Journal: Journal of the American Society of Nephrology

Article Title: CD4+CD25+Regulatory T Cells Inhibit Experimental Anti-Glomerular Basement Membrane Glomerulonephritis in Mice

doi: 10.1681/asn.2004100837

Figure Lengend Snippet: Figure 4. Treg transfer inhibits the infiltration of interstitial macrophages and CD4 and CD8 T cells. The infiltration of inflammatory cells was analyzed by immunohistochemistry. Staining for macrophages (A) and CD4 and CD8 T cells (B) was performed 7 (n 14 per group) and 14 d (n 7 per group) after induction of anti-GBM nephritis in mice that received an injection of Treg () or CD4CD25 T cells (f). Interstitial macrophage accumulation was comparable in both groups at day 7 but significantly diminished at day 14 after transfer of Treg. CD4 T cell infiltration was significantly decreased 14 d after anti-GBM injection, whereas CD8 T cell infiltration was significantly decreased 7 and 14 d after anti-GBM injection in mice that received an injection of Treg (*P 0.05).

Article Snippet: For the detection of CD4 T cells, a rat anti-mouse CD4 mAb (clone YTS191.1; Serotec) was used, and for CD8 T cells, a rat anti-mouse CD8 mAb (clone KT15; Serotec) was used.

Techniques: Immunohistochemistry, Staining, Injection

Journal: Acta Neuropathologica

Article Title: The structural differences between patient-derived α-synuclein strains dictate characteristics of Parkinson’s disease, multiple system atrophy and dementia with Lewy bodies

doi: 10.1007/s00401-020-02157-3

Figure Lengend Snippet: Assessment of immune response after inoculation of different patient-derived homogenates and PMCA-amplified αSYN strains in association with rAAV2/7-mediated αSYN expression. a Representative photomicrographs of the immune response stained for Iba1 (microglia), MHCII (reactive microglia), CD4 (T helper cells) and CD8 (cytotoxic T cells) in the SN 5 months after co-injection of different patient-derived homogenates (left) or PMCA-amplified αSYN assemblies (right) with rAAV2/7-αSYN overexpression. Cells immunoreactive for CD4 and CD8 are marked with black arrows. Scale bar Iba1 = 50 µm, MHCII – CD4 = 100 µm. b Table with scoring system for the different immune markers Iba1, MHCII, CD4 and CD8 in the rat SN 5 months after inoculation with patient-derived homogenates (left) or PMCA-amplified αSYN assemblies (right) combined with human αSYN expression. The scoring system ranges from not present (−) to abundantly present (++++). A total of three animals per group was included. The data were presented as a table with scoring system to demonstrate the presence of an inflammatory response in the rat SN

Article Snippet: IHC was performed on free-floating sections using antibodies against tyrosine hydroxylase (TH, rabbit polyclonal, Ab152, 1:5000, Merck Millipore, Massachusetts, US), phosphorylated αSYN (P-S129 αSYN, mouse 11A5, 1:5000, provided by Elan Pharmaceuticals, Inc., Dublin, Ireland and rabbit, Ab51253, 1:5000, Abcam, Cambridge, UK), Iba1 (goat polyclonal, Ab107159, 1:1000, Abcam), MHC Class II (mouse, MCA46G, 1:250, Serotec), CD4 (mouse, MCA55GA, 1:500, Serotec) and CD8 (mouse, MCA48GA, 1:500, Serotec).

Techniques: Derivative Assay, Amplification, Expressing, Staining, Injection, Over Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Overexpression of IFN-induced protein 10 and its receptor CXCR3 in myasthenia gravis.

doi: 10.4049/jimmunol.174.9.5324

Figure Lengend Snippet: FIGURE 3. mRNA and protein expression levels of ICAM-1 and LFA-1 in LNC and splenocytes of EAMG and healthy rats. LNC and splenocytes from EAMG and healthy CFA-injected control rats were har- vested when rats reached a clinical score of 2. A, mRNA expression levels of ICAM-1 and LFA-1 from EAMG and control CFA healthy rats were determined by Q-RT-PCR, and data are presented as the relative expression value for every EAMG group compared with its control, which was as- signed a value of 100. -Actin was used as an inner control for normal- ization for each gene. , p 0.05 (n 4). The data shown are represen- tative of five experiments. B, Protein levels of ICAM-1 and LFA-1 were determined in splenocytes from EAMG and healthy CFA-injected control rats (two or three rats in each group) that were subjected to FACS analysis with PE-conjugated anti-CD4 or anti-CD8 Abs combined with FITC-con- jugated anti-ICAM-1 or anti-LFA-1 Abs. Shown is a representative exper- iment of five independent experiments performed.

