Journal: Nature cancer

Article Title: CD25-T reg -depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity

doi: 10.1038/s43018-020-00133-0

Figure Lengend Snippet: (A) and (B) Pan T cells were isolated from splenocytes. 200,000 cells were plated and rested for 2 hours at 37°C. Antibodies were added at 50μg/ml and incubated with the cells for 30 mins at 37°C, following which cells were stimulated with IL-2 (50U/ml) for 10 mins at 37°C. Cells were stained for pStat5 as described and % Stat5 phosphorylation by different subsets of cells was recorded. (A) Representative histograms showing percentage of STAT5 phosphorylation observed in Tregs post-incubation with the antibodies listed. (B) Graph showing percentage of Stat5 phosphorylation observed in Tregs post-incubation with the antibodies listed. Data represented as mean of triplicates ±SEM. p<0.0001. (C) (D) (E) and (F) C57BL6 mice were injected with 500,000 MCA205 tumor cells. Once tumors were palpable, mice were injected IP with αCD25 PC61 or αCD25 NIB antibodies (200μg) on days 5, 10 and 14. Tumors were harvested on day 15 post-tumor inoculation (C) . Tumors were processed as described in section. 200,000 cells were plated in a 96-well plate, antibody was either added at 50μg/ml for 30 mins (E and F) or omitted (D) followed by IL-2 stimulation (50U/ml) for 10 mins. Cells were fixed and stained for pSTAT5 as described. (D) Graphs showing percentage of STAT5 phosphorylation by CD8 (p=0.0021 between No Tx and αCD25 PC61 ), CD4 Eff (p=0.0077 between No Tx and αCD25 PC61 ) and Treg cells (p=0.0004 between No Tx and αCD25 PC61 and p=0.0429 between No Tx and αCD25 NIB ) (n=5 mice/group). (E) Representative histograms showing percentage of STAT5 phosphorylation by CD8, CD4 eff and Treg cells post-treatments with antibodies shown in (F) . (F) Graphs showing percentage of STAT5 phosphorylation by CD8, CD4 Eff and Treg cells (p=0.0080) between No Tx and αCD25 NIB (n=5 mice/group). All quantification plots: mean, 1-way ANOVA, Tukey’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Measurement of apparent Kd of binding surrogate murine antibodies αCD25 NIB and αCD25 PC61 to recombinant mouse CD25 (Sino Biological 50292-M08H) was determined using the dextran-based carboxymethylated sensor chip CM5.

Techniques: Isolation, Incubation, Staining, Injection

Journal: Nature cancer

Article Title: CD25-T reg -depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity

doi: 10.1038/s43018-020-00133-0

Figure Lengend Snippet: (A) Balb/C mice were injected with 500,000 CT26 tumor cells. Treatment with αCD25 PC61 /αCD25 NIB was started on day 6 post-tumor inoculation. On the left, growth curves showing growth of tumor. On the right, survival. (B) C57BL6 mice were injected s.c. with 1,000,000 MC38 tumor cells in Matrigel. Treatment started on day 6 post-tumor inoculation. On the left, growth curves showing growth of tumor. On the right, survival . (C) C57BL6 mice were injected with 500,000 MCA205 tumor cells. Treatment started day 6, and continued weekly afterwards for 4 consecutive weeks for the αCD25 NIB weekly group. On the right, survival, p=0.0399 between αCD25 NIB day 6 and αCD25 NIB weekly group. (D) C57BL6 mice were injected with 500,000 MCA205 tumor cells. Treatment started on day 6 post tumor inoculation. Survival of these mice shown on the right. Analysis of Kaplan-Meier survival curves was done using a two-sided log-rank test.

Article Snippet: Measurement of apparent Kd of binding surrogate murine antibodies αCD25 NIB and αCD25 PC61 to recombinant mouse CD25 (Sino Biological 50292-M08H) was determined using the dextran-based carboxymethylated sensor chip CM5.

Techniques: Injection

Journal: Nature cancer

Article Title: CD25-T reg -depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity

doi: 10.1038/s43018-020-00133-0

Figure Lengend Snippet: C57BL6 mice were injected with 500,000 MCA205 tumor cells. Once tumors were palpable, on day 5, mice were injected IP with αCD25 PC61 /αCD25 NIB /αCD25 NIB + αIL2 (200μg). Tumors and LN were harvested on day 12 post-tumor inoculation and processed as described in . (A) Representative FACS plots showing expression of FoxP3 versus CD25 in CD4 + T cells. (B) Graph showing % FoxP3 + cells of total CD4 + cells. (C) Absolute number of Tregs shown as number of Tregs/g of tumor. P-value=0.0086 for No Tx vs αCD25 PC61 and p=0.0204 for No Tx vs aCD25 NIB group. (D) Ratio of effector T cells over Tregs. On the left, p value for CD8/Treg ratio between No Tx and aCD25 NIB = 0.0003. On the right, p value for NK/Treg ratio between No Tx and αCD25 NIB =0.0170. (E) Representative FACS plots showing Granzyme B expression versus Ki67 expression in CD8, CD4 effectors and NK cells. (F) Graph showing percentage of Granzyme B + cells in different effector subsets. In the CD8 subset, p-value between no Tx and αCD25NIB group=0.0452 and 0.0012 for No Tx vs αCD25 NIB + αIL2 group. For the CD4 eff group, p value for No tx vs αCD25 PC61 =0.0221, between No tx and αCD25 NIB =0.0059, between No tx and αCD25 NIB +αIL2=0.0276. For the NK subset, p value between the no tx and αCD25 NIB group=0.0232. (G) Graph showing the Mean Fluorescence Intensity of Granzyme B of the effector cells plotted in (F) . In the CD8 subset, p value for no tx vs αCD25 PC61 =0.0222, and 0.0190 for No tx vs αCD25 NIB + αIL2 group. For the CD4 eff subset, p value between No tx and αCD25NIB=0.0112. For the NK subset, p-value for No tx vs αCD25 NIB =0.0057 and between αCD25 PC61 vs αCD25 NIB =0.0295. All quantification plots: mean, 1-way ANOVA, Tukey’s multiple comparison test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Measurement of apparent Kd of binding surrogate murine antibodies αCD25 NIB and αCD25 PC61 to recombinant mouse CD25 (Sino Biological 50292-M08H) was determined using the dextran-based carboxymethylated sensor chip CM5.

Techniques: Injection, Expressing, Fluorescence

Journal: Nature cancer

Article Title: CD25-T reg -depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity

doi: 10.1038/s43018-020-00133-0

Figure Lengend Snippet: C57BL6 mice were injected with 500,000 MC38 tumor cells. Once tumors were palpable, on day 7, mice were injected IP with αPC61/αCD25 NIB /αCD25 NIB + αIL2 (200μg). Tumors and LN were harvested on day 15 post-tumor inoculation and processed as described in section. (A) Graph showing % FoxP3 + cells of total CD4 + cells. (B) Absolute number of Tregs shown as number of Tregs/g of tumor. p-value=0.0003 between No Tx and αCD25 NIB group. ****=p-value <0.0001 (C) Ratio of effector T cells over Tregs. For CD8/Treg ratio, P-value between No tx versus αCD25 NIB =0.0008, and between No tx and αCD25 NIB + αIL2 =0.0045. For CD4 Eff/Treg ratio, p-value between no tx and /αCD25 PC61 = 0.0056, between No tx and αCD25 NIB =0.0002, between no tx and αCD25 NIB + αIL2=0.0001. For NK/Treg ratio, p-value=0.0001 for no tx versus αCD25 PC61 , 0.0010 for no tx versus αCD25 NIB and 0.0004 for No tx versus αCD25 NIB + αIL2. (D) Representative FACS plots showing Granzyme B expression versus Ki67 expression in CD8, CD4 effectors and NK cells. (E) Graph showing percentage of Granzyme B + cells in different effector subsets. (F) Graph showing the Mean Fluorescence Intensity of Granzyme of the effector cells plotted in (E) . For CD8 cells, p-value between No tx group and αCD25 NIB =0.0001. For CD4 Eff, p-values between No tx versus αCD25 NIB =0.0007, for αCD25 NIB versus αCD25 PC61 =0.0009, and between αCD25 NIB and αCD25 NIB + αIL2 group= 0.0002. For NK cells, p-value between No tx group and αCD25 NIB group=0.0164 and between αCD25 NIB and αCD25 NIB + αIL2 group=0.0280. Quantification plots: mean ± SEM, 1-way ANOVA, Tukey’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Measurement of apparent Kd of binding surrogate murine antibodies αCD25 NIB and αCD25 PC61 to recombinant mouse CD25 (Sino Biological 50292-M08H) was determined using the dextran-based carboxymethylated sensor chip CM5.

Techniques: Injection, Expressing, Fluorescence

Journal: Nature cancer

Article Title: CD25-T reg -depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity

doi: 10.1038/s43018-020-00133-0

Figure Lengend Snippet: (A) C57BL6 mice were injected with 500,000 MCA205 tumor cells. Treatment with αPD-L1 (200μg) was started day 6, continued on days 9, 12 and 18. Groups receiving αCD25 NIB received either 1 dose at day 10 or an additional dose at day 15. Representative growth curves on the left, cumulative survival from 2 independent experiments of those mice is shown on the right. (B) C57BL6 mice were injected with 50,000 B16BL6 tumor cells. Treatment with Gvax was on days 6,9 and 12. αCD25 NIB (200μg) was administered on day 5. Cumulative survival of 2 independent experiments shown on the right, representative growth curves on the left. Analysis of Kaplan-Meier survival curves was done using a two-sided log-rank test, p value between αCD25 NIB and αPD-L1+ αCD25 NIB group=0.0073. (C) (D) and (E) C57BL6 mice were injected with 50,000 B16BL6 tumor cells and treatment was administered as described in (B) . Tumors and LN were harvested on day 15 post-tumor inoculation and processed as described in section. (C) Lymphoid cell populations integrated across all samples. Heatmap on the left shows min-max scaled, median marker expression of 20 subpopulations identified by unsupervised clustering. The identities of each cluster are annotated based on previously published analyses. The relationship between these clusters in UMAP dimension reduced space is shown in the plot on the right, and the effects of treatment on the lymphoid landscape are shown in (D) . Volcano plots in (E) represent differential cluster abundance in comparisons of each treatment group against control.

Article Snippet: Measurement of apparent Kd of binding surrogate murine antibodies αCD25 NIB and αCD25 PC61 to recombinant mouse CD25 (Sino Biological 50292-M08H) was determined using the dextran-based carboxymethylated sensor chip CM5.

Techniques: Injection, Marker, Expressing

Journal: Nature cancer

Article Title: CD25-T reg -depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity

doi: 10.1038/s43018-020-00133-0

Figure Lengend Snippet: For binding experiments with RG6292 and Daclizumab, SU-DHL1 tumor cells (human CD25 + , (A) ) and HSC-F cells (cynomolgus CD25 + , (B) ) were used. To quantify binding of αCD25 NIB , splenocytes were isolated of spleens resected from female C57BL/6-Foxp3tm1Flv/J mice (C) Cells were incubated with indicated serial dilutions of the test antibody detected then by fluorescently labeled 2 nd antibody against human and mouse Fcγ, respectively. Living mouse Treg cells (Aqua - , mRFP + singlets) and tumor cells (Aqua - , singlets), respectively, were gated and the mean fluorescence intensity of the secondary antibody was plotted. EC50 values were calculated by as described in the data analysis section in . Shown are technical duplicates of one representative experiment out of several independent ones conducted (N>2). (D) RG6292 (and the fully fucosylated version RG6292 (FF)) depleted via ADCC in-vitro differentiated Treg cells using purified, IL-2 activated NK cells. Shown are technical duplicates of one representative experiment out of several independent ones conducted (N>2). (E) RG6292 and RG6292 (FF) mediated ADCP of in-vitro differentiated Treg cells when co-cultured with MCSF differentiated macrophages. Flow cytometric analysis was performed to determine percentage of phagocytosis. Shown are technical duplicates of one representative experiment out of several independent ones conducted (N>2). (F) Schematics of binder selection.

Article Snippet: Measurement of apparent Kd of binding surrogate murine antibodies αCD25 NIB and αCD25 PC61 to recombinant mouse CD25 (Sino Biological 50292-M08H) was determined using the dextran-based carboxymethylated sensor chip CM5.

Techniques: Binding Assay, Isolation, Incubation, Labeling, Fluorescence, In Vitro, Purification, Cell Culture, Selection

Journal: The Journal of Experimental Medicine

Article Title: Tumoral EHF predicts the efficacy of anti-PD1 therapy in pancreatic ductal adenocarcinoma

doi: 10.1084/jem.20180749

Figure Lengend Snippet: Tumoral EHF associates with the immune profile in human PDAC tissue. (A–C) IF staining (left) of EHF expression and the accumulation of FOXP3 + T reg cells (A), CD33 + MDSCs (B), and CD8 + T cells (C) in tumor tissues. An example from the 96 cases is shown. The arrows indicated tumor-infiltrating Foxp3 + T reg cells, CD33 + myeloid cells, and CD8 + T cells. Bars, 200 µm. Nonpaired Student’s t test was used as statistical analysis; n = 96, ***, P < 0.001. (D and E) Kaplan–Meier OS (D) and RFS (E) for different levels of EHF based on the log-rank statistic test (P < 0.001). (F–I) Single-cell suspensions were prepared from 45 cases of fresh PDAC tissues and stained with specific antibodies against human T reg cells (CD4 + , CD25 + , and FOXP3 + ), MDSCs (HLA-DR − , CD33 + , and CD11b + ), and CD8 + T cells (CD8 + ). Representative IHC staining of EHF is shown. Bars, 200 µm. Representative dot plots or histograms of T reg cells (gated on CD4 + T cells; G, left), MDSCs (gated on HLA-DR − cells; H, left), and CD8 + T cells (I, left). Spearman correlation analyses between EHF IHC score and the proportions of tumor-infiltrating T reg cells (G, right), MDSCs (H, right), and CD8 + T cells (I, right); n = 45.

Article Snippet: Anti-CD25 Abs (BioXcell; PC61) combined with anti-Gr1 Abs (BioLegend; 108414, RB6-8C5) or combined isotype IgG were intraperitoneal injected (200 μg per mouse twice a week).

Techniques: Staining, Expressing, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Tumoral EHF predicts the efficacy of anti-PD1 therapy in pancreatic ductal adenocarcinoma

doi: 10.1084/jem.20180749

Figure Lengend Snippet: Tumor cells expressing low levels of EHF induce the conversion, expansion, and function in vitro of T reg cells and MDSCs. (A) Representative plots of T reg cell conversion from CD4 + CD25 − T cell induced by PANC-1-EHF cells relative to PANC-1-vector and T reg cell conversion induced by BxPC-3-EHF-KD cells relative to BxPC-3-scramble. TGFβ1 was used as positive control. RPMI 1640 was used as negative control. CD25 and FOXP3 expression was determined by flow cytometry after 3 d of coculture (left). Percentage of T reg cell conversion from CD4 + CD25 − T cells. CD4 was gated (right). (B) Representative histogram of T reg cell proliferation. Flow cytometry was performed after 5 d of coculture by gating on live cells to determine the percentage of T reg cells that diluted CFSE (left). Statistical analysis of the percentage of T reg cell division (right). (C) T reg cell suppression assay compared the percentage of CD8 + T cells division cocultured with PANC-1-EHF (BxPC-3-EHF-KD)–generated T reg cells and PANC-1-vector (BxPC-3-scramble)–generated T reg cells. Statistical analyses of the responder T cells division (right). (D) Human PBMCs were cultured in vitro under different conditions (negative control: RPMI 1640; positive control: IL6 and GM-CSF; experimental group: PANC-1-vector, PANC-1-EHF, BxPC-3-scramble, and BxPC-3-EHF-KD) and analyzed on day 6 to determine the MDSC marker expression by flow cytometry. Representative plots show the surface CD11b + and CD33 + costaining of MDSCs. HLA-DR − cells were gated (left). The frequency of CD11b + CD33 + cells among HLA-DR − cells was quantified in the bar graph (right). (E) Mouse BMDCs were cultured in vitro under different conditions (negative control: RPMI 1640; positive control: IL6 and GM-CSF; experimental group: PANC02-EHF and PANC02-vector) and analyzed on day 6 to determine the MDSC marker expression by flow cytometry. Representative plots show the surface CD45 + CD11b + Gr-1 + costaining of MDSCs (left). The frequencies of CD11b + Gr-1 + cells among CD45 + cells were quantified in the bar graph (right). (F) Representative histogram of MDSC expansion. IL-6 and GM-CSF were used as positive controls. Flow cytometry was performed after 3 d of coculture by gating on live cells to determine the percentage of MDSCs that diluted CFSE (left). Statistical analysis of the percentage of MDSCs division (right). (G) MDSCs induced by PANC02-vector or PANC02-EHF cells were isolated to evaluate their suppressive activities in vitro. Histograms were gated on CFSE + cells to determine the percentage of CD8 + T cells that diluted CFSE (left). The percentage of division of responder T cells is summarized and quantified in the bar graph (right). The coculture experiments (A–G) were repeated five times independently. Representative data are shown. Data are presented as mean ± SD. Paired Student’s t test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Anti-CD25 Abs (BioXcell; PC61) combined with anti-Gr1 Abs (BioLegend; 108414, RB6-8C5) or combined isotype IgG were intraperitoneal injected (200 μg per mouse twice a week).

Techniques: Expressing, In Vitro, Plasmid Preparation, Positive Control, Negative Control, Flow Cytometry, Suppression Assay, Generated, Cell Culture, Marker, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Tumoral EHF predicts the efficacy of anti-PD1 therapy in pancreatic ductal adenocarcinoma

doi: 10.1084/jem.20180749

Figure Lengend Snippet: Tumor EHF deficiency induces immune suppression through TGFβ1 and GM-CSF. (A) Statistical analysis of CD4 + CD25 − T cells to T reg cell conversion (left) and T reg cell proliferation (right) induced by PANC-1-EHF cells relative to PANC-1-vector with or without TGFβ1 neutralization. CD4 + CD25 − T cells cultured in RPMI 1640 were used as negative controls. (B) Statistical analysis of mouse BMDC to MDSC conversion and MDSC expansion induced by PANC02-vector cells relative to PANC02-EHF with or without GM-CSF neutralization. BMDCs cultured in RPMI 1640 were used as negative controls. Experiments were repeated three times independently. Paired Student’s t test was used for statistical analysis. (C) PANC02-vector or PANC02-EHF was injected subcutaneously in a Matrigel plug containing neutralizing anti–GM-CSF mAb and anti-TGFβ1 mAb (vs. isotype IgGs as control) into the flanks of C57BL/6 mice. After 8 d, TGFβ1 and GM-CSF antibodies or isotype IgGs were intratumorally injected at 20 µg/mouse two times a week. (D and E) Representative dot plots of the proportion of tumor infiltration by T reg cells (D, left) and MDSCs (E, left) in the PANC02-vector and PANC02-EHF groups with or without combined TGFβ1 and GM-CSF depletion. Statistical analysis of the proportion of tumor-infiltrating T reg cells (D, right) and MDSCs (E, right) in the PANC02-vector and PANC02-EHF groups with or without combined TGFβ1 and GM-CSF depletion. The mouse experiments were repeated three times independently, using seven mice per experimental group. Representative data are shown. Nonpaired Student’s t test was used for statistical analysis. (F) Schematic of the roles of EHF in tumor immune modulation. EHF decreased the tumor-infiltrating T reg cells and MDSCs by transcriptionally suppressing the expression of TGFβ1 and GM-CSF. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

Article Snippet: Anti-CD25 Abs (BioXcell; PC61) combined with anti-Gr1 Abs (BioLegend; 108414, RB6-8C5) or combined isotype IgG were intraperitoneal injected (200 μg per mouse twice a week).

Techniques: Plasmid Preparation, Neutralization, Cell Culture, Injection, Control, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Tumoral EHF predicts the efficacy of anti-PD1 therapy in pancreatic ductal adenocarcinoma

doi: 10.1084/jem.20180749

Figure Lengend Snippet: EHF-low tumors can benefit from T reg cell and MDSC depletion treatment. (A) Anti-mouse CD25 antibody combined with anti-mouse Gr-1 antibody (or combined isotype control) was injected intraperitoneally two times a week, 7 d after tumor inoculation (red plots represent the time points of drug administration). T reg cell and MDSCs depletion therapy efficacy was evaluated in PANC02-vector and PANC02-EHF tumors, respectively. Tumor volumes were measured every 4 d using calipers. (B) Tumor growth curves of the four groups. Repeated measure two-way ANOVA (time × tumor volume) and post hoc analysis were used to test mouse tumor growth between groups. (C) Effects of T reg cell and MDSC depletion therapy on tumor growth in the PANC02-vector and PANC02-EHF groups. Tumors at the end point of the experiment are also shown (left, PANC02-vector; right, PANC02-EHF). (D and E) Representative plots (D) and statistical analyses (E) of tumor-infiltrating CD8 + T cells in tumors that received T reg cell and MDSCs depletion therapy or isotype IgG (left, vector group; right, EHF group). (F and G) Representative plots (F) and statistical analyses (G) of tumor-infiltrating IFNγ + CD8 + T cells. (H and I) Representative plots (H) and statistical analyses (I) of tumor-infiltrating CD8 + T cell apoptosis. (J) Histogram of tumor infiltration by PD1 + CD8 + T cells, PD1 int CD8 + T cells, and PD1 hi CD8 + T cells in the four groups. (K–M) Percentage of PD1 + CD8 + T cells, PD1 int CD8 + T cells, and PD1 hi CD8 + T cells in tumors that received T reg cell and MDSC depletion therapy or isotype IgG. Data are presented as mean ± SD. Nonpaired Student’s t test was used for statistical analysis; **, P < 0.01, ***, P < 0.001; n.s., not significant. All mouse experiments were repeated three times independently, using seven mice per experimental group. Representative data are shown.

Article Snippet: Anti-CD25 Abs (BioXcell; PC61) combined with anti-Gr1 Abs (BioLegend; 108414, RB6-8C5) or combined isotype IgG were intraperitoneal injected (200 μg per mouse twice a week).

Techniques: Control, Injection, Plasmid Preparation

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The structure and characterization of CD25 aptamer. ( A ) The secondary structure of the CD25 aptamer was estimated by RNAstructure software v6.4 of Mathews Lab. The sequence of CD25 aptamer is shown with modifications indicated (Z, 5-[ N -(1-naphthylmethyl)carboxamide]-2′-deoxyuridine; N me , 2′- O -methyl nucleosides). ( B ) The binding affinity of CD25 aptamer to the CD25 recombinant protein was determined by the BLI method. Ni-NTA probes were immobilized with His-tag CD25 protein, followed by incubation with the aptamer 125 (green), 250 (yellow), or 500 nM (red). The binding signal over time is shown. Kd is expressed as mean ± SD. ( C ) The cells were stained with biotin-aptamer combined with NeutrAvidin DyLight 650 or APC-conjugated anti-CD25 monoclonal antibody (mAb), and then the specificity of the antibody and the aptamer to the cells was examined by flow cytometry (control for aptamer: DyLight 650 only; control for antibody: not stained).

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Software, Sequencing, Binding Assay, Recombinant, Incubation, Staining, Flow Cytometry, Control

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The CD25 aptamer specifically binds and internalizes into CD25-positive cells. ( A ) The cell internalization of Cy-5-labeled CD25 aptamer (red) was visualized for 0, 1, and 4 h using confocal fluorescence microscopy using CD25-positive Karpas299 and CD25-negative Daudi cell lines. The nuclei were stained with DAPI (blue). ( B ) The rate of internalized CD25 aptamer was determined using the MFI value of flow cytometry analysis at 0 to 240 min. ( C ) Cellular trafficking of the CD25 aptamer. Fluorescence microscopy visualized the lysosomal delivery of pHrodo-labeled CD25 aptamer (red) for up to 4 h.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Labeling, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: Effects of the CD25 aptamer on CD25/IL-2 signaling. ( A ) A competitive binding assay was performed by adding biotinylated IL-2 proteins to 96-well plates coated with CD25 proteins, in the presence or absence of the CD25 aptamer. ( B , C ) Karpas299 cells were pre-treated with the CD25 aptamer for 30 min, followed by stimulation with IL-2 for 15 min. The levels of pSTAT5 protein and TGF-β mRNA were analyzed by Western blotting and quantitative RT-PCR, respectively. ( D , E ) HuT78 cells were treated with IL-2 in the presence of either the CD25 aptamer or the anti-CD25 antibody Daclizumab. The expression of pSTAT5 was then assessed by Western blot analysis. ( F ) HuT78 cells were pre-treated with the indicated concentrations of the CD25 aptamer, stimulated with IL-2, and the secretion of IL-4 was measured as described in the Materials and Methods. Results are expressed as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Competitive Binding Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vitro cytotoxicity of CD25 aptamer–MMAE conjugates. Karpas299 and Daudi Cells were treated with CD25-ApDC MMAE1 ( A ) or CD25-ApDC MMAE3 ( B ) for 3 days, after which cell viability was assessed, as described in the Materials and Methods. ( C ) Karpas299 and HuT78 cells were co-cultured at a 1:1 ratio for 24 h, stained with anti-CD4 and anti-CD25 antibodies, and analyzed by flow cytometry. The co-cultured cells were subsequently incubated with 45 nM CD25-ApDC MMAE3 for 24, 48, or 72 h, and analyzed again using flow cytometry. ( D ) Cells were treated with increasing concentrations of MMAE or CD25-ApDC MMAE3 for 24 h. Western blot analysis of total PRAP, cleaved PARP, total caspase-3, and cleaved caspase-3 was performed. ( E ) The cell cycle was analyzed using flow cytometry after staining with PI. Results are expressed as mean ±SD. * p < 0.05, *** p < 0.001.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: In Vitro, Cell Culture, Staining, Flow Cytometry, Incubation, Western Blot

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vivo antitumor efficacy of CD25 aptamer–MMAE conjugates in xenograft models. Tumor growth curves were generated by measuring tumor volumes in Karpas299 tumor-bearing mice following intravenous administration of CD25 aptamer–MMAE conjugates when tumors reached an average volume of 150 mm 3 . ( A ) Red arrows indicate the time points of injection with CD25-ApDC MMAE1 at doses of 1, 2, or 4 mg/kg. ( B ) Mice were treated either four times with 4 mg/kg (red arrows) or twice with 12 mg/kg (green arrows). ( C ) Tumor-bearing mice received a single dose of 0.4, 0.8, or 1.6 mg/kg, or were administered doses three times (once per week) with 0.8 or 1.6 mg/kg CD25-ApDC MMAE3 . Data are the mean tumor volume ±SE of eight animals per group. ( D ) NOD/SCID mice were systemically inoculated with Karpas299 cells and treated intravenously with the indicated dose of CD25-ApDC MMAE1 or CD25-ApDC MMAE3 twice per week for 3 weeks. Kaplan–Meier survival curves show the percentage of survival for each group, with statistical comparison performed using log-rank tests.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: In Vivo, Generated, Injection, Comparison

Journal: Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation

Article Title: Functional divergence of antigen-specific T-lymphocyte responses in syngeneic graft-versus-host disease.

doi: 10.1016/j.bbmt.2004.05.003

Figure Lengend Snippet: Figure 2. Flow cytometric selection of CLIP antigen-specific T-cell subsets. Splenic T lymphocytes harvested on day 14 after cessation of cyclosporine treatment were subjected to 3-color immunofluorescence. The cells were stained with the biotinylated soluble MHC class II immunoglobulin construct loaded with the N- or C-terminal CLIP variants (counterstained with CyChrome/streptavidin) and with fluorescein isothiocyanate (FITC)–and phycoerythrin (PE)–conjugated antibodies to CD8 and CD25, respectively. The percentage of cells staining with the construct is shown in the histogram; the dot plot displays CD8 and CD25 expression of the cells reacting with the peptide-loaded construct.

Article Snippet: The shaded area, however, repr etween N- and C-terminal flanking domain dependence. eric molecule loaded with unrelated peptides was [ B & M T neffective in isolating CLIP-specific T cells [23]. low cytometric multicolor analysis was used to isoate the antigen-specific T cells and to confirm expresion of the CD4, CD8, and CD25 cell-surface markrs (phycoerythrin- or fluorescein isothiocyanate– onjugated mouse anti-rat CD8, anti-CD4, and CD25 onoclonal antibodies; Serotec, Harlan Bioproducts or Science, Indianapolis, IN).

Techniques: Selection, Staining, Construct, Expressing

Journal: The Journal of Neuroscience

Article Title: Combined Active Humoral and Cellular Immunization Approaches for the Treatment of Synucleinopathies

doi: 10.1523/JNEUROSCI.1170-17.2017

Figure Lengend Snippet: Effects of combined GP+RAP/α-syn vaccination on the trafficking of T and B cells in the brains of α-syn tg mice. Brain sections from α-syn tg mice immunized with GP, GP-α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control mice immunized with GP were analyzed immunohistochemically for markers of T cells (CD4), Tregs (CD25, FOXP3), and B cells (CD20). A–F, There was a significant increase in the numbers of the CD4-immunoreactive T cells (A, B), the CD25 cells (C, D), and the FOXP3 markers of Tregs (E, F) in α-syn tg mice treated with either GP+RAP or GP+RAP/α-syn compared with non-tg mice treated with GP-alone. G, H, There was no change in the CD20 marker for B cells across any of the treatment groups. p < 0.05 using ANOVA followed by Dunnett's post hoc test comparing GP+RAP/α-syn (&) or GP+RAP (#) treatment with all other groups. N = 6 mice/group. Scale bar, 25 μm.

Article Snippet: To evaluate the effects of the combined vaccine of immunological markers, the blots were probed with antibodies against CD25 (1:1000; monoclonal, MBL); TGF-β1 (polyclonal, 1:2500, Abcam); CD68 (1:1000; monoclonal, Abcam), TNF-α (1:2000; polyclonal, Abcam), and IL-6 (1:1000; polyclonal, Santa Cruz Biotechnology).

Techniques: Marker

Journal: The Journal of Neuroscience

Article Title: Combined Active Humoral and Cellular Immunization Approaches for the Treatment of Synucleinopathies

doi: 10.1523/JNEUROSCI.1170-17.2017

Figure Lengend Snippet: Verification of the effects of combined GP+RAP/α-syn vaccination on α-synuclein and immune activation markers by immunoblot. Brain homogenates from α-syn tg mice immunized with GP, GP-α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control mice immunized with GP were fractioned and analyzed by Western blot. A–G, Representative Western blot images (A) and quantitative analysis for CD25 (B), FOXP3 (C), α-syn (D), TNF-α (E), IL-6 (F), and TGF-β1 (G) normalized to actin. N = 6 mice/group. p < 0.05 using ANOVA followed by Dunnett's post hoc test comparing each of the other groups with GP-alone treated tg mice (*), GP+RAP/α-syn (&), or GP+RAP (#) treatment.

Article Snippet: To evaluate the effects of the combined vaccine of immunological markers, the blots were probed with antibodies against CD25 (1:1000; monoclonal, MBL); TGF-β1 (polyclonal, 1:2500, Abcam); CD68 (1:1000; monoclonal, Abcam), TNF-α (1:2000; polyclonal, Abcam), and IL-6 (1:1000; polyclonal, Santa Cruz Biotechnology).

Techniques: Activation Assay, Western Blot

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: Phenotpyes of primed CD8 + Foxp3 + T cells. (A) Surface staining of CD8, CD25, GITR, CTLA4, and PD-1 on the splenic cells taken from Foxp3-GFPtg mice on day 6 after infection with H5N1 virus. The dot plots represent one of four independent experiments with similar results ( n = 5 mice). (B) Splenic CD8 + CD25 + T cells from Foxp3-GFPtg mice on day 6 after H5N1 viral infection were purified for quantitative RT-PCR. Naïve CD8 + T cells were purified from naïve Foxp3-GFPtg mice as control. Data are shown as mean + SEM and are pooled from three independent experiments. ** p < 0.01. n.s., p > 0.05, unpaired two-tailed t -test. (C) Splenic cells were isolated on day 6 from Foxp3-GFPtg mice infected by H5N1 virus and were stimulated with 5 μg/mL NP peptide (NP366–374) or no peptide. Cells were incubated for 6 h with 2 μM monensin before intracellular staining. CD8 + T cells were gated to analyze the expression of Foxp3 and IL-10. The dot plots represent one of three independent experiments with similar results ( n = 5 mice).

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Staining, Infection, Virus, Purification, Quantitative RT-PCR, Control, Two Tailed Test, Isolation, Incubation, Expressing

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: Primed CD8 + CD25 + T cells expressed IL-10. Splenic cells were isolated on day 6 from IL-10-GFPtg mice infected with H5N1 virus and surface stained for CD8 and CD25. CD8 + T cells were separated into CD25 positive and negative cells. These cells were further divided into GFP positive or negative, with GFP serving as a marker for IL-10 positivity. The percentages of CD8 + CD25 + T cells and CD8 + CD25 − T cells that were IL-10 + or IL-10 − are shown together with results of statistical analysis. The dot plots represent one of five independent experiments with similar results ( n = 5 mice).

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Isolation, Infection, Virus, Staining, Marker

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: CD8 + Treg cells resulted in enhanced mortality and increased virus load in the lung. (A) Splenic CD8 + CD25 + T cells were isolated on day 6 from Foxp3-GFPtg mice infected with H5N1 virus. The cells were transferred into BALB/c mice (1 × 10 5 or 5 × 10 5 CD8 + CD25 + T cells per mouse) and the mice ( n = 10 mice per group) were then immediately challenged with H5N1 virus. (B) Survival of mice was monitored from day 7 to 16 after virus infection. Log-rank test for comparisons of survival curves between control mice and CD8 + Treg cells recipient mice; ** p < 0.01. *** p < 0.001, log-rank test. (C) Lung viral load was assayed on day 6 after virus infection ( n = 5 mice per group). (D) Total RNA was extracted from lung for real-time PCR for IFN-β and Mx-1. Data are shown as mean + SEM and are pooled from three independent experiments. n.s., p > 0.05. * p < 0.05. ** p < 0.01. *** p < 0.001, unpaired two-tailed t -test.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Virus, Isolation, Infection, Control, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: CD8 + T-cell immunity was inhibited by CD8 Treg cells in vivo. (A) CD8 + CD25 + T cells, CD8 + CD25 − T cells, total CD8 + T cells were isolated from spleens of C57BL/6 mice on day 6 after infection with H5N1 virus and were transferred to CD8 KO mice. 5 × 10 5 CD8 + CD25 + T cells, 1 × 10 7 CD8 + CD25 − T cells, or 1 × 10 7 CD8 + T cells were transferred into each mouse ( n = 10 mice per group). (B) Mouse survival was monitored from day 4 to 16 after virus infection. Log-rank test for comparisons of survival curves between CD8 + T-cell recipient mice and CD8 + CD25 − T-cell recipient mice; ** p < 0.01 ( n = 10 mice per group), log-rank test. (C) Lung viral loads were assayed on day 6 after virus infection ( n = 5 mice per group). (D). Lung cells taken on day 8 of infection were stimulated with PMA and ionomycin for 5 h in the presence of monensin and then stained to detect intracellular IFN-γ expression in the CD4 + and CD8 + T cells ( n = 4 mice per group). (E) Statistical analysis of IFN-γ + cells among CD4 + and CD8 + T cells (%). (F) Serum total H5N1 virus-specific IgG titers, assayed by ELISA on day 8 post virus infection ( n = 4 mice per group). Mice receiving CD8 + CD25 − T cells served as CD8nonTreg-cell controls. Data are presented as means ± SEM and are representative of three independent experiments. * p < 0.05. ** p < 0.01. *** p < 0.001, unpaired two-tailed t -test.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: In Vivo, Isolation, Infection, Virus, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: CD8 + CD25 − T-cell proliferation was inhibited by CD8 + Treg cells through IL-10 in vitro. (A) CD8 + CD25 + T cells, CD8 + CD25 − T cells, CD11c + cells were isolated on day 6 from spleens of H5N1-infected C57BL/6 mice. CFSE-stained CD8 + CD25 − T cells were stimulated to proliferate in vitro; 2 × 10 5 CD8 + CD25 − T cells and 5 × 10 4 CD11c + cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of different numbers of CD8 + Treg cells for 5 days. (B) CD4 + CD25 + T cells, CD8 + CD25 + T cells, CD8 + CD25 − T cells, and CD11c + cells were isolated from spleens of C57BL/6 mice on day 6 after infection with H5N1 virus. CFSE-stained CD8 + CD25 − T cells were stimulated to proliferate in vitro; 2 × 10 5 CD8 + CD25 − T cells and 5 × 10 4 CD11c + cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of different numbers of CD4 + or CD8 + Treg cells for 5 days. (C) 2 × 10 5 CD8 + CD25 − T cells and 5 × 10 4 CD11c + cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of 1 × 10 5 CD8 + Treg cells in transwell plates for 5 days. (D) T-cell proliferation was done with 50 μg/mL anti-IL-10 mAb or isotype control antibodies in the wells. The number of CD8 + Treg cells used in the system (C and D) was 1 × 10 5 . (E) CD8 + CD25 + T cells and CD11c + cells from C57BL/6 mice and CD8 + CD25 − T cells from DNIL-10R mice were isolated 6 days after infection with H5N1 virus and T-cell proliferation assays were performed. (A–E) Data shown are representative of at least three independent experiments with four mice per group.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: In Vitro, Isolation, Infection, Staining, Virus, Control

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: The antiviral activity of CD8 + CD25 − T cells was inhibited by CD8 + Treg cells in vivo through IL-10. (A) CD8 + CD25 + T cells and CD8 + CD25 − T cells from the spleens of WT mice and CD8 + CD25 − T cells from the spleens of DNIL-10R mice were isolated on day 6 after H5N1 infection and transferred into CD8 KO mice (5 × 10 5 CD8 + CD25 + T cells and 1 × 10 7 CD8 + CD25 − T cells per mouse) that were then immediately infected with H5N1 virus (day 0 of challenge). (B) Survival of mice ( n = 10 per group) was monitored from day 4 to 16 after virus infection. Log-rank test for comparisons of survival curves between DNIL-10R CD8 + CD25 − T cells recipient mice and DNIL-10R CD8 + CD25 − T cells plus WT CD8 + Treg cells recipient mice, or between DNIL-10R CD8 + CD25 − T cells plus WT CD8 + Treg cells recipient mice and WT CD8 + CD25 − T cells plus WT CD8 + Treg cells recipient mice; n.s., p > 0.05. **, p < 0.01, log-rank test. (C) Viral load in mouse lungs were assayed on day 6 after virus infection ( n = 5 mice per group). Data are presented as means ± SEM and are representative of four independent experiments. n.s., p > 0.05. * p < 0.05, unpaired two-tailed t -test.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Activity Assay, In Vivo, Isolation, Infection, Virus, Two Tailed Test