNA is shown in the insert. The results are expressed as mean ± SD of data from 4 independent experiments performed with cells from donors # 1, 2, 3, 8, respectively. *: P = 0.04. e Histogram showing CD39 expression on control CD8+ Treg untreated (dark gray) or treated with the liposomal transfection reagent in the absence of the CD39 specific siRNA (light gray)

Article Snippet: The following mAbs were used: phycoerythrin (PE) or fluorescein isothiocynate (FITC)-conjugated anti-CD127, allophycocianin (APC)-cyanin (Cy) 7-conjugated anti-CD3 (e-Biosciences, San Diego, CA), FITC-conjugated anti-CD25 (Miltenyi, Bergisch Gladbach, Germany), PE-Cy7 or eFluor 450-conjugated anti-CD8 (Biolegend, San Diego, CA or e-Biosciences, San Diego, CA, respectively), Peridinin Chlorophyll Protein Complex-cyanin 5.5 (PerCP-Cy5.5)-conjugated anti-CD28 (eBioscience), APC-conjugated anti-CD39 (Serotec, MorphoSys US Inc), FITC-conjugated anti-CD103 (Becton–Dickinson, BD Biosciences, San Josè CA), PE-conjugated anti-NKG2A (IL, Milan, Italy), FITC-conjugated anti-HLA-G (Exbio Praha a.s., Vestec, CZ), PE-conjugated anti-LAG3 (Bioss Inc., Vobum, MA), PE-conjugated anti-CD122 (BD Biosciences, San Josè CA), FITC-conjugated anti-PD1 (e-Biosciences, San Diego, CA), PE-conjugated anti-TGFβ (BD Biosciences, San Josè CA).

Techniques: Activity Assay, Expressing, Generated, In Vitro, Purification, Cell Culture, Transfection, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

doi: 10.1007/s00262-013-1392-z

Figure Lengend Snippet: Expression of CD39 molecule on Treg from healthy subjects

Article Snippet: The following mAbs were used: phycoerythrin (PE) or fluorescein isothiocynate (FITC)-conjugated anti-CD127, allophycocianin (APC)-cyanin (Cy) 7-conjugated anti-CD3 (e-Biosciences, San Diego, CA), FITC-conjugated anti-CD25 (Miltenyi, Bergisch Gladbach, Germany), PE-Cy7 or eFluor 450-conjugated anti-CD8 (Biolegend, San Diego, CA or e-Biosciences, San Diego, CA, respectively), Peridinin Chlorophyll Protein Complex-cyanin 5.5 (PerCP-Cy5.5)-conjugated anti-CD28 (eBioscience), APC-conjugated anti-CD39 (Serotec, MorphoSys US Inc), FITC-conjugated anti-CD103 (Becton–Dickinson, BD Biosciences, San Josè CA), PE-conjugated anti-NKG2A (IL, Milan, Italy), FITC-conjugated anti-HLA-G (Exbio Praha a.s., Vestec, CZ), PE-conjugated anti-LAG3 (Bioss Inc., Vobum, MA), PE-conjugated anti-CD122 (BD Biosciences, San Josè CA), FITC-conjugated anti-PD1 (e-Biosciences, San Diego, CA), PE-conjugated anti-TGFβ (BD Biosciences, San Josè CA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Lung structural cell dynamics are altered by influenza virus infection experience leading to rapid immune protection following viral re-challenge

doi: 10.1101/2024.07.20.604410

Figure Lengend Snippet: C57BL/6 mice were infected i.n with IAV (X31 strain, either wild type or single cycle (S-FLU)) for 30 days. (A) Weight loss graph showing percentage of total body weight over time post infection (n=9) mice per group (naïve, WT-IAV and S-FLU). Data are combined from two independent experiments. (B) Histological analysis of inflammation in naïve, WT-IAV and S-FLU infected mice at day 30. Data are normally distributed and shown as mean +/- SEM. One way-ANOVA with Šidák’s multiple comparison, **P <0.01, ***P <0.001. (C) H&E staining showing airways (Aw), blood vessels (Bv) inflammatory foci/immune cell clusters (labelled with white stars) in naïve, WT-IAV and S-FLU infected mice at day 30. All images taken at 200x magnification, scale bar 200µm. Immunofluorescent staining shows the localization of CD4 T cells (green), B cells (B220+, red) and antigen presenting cells (MHCII, blue) in lung sections from naïve and IAV infected mice culled at day 30 post infection. All images taken at 200x magnification, scale bar 200µm. Airways (Aw), blood vessels (Bv) inflammatory foci/immune cell clusters (labelled with dashed white box). Positive cells are indicated by white arrows.

Article Snippet: For T cells, anti-CD4 APC-Alexa647 (RM4-5, ThermoFisher), anti-CD8 BUV805 (BD 53-6.7), anti-CD44 BUV395 (BD, IM7), anti-CD25 BV711 (Biolegend, PC61), anti-CD69 PE (BD, H1.2F3) anti-B220 eFluor 450 (RA3-6B2), anti-MHCII eFluor 450 (M5114) and anti- F480 eFluor 450 (BM8) all ThermoFisher.

Techniques: Infection, Comparison, Staining

Journal: bioRxiv

Article Title: Lung structural cell dynamics are altered by influenza virus infection experience leading to rapid immune protection following viral re-challenge

doi: 10.1101/2024.07.20.604410

Figure Lengend Snippet: (A) Schematic showing experimental design of IAV infection and in vitro re-challenge and co-culture. Lung CD45 negative cells were isolated from naïve or C57BL/6 mice infected 30 days earlier with IAV- WSN. After 24hours, the cells were infected with IAV-X31 and the cells examined by flow cytometry after a further 24hours or co-cultured with T cells isolated from the spleens of mice infected with IAV- X31 9 days earlier. (B) The percentage of CD44hi CD4 or CD8+ T cells that were CD25 or CD69 positive, normalized to the mean of the naïve- no infection control within each of the 2 experiments. CD4 data not normally distributed and tested via a ONE-WAY ANOVA and a Dunn’s multiple comparison test. CD8 data are normally distributed and tested via ONE-WAY ANOVA with Šidák’s multiple comparison test. Data are from two experiments with 4 mice within each naïve and IAV-memory group per experiment. (C) Representative FACS plots for IAV-NP for each cell type are shown for each group. Cells were gated based on live, single, CD45 negative populations that were CD31+ (endothelial cells), CD140+ (fibroblasts) and EpCAM1+ (epithelial cells). Numbers in plots indicate the percentages of cells that are IAV-NP+. Data are normally distributed and are from two combined experiments, with n=8 samples per timepoint. ONE-WAY ANOVA with Šidák’s multiple comparison test. (D) Cytokine levels were measured by Luminex assay in the supernatants of in vitro IAV infected and re-challenged groups taken 24hours after the in vitro infection. Data were normally distributed, are mean +/- SEM and analyzed by T test. Data combined from the same two experiments. In all graphs, each symbol represents a mouse, the horizontal line shows the mean and error bars are SEM and *:p<0.05,**: p<0.001,***: p <0.001, ****: p<0.0001.

Article Snippet: For T cells, anti-CD4 APC-Alexa647 (RM4-5, ThermoFisher), anti-CD8 BUV805 (BD 53-6.7), anti-CD44 BUV395 (BD, IM7), anti-CD25 BV711 (Biolegend, PC61), anti-CD69 PE (BD, H1.2F3) anti-B220 eFluor 450 (RA3-6B2), anti-MHCII eFluor 450 (M5114) and anti- F480 eFluor 450 (BM8) all ThermoFisher.

Techniques: Infection, In Vitro, Co-Culture Assay, Isolation, Flow Cytometry, Cell Culture, Control, Comparison, Luminex

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 1. Immunohistochemistry and immunofluorescence of coronary thrombus. (A) Aspirated material in a patient with STEMI due to total occlusion of coronary artery. (B) H&E staining of coronary artery thrombus. (C and D) Immunohistochemistry of coronary thrombus with MMP12 antibody. Arrows indicate examples of MMP12 positive cells. (E and F) Immunofluorescence staining of coronary thrombus with MMP12 antibody. Chartreuse: MMP12, Blue: Counterstain nucleus with DAPI. (E) Negative control. (F) Experimental group (magnification 400). Analysis of staining from 3 independent experiments showed similar results. STEMI=ST- segment elevation myocardial infarction.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Immunohistochemistry, Staining, Negative Control

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 3. The plasma levels of MMP12 and TIMP1 in STEMI, SAP, and control groups. (A) Columns and lines show significant increase in MMP12 concentration but lower activity in STEMI compared with SAP group and normal coronary artery control group. (B) The STEMI group had significantly higher TIMP1 concentration than SAP group and control group. (C) The imbalance in MMP/TIMP ratio was observed in STEMI group compared with SAP control group. The assays were performed in triplicate and the results expressed as the mean plus or minus SEM. ∗P<.05, means significant difference between 2 groups by Mann–Whitney test. SAP=stable angina pectoris, STEMI=ST-segment elevation myocardial infarction.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Clinical Proteomics, Control, Concentration Assay, Activity Assay, MANN-WHITNEY

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 2. MMP12 was present in human coronary thrombi. (A) RT-PCR analysis of MMP12 gene fragment. Lane 1: DNA ladder; lanes 2–6: coronary thrombus; GAPDH blot (bottom panel) was included as loading control. (B) WB analysis of MMP12 expression in coronary artery thrombus by SDS–PAGE. Lane 1: molecular weight markers; lanes 2–6: WB products from coronary artery thrombus of STEMI-LTB; lane 7: recombinant human MMP12 with molecular weights of 45kDa. (C) The caseinolytic activity of MMP12 in coronary thrombi. Lane 1: molecular weight markers; lanes 2–4: zymography products from the coronary artery thrombus of STEMI patients; lane 5: zymography products from the coronary artery thrombus of STEMI patients incubated with antihuman MMP12 antibody. Lane 6: Recombinant human MMP12 with molecular weights of 45kDa. Analysis of the blots from 3 independent experiments showed similar results. LTB=large thrombus burden, RT-PCR=reverse transcription polymerase chain reaction, STEMI=ST-segment elevation myocardial infarction, WB=Western blot.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, SDS Page, Molecular Weight, Recombinant, Activity Assay, Zymography, Incubation, Reverse Transcription, Polymerase Chain Reaction, Western Blot

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 4. The plasma levels of MMP12 and TIMP1 in LTB and STB subgroups. (A) Columns and lines show significant increase in MMP12 concentration (P<.05) but lower activity (P>.05) in LTB compared with STB subgroup. (B) The LTB subgroup had significantly higher TIMP1 concentration than STB subgroup. (C) Although the difference did not reach statistical significance, the imbalance in MMP/TIMP ratio were higher in LTB than patients with STB. The assays were performed in triplicate and the results expressed as the mean plus or minus SEM. ∗P<.05, means significant difference between 2 groups by Mann–Whitney test. LTB=large thrombus burden, STB=small thrombus burden.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Clinical Proteomics, Concentration Assay, Activity Assay, MANN-WHITNEY