Article Snippet: The following primary Abs were used for flow cytometry: PE-conjugated mouse anti-rat CD8 (MCA938 PE), PE-conjugated mouse IgG anti-rat CD4 (MCA55 PE), FITC-conjugated mouse IgG2a anti-rat LFA-1 (MCA774F), FITC-conjugated mouse IgG1 anti-rat VLA-4 (MCA1383F; all from Serotec), and FITC-conjugated mouse IgG1 anti-rat ICAM-1 (554969; BD Biosciences).

Techniques: Expressing, Injection, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Overexpression of IFN-induced protein 10 and its receptor CXCR3 in myasthenia gravis.

doi: 10.4049/jimmunol.174.9.5324

Figure Lengend Snippet: FIGURE 5. CXCR3 surface expression in PBMC of MG patients and controls. PBMC from MG patients (n 6) and age-matched controls (n 7) were incubated first with anti-CXCR3 Abs, followed by FITC-conju- gated goat anti-mouse Ig. The cells were further incubated with mouse IgG1 to saturate the free sites of the FITC-goat anti-mouse Ig. After wash- ings, the cells were incubated with one of the following mAbs: anti-CD19- R-PE, anti-CD4-R-PE-Cy5, or anti-CD8-R-PE. The results are shown as the mean SEM. The p values were obtained using the nonparametric Mann-Whitney U test.

Article Snippet: The following primary Abs were used for flow cytometry: PE-conjugated mouse anti-rat CD8 (MCA938 PE), PE-conjugated mouse IgG anti-rat CD4 (MCA55 PE), FITC-conjugated mouse IgG2a anti-rat LFA-1 (MCA774F), FITC-conjugated mouse IgG1 anti-rat VLA-4 (MCA1383F; all from Serotec), and FITC-conjugated mouse IgG1 anti-rat ICAM-1 (554969; BD Biosciences).

Techniques: Expressing, Incubation, 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 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

Journal:

Article Title: Anti-T-cell humoral and cellular responses in healthy BALB/c mice following immunization with ovalbumin or ovalbumin-specific T cells

doi: 10.1046/j.1365-2567.2003.01604.x

Figure Lengend Snippet: Phenotypic changes of splenocytes responding to T-cell restimulation in vitro. Splenocytes from mice immunized with phosphate-buffered saline (PBS) (PBS-Spl), ovalbumin (OVA) (OVA-Spl), or OVA-specific T-cell clone 3 (OVA-T3) cells (TCV-Spl) were co-cultured with irradiated OVA-T3 cells on 24-well plates for 5 days and then stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 or anti-CD8 monoclonal antibodies (mAbs) for flow-cytometric analysis. Results are expressed as percentage CD4+ or CD8+ cells in the cultured splenocyte populations.

Article Snippet: FITC- or R-phycoerythrin (PE)-conjugated rat anti-mouse TCRVβ2, CD4 or CD8 mAbs were purchased from PharMingen (San Diego, CA).

Techniques: In Vitro, Cell Culture, Irradiation, Staining

Journal:

Article Title: Anti-T-cell humoral and cellular responses in healthy BALB/c mice following immunization with ovalbumin or ovalbumin-specific T cells

doi: 10.1046/j.1365-2567.2003.01604.x

Figure Lengend Snippet: Indirect staining of T cells using antisera from mice after ovalbumin (OVA) immunization or T-cell vaccination (TCV). Activated (panels a and d), or resting (panels b and e), OVA-specific T-cell clone 3 (OVA-T3) cells and activated BXSB T-cell clone (panels c and f) were sequentially treated with serum samples (diluted 1 : 20) from mice immunized with OVA (panels a, b and c) or OVA-T3 cells (panels d, e and f) and fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The cells were then washed and subjected to flow cytometry analysis. For each sample, a total of 10 000 cells was counted per sample. These data are from an experiment representative of four experiments.

Article Snippet: FITC- or R-phycoerythrin (PE)-conjugated rat anti-mouse TCRVβ2, CD4 or CD8 mAbs were purchased from PharMingen (San Diego, CA).

Techniques: Staining, Flow Cytometry

Journal:

Article Title: Simultaneous blockade of multiple immune system inhibitory checkpoints enhances anti-tumor activity mediated by interleukin-15 in a murine metastatic colon carcinoma model

doi: 10.1158/1078-0432.CCR-10-1966

Figure Lengend Snippet: Expression of MHC class I on CT26 cells was analyzed by flow cytometry using FITC-anti-mouse-H-2Kd (open area). An antibody of the same isotype was used as a negative control (filled area). MHC class I mean fluorescence intensity (MFI) of CT26 cells either A; in vitro or B; obtained ex vivo from lung nodules of tumor-bearing animals treated with PBS is shown. C; Survival of CT26 tumor-bearing mice treated with mIL-15 with and without CD8+ T-cells depletion is shown. One day after tumor inoculation, mice received 5 µg/day of mIL-15 injected five times a week for three weeks (mIL-15, diamonds). One group of mice were depleted of CD8+ T-cells (mIL-15 + anti-CD8, squares) using anti-mouse-CD8 antibody administered on days 0 and 1, and then three time a week for three weeks. PBS treated animals served as controls (PBS, dots). The data represent three independent experiments.

Article Snippet: Groups of animals received 200 µg of either rat-anti-mouse-CD8 (clone 2.43, Bio-express) or an isotype control rat IgG (clone LTF-2, Bio-express) intraperitonelly beginning on days 0 and 1, and then three times a week for three weeks.

Techniques: Expressing, Flow Cytometry, Negative Control, Fluorescence, In Vitro, Ex Vivo, Injection

Journal:

Article Title: Simultaneous blockade of multiple immune system inhibitory checkpoints enhances anti-tumor activity mediated by interleukin-15 in a murine metastatic colon carcinoma model

doi: 10.1158/1078-0432.CCR-10-1966

Figure Lengend Snippet: Flow cytometry was used to analyze the PD-1 expression on splenic CD8+ T-cells as well as on the CD8+CD44high population from the CT26-bearing mice following treatment with PBS, mIL-15, mIL-15 and anti-PD-L1, or mIL-15, anti-PD-L1 and anti-CTLA-4 on day 10 after CT26 tumor inoculation. Surface expression of PD-1 on CD8+ T-cells was detected using APC-anti-CD8 and PE-anti-PD-1 (open area, Fig. 2A). The CD8+CD44high population was stained with APC-anti-CD8 and PE-Cy5.5-anti-CD44 antibodies (open area, Fig. 2B). An isotype matched IgG was used as a negative control (filled area). All the samples were analyzed on gated CD8+ T-cells or CD8+CD44high cells as indicated. MFI is shown. The figure is representative of two independent experiments wherein each group contained three to five animals. Statistical analysis was performed based on PD-1 expression on CD8+ T cells (Fig. 2C) and CD8+ CD44high population (Fig. 2D). * P<0.05 and ** P<0.01.

Article Snippet: Groups of animals received 200 µg of either rat-anti-mouse-CD8 (clone 2.43, Bio-express) or an isotype control rat IgG (clone LTF-2, Bio-express) intraperitonelly beginning on days 0 and 1, and then three times a week for three weeks.

Techniques: Flow Cytometry, Expressing, Staining, Negative Control

Journal:

Article Title: Simultaneous blockade of multiple immune system inhibitory checkpoints enhances anti-tumor activity mediated by interleukin-15 in a murine metastatic colon carcinoma model

doi: 10.1158/1078-0432.CCR-10-1966

Figure Lengend Snippet: A and B; Splenic CD8+ T-cells obtained ex vivo from CT26 tumor-bearing mice treated with PBS, mIL-15, mIL-15 and anti-PD-L1, or mIL-15 combined with anti-PD-L1 and anti-CTLA-4 were stimulated with 10 µg/ml plate-bound anti-CD3 plus 1 µg/mL of a soluble anti-CD28 antibody and the supernatants were collected after 72 hours. IFNγ and IL-10 secretion was quantitated by ELISA. Cells were co-cultured with the same concentration of isotype-matched antibodies as a background control; *P<0.05; C, Naïve splenic CD8+ T-cells were stimulated with anti-CD3 (10 µg/mL, black dots; 4 µg/mL, grey diamonds) plus 1 µg/mL of a soluble anti-CD28 antibody in the presence of different amounts of mIL-15 as indicated. IL-10 secretion was quantitated by ELISA. Cells cultured with the same concentration of an isotype control antibody were set up as controls. D, anti-PD-L1 and anti-CTLA-4 (10 ng/mL) were added with 20 ng/mL mIL-15 present in wells at the same stimulation conditions as above (black bars). IL-10 secretion in the supernatants was quantitated by ELISA. Media alone with no mIL-15 was set up as a control, *P<0.05. ND: non-detectable (>16pg/ml). The data is representative of three independent experiments.

Article Snippet: Groups of animals received 200 µg of either rat-anti-mouse-CD8 (clone 2.43, Bio-express) or an isotype control rat IgG (clone LTF-2, Bio-express) intraperitonelly beginning on days 0 and 1, and then three times a week for three weeks.

Techniques: Ex Vivo, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

Journal:

Article Title: Simultaneous blockade of multiple immune system inhibitory checkpoints enhances anti-tumor activity mediated by interleukin-15 in a murine metastatic colon carcinoma model

doi: 10.1158/1078-0432.CCR-10-1966

Figure Lengend Snippet: On day 21, CT26-bearing mouse splenocytes were harvested and incubated with irradiated CT26 cells at a ratio of 50:1. Cultures were incubated for 4 days. A, 1 × 106 effector cells were cultured with CT26-tumor cells at a ratio of 20:1 for 6 h. Brefeldin A (10 µg/mL) was added to the cultures for the last 5 h. Cells from each group were first stained with APC conjugated anti-CD8, then intracellularly stained with PE-anti-IFNγ. Five hundred thousand events were collected for each sample by a flow cytometer. B; tumor-specific CD8+ T-cell target cell killing activities were detected by a non-radioactive cytotoxicity assay. PBS (open triangles), mIL-15 (open squares), mIL-15 + anti-PD-L1 (filled triangles) and mIL-15 + anti-PD-L1 + anti-CTLA4 (filled diamonds) groups are shown in panel B. The figures are representative of three independent experiments.

Article Snippet: Groups of animals received 200 µg of either rat-anti-mouse-CD8 (clone 2.43, Bio-express) or an isotype control rat IgG (clone LTF-2, Bio-express) intraperitonelly beginning on days 0 and 1, and then three times a week for three weeks.

Techniques: Incubation, Irradiation, Cell Culture, Staining, Flow Cytometry, Cytotoxicity Assay

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: List of mAb used for the analysis of lymphocyte subsets. BD (BD Biosciences, San Jose, CA); eB (eBioscience, San Diego, CA); DN: double negative; DP: double positive; SP: single positive; T CM : central memory T cell; T EM : effector memory T cell; CD26 expression was assessed in all lymphocyte subsets.

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Expressing, Marker

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: Splenocytes were obtained at 4 and 6 weeks after treatment. A) Percentage of T effector memory (EM) , T central memory (CM) and naïve T lymphocytes (CD8 + and CD4 + ) in control (white circles) and treated mice (black circles). B) Representative FACS plots of the naïve (CD3 + CD4 + /CD8 + CD44 low CD62L + ), T CM (CD3 + CD4 + /CD8 + CD44 high CD62L + ) and T EM (CD3 + CD4 + /CD8 + CD44 high CD62L neg ) both for CD4 + and CD8 + T lymphocytes from spleen in treated and control mice at 4 weeks of treatment. C) MFI for CD26 expression on T EM, T CM and naïve T lymphocytes (CD8 + and CD4 + ) in control (white circles) and treated mice (black circles). Lines represent the mean of 8–10 mice. Comparison between treated and untreated mice showed an increased expression of CD26 on CD8 + T EM cells at any of the study’s time-points. (*p<0.05, **p<0.01, three way Anova).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Control, Expressing, Comparison

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: T cells from PLNs were obtained from control (white circles) and treated (black circles) NOD mice at 4 weeks after treatment. A) Percentage of T effector memory (EM) , T central memory (CM) and naïve T lymphocytes (CD8 + and CD4 + ). B) MFI for CD26 expression on T EM, T CM and naïve T lymphocytes (CD8 + and CD4 + ). The expression of CD26 was significantly increased on CD4 + T EM and CD8 + T EM cells in MK626-treated mice when compared to controls. Lines represent the mean of 6 mice (*p<0.05, ***p<0.001, three way Anova).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Control, Expressing

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: A) Percentage of CD8 + and CD4 + T lymphocytes infiltrating pancreatic islets in untreated (white circles) and treated (black circles) mice assessed by flow cytometry. Lines represent the mean of 6–9 mice. No statistical differences were observed between groups (Mann-Whitney test). B) Expression of CD26 determined by flow cytometry (MFI) in CD8 + and CD4 + T lymphocytes from control (white circles) and treated (black circles) mice. Lines represent the mean of 4–8 mice. (*p≤0.05, Mann-Whitney test).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Flow Cytometry, MANN-WHITNEY, Expressing, Control

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: CD8 + T EM cell from control (n = 9) and treated (n = 9) mice obtained at 4 weeks of treatment were stimulated with anti–CD3 in vitro . A) Proliferative rate in CD8 + T EM lymphocytes from MK626-treated mice (black circles) and in control group (white circles). T cell proliferation was expressed as counts per minute (c.p.m) using 3 H-TdR. B) Cytokine concentration (IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IFN-γ, TNF-α and TGF-β) in supernatant from proliferation assays in treated mice (black bars) compared to control mice (white bars). Results are expressed as mean ± SEM. Mann-Whitney test was used for the evaluation of statistical significance (*p<0.05;**p<0.01).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Control, In Vitro, Concentration Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: A) Representative dot plots showing CFSE-T lymphocytes, stimulated with insulin-loaded mDCs, in the presence or absence of CTV-CD8 + T EM lymphocytes (ratio 1:1) and MK626 (10μM). B) Percentage of proliferating CFSE low T lymphocytes versus all T lymphocytes, with insulin-loaded mDCs (circles and triangles), in the presence of CTV-CD8 + T EM lymphocytes (triangles) and MK626 (open symbols) for 3 days. Plots show the mean (line) of three independent experiments.

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: