Journal: Cancer gene therapy

Article Title: Adenovirus-mediated intratumoral expression of immunostimulatory proteins in combination with systemic Treg inactivation induces tumor-destructive immune responses in mouse models

doi: 10.1038/cgt.2011.8

Figure Lengend Snippet: MMC cells were subcutaneously transplanted into neu -tg mice. A–D) Two weeks after transplantation, tumor sections were stained for E-cadherin (A) , laminin (B) , FoxP3 (red) and CD4 (green) (C) , and CD8 (green) (D) . Cell nuclei are stained with DAPI (blue). E) β-galactosidase expression 2 days after intratumoral injection of 1×10 9 pfu of Ad.bGal.

Article Snippet: CD4 + /CD8 + T cells and NK cells were depleted using i.p. injection of the following antibodies diluted in 500 μl PBS: 200 μg rat anti-mouse CD4 IgG (GK1.5, ATTC), 200 μg rat anti-mouse CD8 IgG (169.4; ATTC) or 20 μl rabbit anti-mouse asalio GM1 IgG (Cedarlane, Ontario, Canada).

Techniques: Transplantation Assay, Staining, Expressing, Injection

Journal: Cancer gene therapy

Article Title: Adenovirus-mediated intratumoral expression of immunostimulatory proteins in combination with systemic Treg inactivation induces tumor-destructive immune responses in mouse models

doi: 10.1038/cgt.2011.8

Figure Lengend Snippet: TC-1 cells were subcutaneously transplanted into C57Bl/6. Two weeks after transplantation, tumor sections were stained for E-cadherin (A) , laminin (B) , FoxP3 (red) and CD4 (green) (C) , and CD8 (green) (D) . Cell nuclei are stained with DAPI (blue). E) β-galactosidase expression 2 days after intratumoral injection of 1×10 9 pfu of Ad.bGal. The scale bar represents 40μm.

Article Snippet: CD4 + /CD8 + T cells and NK cells were depleted using i.p. injection of the following antibodies diluted in 500 μl PBS: 200 μg rat anti-mouse CD4 IgG (GK1.5, ATTC), 200 μg rat anti-mouse CD8 IgG (169.4; ATTC) or 20 μl rabbit anti-mouse asalio GM1 IgG (Cedarlane, Ontario, Canada).

Techniques: Transplantation Assay, Staining, Expressing, Injection

Journal: Cancer gene therapy

Article Title: Adenovirus-mediated intratumoral expression of immunostimulatory proteins in combination with systemic Treg inactivation induces tumor-destructive immune responses in mouse models

doi: 10.1038/cgt.2011.8

Figure Lengend Snippet: A total of 1×10 4 TC-1 cells were subcutaneously injected into the left inguinal flank of C57Bl/6 mice at day 0. At day 5, mice received an intraperitoneal injection of CY. At day 9, 1×10 6 Ad.zero or Ad.αCTLA4 transduced TC-1 cells were injected into the right inguinal flank. A) At day 22, tumors, tumor-draining lymph nodes, and spleen were harvested and analyzed for CD4, CD8, FoxP3, CD3 and NK1.1 by flow cytometry. White bars: Ad.zero transduced TC-1-cells, back bars: Ad.αCTLA4 transduced cells. N=4, *p<0.05 B and C) Four days before vaccination with Ad. αCTLA4 (B) or Ad.zero (C) transduced cells, mice received intraperitoneal injection of CD4, CD8 or NK-cell depleting antibodies. Injections were repeated every 3 days for the time of monitoring. Shown is the tumor volume of individual mice. Statistical analysis was performed using tumor volumes measured at the end of the observation period (day 22); see also . Ad.aCTLA4/CY: PBS vs CD4 depletion: p=0.964 PBS vs CD8 depletion: p=0.029 PBS vs NK depletion: p=0.001 Ad.zero/CY: PBS vs CD4 depletion: p=0.678 PBS vs CD8 depletion: p=0.191 PBS vs NK depletion: p=0.00003 Ad.aCTLA4/CY-PBS vs. Ad.zero/CY-PBS: p=0.020

Article Snippet: CD4 + /CD8 + T cells and NK cells were depleted using i.p. injection of the following antibodies diluted in 500 μl PBS: 200 μg rat anti-mouse CD4 IgG (GK1.5, ATTC), 200 μg rat anti-mouse CD8 IgG (169.4; ATTC) or 20 μl rabbit anti-mouse asalio GM1 IgG (Cedarlane, Ontario, Canada).

Techniques: Injection, Flow Cytometry

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling

doi: 10.1038/s41467-021-21737-9

Figure Lengend Snippet: A Representative examples of CD4 + and CD8 + T cell staining in the heart of C57BL/6J mice following coronary ligation (MI) or sham surgery. B Kinetic of CD8 + T cell infiltration in the myocardium, performed at days 0, 1, 3, 7, and 14 after surgery in MI (white circle, n = 9/12/12/5, respectively, at day 1/3/7/14) and sham-operated mice (black box, n = 6/time point); data are presented as mean ± SEM. C Immunostaining in the ischemic myocardium at day 3 after MI or sham showing CD8 + T infiltration (green) in the ischemic heart tissue, scale bar 40 μm. D Kinetic of CD4 + T cell infiltration in the myocardium, performed at days 0, 1, 3, 7, and 14 after surgery in MI (white circle, n = 10/9/12/5, respectively, at day 1/3/7/14) and sham-operated mice (black box, n = 6/time point); data are presented as mean ± SEM. E Flow cytometry characterization of CD8 + T cell subsets in the ischemic heart at days 1, 3, and 7 after MI including naïve (CD3 + CD8 + CCR7 High CD44 − ), effector memory (CD3 + CD8 + CCR7 Low CD44 + ), and central memory (CD3 + CD8 + CCR7 High CD44 + ) subsets. F Representative examples and quantitative analysis of CD8 + T cells expressing CD69 or CD107a within the ischemic heart tissue in MI (white circle, n = 6/9/12, respectively, at day 1/3/7) and sham-operated mice (black box, n = 6/time point); data are presented as mean ± SEM. G Immunostaining in the ischemic myocardium at day 3 after MI showing CD8 + T cells (green), and Granzyme B (Red) and merged area (Yellow), Inf for infarct area; scale bar 40 μm. H mRNA levels of Granzyme B within the injured myocardium on days 1, 3, and 7 after coronary ligation (gray, n = 5/4/5 at day 1/3/7) or sham (white, n = 4/8/6 at day 1/3/7). I C57Bl6 WT mice received intraperitoneal injection of isotype control (CTR, blue borderline, n = 4/time point) or anti-CD4 depleting monoclonal antibody (blue filled, n = 5/time point) (150 μg/mice) 1 day before coronary occlusion. CD4 + T cell depletion was confirmed in the heart by flow cytometry at day 1 and day 3 following MI. J , K Quantification and representative example of CD8 + T cell count in the heart of control ( n = 4/time point) or CD4-depleted ( n = 5/time point) mice at day 1 and day 3 after MI. P values were calculated using two-tailed Mann-Whitney test ( B , D , F , H , I , J ). Inf, infarct; MI, myocardial infarction; FMO, fluorescent minus one.

Article Snippet: OT-I mice were injected i.p. with a rat anti-mouse CD8 monoclonal antibody (100 μg/mouse, Biotem Clone YTS 169.4) or with a rat IgG2b isotype control (R&D Systems; Abingdon, UK) 1 h after MI.

Techniques: Staining, Ligation, Immunostaining, Flow Cytometry, Expressing, Injection, Control, Cell Counting, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling

doi: 10.1038/s41467-021-21737-9

Figure Lengend Snippet: A Representative examples (left) and quantitative analysis (right) of CD8 + T cell staining in the blood of C57BL/6J mice treated with isotype control (CTR, black box) or with the CD8 mAb (CD8 depleted, white circle) ( n = 6 mice per group/time point); data are presented as mean ± SEM, *** P < 0.001. B Representative examples (left) and quantitative analysis (right) of CD8 + T cell staining in the heart of C57BL/6J mice treated with isotype control (CTR) or with the CD8 mAb (CD8 depleted) ( n = 6 mice per group/time point); data are presented as mean ± SEM, *** P < 0.001. C Representative examples of CD8 + T cell staining in the peri-infarct area of C57BL/6J mice treated with isotype control (CTR) or with the CD8 mAb (CD8 depleted) at day 3, scale bar 40 μm. D Echocardiography analysis after anti-CD8 therapy. We measured LV shortening fraction (SF), LV volume at end systole and end diastole * (CTR white, n = 8 and CD8 depleted gray n = 9–10). E Systolic and diastolic pressure measured in the LV, and F related function parameters using intracardiac probe at day 28 (CTR n = 7 and CD8 depleted n = 9–10). G Representative photomicrographs and quantitative analysis of infarct size evaluation evaluated by Masson trichrome staining, in the 2 groups of mice (CTR n = 8 and CD8 depleted n = 10). H Representative photomicrographs and quantitative analysis of myocardial fibrosis evaluated by Sirius Red staining, in the 2 groups of mice (CTR n = 8 and CD8 depleted n = 9). LV, left ventricle. P values were calculated using two-tailed Mann-Whitney test ( A , B , D , E , F , G , H ).

Article Snippet: OT-I mice were injected i.p. with a rat anti-mouse CD8 monoclonal antibody (100 μg/mouse, Biotem Clone YTS 169.4) or with a rat IgG2b isotype control (R&D Systems; Abingdon, UK) 1 h after MI.

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

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling

doi: 10.1038/s41467-021-21737-9

Figure Lengend Snippet: A Male 9-week-old OT-I mice were treated with isotype control (CTR, white) or the CD8 mAb (CD8 depleted, gray). B CD8 depletion (red) was confirmed in the spleen at day 21 using flow cytometry. C , D Representative photomicrographs and quantitative analysis of infarct size evaluation using Masson trichrome staining, in the 2 groups of OT-I mice (CTR n = 10 and CD8 depleted n = 9). E Rag1 −/− mice injected with CD8-depleted splenocytes re-supplemented with WT (white) or OT-I (pink) CD8 + T cells, 3 weeks before MI. F Survival rate following MI (from 2 experiments, WT n = 16 and OT-I n = 13). G Quantitative analysis of infarct size evaluation assessed by Masson trichrome staining in the 2 groups of mice (WT n = 8 and OT-I n = 7). H Echocardiography analysis 21 days after MI and assessment of LV shortening fraction (SF) in the 2 groups of re-supplemented mice (WT n = 8 and OT-I n = 7). I CMy-mOva mice were injected with WT (white) or OT-I (pink) CD8 + T cells, 3 days before MI. J Granzyme B mRNA expression in the ischemic heart at day 2 after MI in CMy-mOva mice injected with WT or OT-I CD8 + T cells ( n = 4/group). K Survival rate following MI (pooled 2 experiments, WT n = 10 and OT-I n = 12). L Representative examples of infarct size after Masson trichrome staining. P values were calculated using two-tailed Mann-Whitney test ( C , G , H , J ). Difference in survival was evaluated using log-rank test ( F , K ).

Article Snippet: OT-I mice were injected i.p. with a rat anti-mouse CD8 monoclonal antibody (100 μg/mouse, Biotem Clone YTS 169.4) or with a rat IgG2b isotype control (R&D Systems; Abingdon, UK) 1 h after MI.

Techniques: Control, Flow Cytometry, Staining, Injection, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling

doi: 10.1038/s41467-021-21737-9

Figure Lengend Snippet: A Representative histograms of mRNA levels of Granzyme B within the injured myocardium on day 3 after MI in CTR (white) and CD8-depleted (gray) mice ( n = 5/group). B Representative examples (right) and quantitative analysis (left) of Granzyme B staining in the ischemic heart of C57BL/6J mice with or without CD8 depletion ( n = 5/group); scale bars 50 and 25 μm. C Representative examples (right) and quantitative analysis (left) of TUNEL+ cells (Red) in the peri-infarct area of C57BL/6J mice ( n = 5/group); scale bars 50 and 25 μm. D Representative histograms of mRNA levels of Il-1β, I-6, Tnf-α, and Il-10 within the injured myocardium on day 7 after MI ( n = 5/group). E Representative histograms of mRNA levels of Mmp9 within the injured myocardium on day 7 after MI ( n = 5/group). F Quantification (left) and representative photomicrographs (right) of matrix metalloproteinase (MMP)-sense 680 activity in the ischemic heart measured by ex vivo reflectance epifluorescence imaging at day 7 (CTR n = 5 and CD8 depleted n = 6), scale bar 2.5 mm. G Acute MI was induced on C57bl6 wild-type (WT, white) mice or Granzyme B deficient ( GzmB −/− , green) mice. Representative examples (right) and quantitative analysis (left) of TUNEL+ cells in the peri-infarct area of WT C57BL/6J or GzmB −/− mice at day 3 after MI (WT n = 8 and GzmB −/− n = 9); scale bars 50 and 25 μm. H Il-1β, Il-6, Tnf-α, and Il-10 mRNA levels measured by qPCR in infarcted heart at day 3 after MI ( n = 9/group). I Representative photomicrographs (left) and quantitative analysis (right) of infarct size evaluation using Masson trichrome staining, in the 2 groups of mice ( n = 7/group); scale bar 2.5 mm. J Purified non-activated or activated WT CD8 + T cells were co-cultured with cardiomyocytes at different ratios (Cardiomyocyte/CD8 + T cells) for 24 h before their removal. Apoptotic cardiomyocytes labeled with an active caspase-3 fluorescent dye was monitored for 24 h ( n = 4–5/conditions), Data are presented as mean ± SEM, # P < 0.001 for activated CD8 + T cells 1/5 versus non-activated CD8 + T cells or activated CD8 + T cells 1/1 or activated CD8 + T cells 1/3; # P < 0.001 for activated CD8 + T cells 1/10 versus non-activated CD8 + T cells or activated CD8 + T cells 1/1 or activated CD8 + T cells 1/3. K WT or GzmB −/− CD8 + T cells were co-cultured with cardiomyocytes for 24 h at 1/5 and 1/10 ratio and cardiomyocyte apoptosis using an active caspase-3 fluorescent dye was quantified. Cardiomyocyte and non-activated CD8 + T cells co-culture was named CTR condition ( n = 4/condition) *** P < 0.001. L Isolated cardiomyocytes were co-cultured overnight with CD8 + T cells isolated from WT or GzmB −/− mice and cardiomyocyte sarcomere shortening was measured (non-activated n = 14, activated n = 17 and GzmB −/− n = 10) at a ratio 1/3. P values were calculated using two-tailed Mann-Whitney test ( A , B , C , D , E , F , G , H , I , J ) or Kruskal - Wallis test ( K , L ). TUNEL, terminal deoxynucleotidyl transferase dUTP Nick End Labeling; MMP, matrix metalloprotease.

Article Snippet: OT-I mice were injected i.p. with a rat anti-mouse CD8 monoclonal antibody (100 μg/mouse, Biotem Clone YTS 169.4) or with a rat IgG2b isotype control (R&D Systems; Abingdon, UK) 1 h after MI.

Techniques: Staining, TUNEL Assay, Activity Assay, Ex Vivo, Imaging, Purification, Cell Culture, Labeling, Co-Culture Assay, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling

doi: 10.1038/s41467-021-21737-9

Figure Lengend Snippet: A Rag1 −/− mice injected with either CD8-depleted splenocytes (White) or CD8 cell-depleted splenocytes re-supplemented with WT (gray) or GzmB −/− CD8 + T cells (green), 3 weeks before MI. B Survival curves following MI (from 3 experiments, No CD8 n = 15, WT CD8 n = 18, and GzmB −/− CD8 n = 8). C Representative photomicrographs and quantitative analysis of infarct size. D Collagen content in the peri-infarct area in the 3 groups of mice, scale bar 100 μm. Results are pooled from three independent experiments including surviving mice (no CD8 n = 9, WT CD8 n = 7, and GzmB −/− CD8 n = 7). E Echocardiography analysis after 21 days of MI and assessment of LV shortening fraction (SF) in the 3 groups of mice (no CD8 n = 9, WT CD8 n = 7, and GzmB −/− CD8 n = 7). F Correlation between CD8 + T cell number in the spleen at day 21 and LV shortening fraction. Data from CD8-depleted splenocytes or CD8 cell-depleted splenocytes re-supplemented with WT CD8 + T cells have been included ( n = 16). P values were calculated using two-tailed Kruskal - Wallis test ( C , D , E ). Difference in survival was evaluated using log-rank test ( B ) and correlation was studied using Spearman’s test ( F ).

Article Snippet: OT-I mice were injected i.p. with a rat anti-mouse CD8 monoclonal antibody (100 μg/mouse, Biotem Clone YTS 169.4) or with a rat IgG2b isotype control (R&D Systems; Abingdon, UK) 1 h after MI.

Techniques: Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling

doi: 10.1038/s41467-021-21737-9

Figure Lengend Snippet: A Flow cytometry analysis of blood CD4 + T and CD8 + T subsets at day 1 after MI in control (PBS, CTR, n = 6), low (light gray, n = 5), and high (dark gray, n = 5) CD8-depleted groups. B Quantification of CD8 + T cell count in the blood at baseline, day 0, day 1, and day 3 after MI . C Representative picture and quantification of infarct size at day 14 in control, low, and high CD8-depleted groups. D Quantitative evaluation of left ventricle ejection fraction (Simpson) of CTR or CD8-depleted pigs. E , F Detection of CD8 + T cells (brown) in human heart biopsy of MI patients, using immunohistochemistry at day 3 ( E , upper) and day 8 ( F , lower) after MI, scale bar 50 μm. G Detection of GRANZYME B+ cells (brown) in human heart biopsy of MI patients, using immunohistochemistry, scale bar 20 μm. H Quantification of GRANZYME B+ cells (brown) in human heart biopsy of MI patients, using immunohistochemistry at different time points ( n = 9 day ≤ 7, n = 8 day ≥ 8). I Survival according to baseline circulating GRANZYME B level (< or > median value) in patients with acute MI ( n = 1046). High level of GRANZYME B at the admission for acute MI were independently predictive of death after 1 year of follow-up after multiple adjustments (see Methods and Supplementary Table ). HR = hazard ratio. P values were calculated using two-tailed Mann-Whitney test ( H ) or Kruskal - Wallis test ( B – D ).

Article Snippet: OT-I mice were injected i.p. with a rat anti-mouse CD8 monoclonal antibody (100 μg/mouse, Biotem Clone YTS 169.4) or with a rat IgG2b isotype control (R&D Systems; Abingdon, UK) 1 h after MI.

Techniques: Flow Cytometry, Control, Cell Counting, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY