Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Maturation of DCs in vCP172-infected cultures. (A) Immature human DCs were infected with vCP172 (MOI of 10). Infected (+) and uninfected (−) DCs were cultured for 4 days. After culture, the cells were harvested and monitored for the expression of CD25-, CD83-, and CD86-PE (log PE y axes) versus HLA-DR–FITC (x axes). The percentage of large cells expressing CD25 and CD83 above the isotype control are indicated. (B) Immature rhesus macaque DCs were infected with vCP172 (+) (MOI of 10) or not (−) and cultured for 1 to 3 days before being harvested and analyzed. FACS analysis was performed on cells stained with FITC–anti-HLA-DR versus PE-immunoglobulin, -anti-CD25, -CD80, -CD83, or -CD86. Similar data were obtained from more than five experiments with human DCs and three different monkey donors.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Cell Culture, Expressing, Staining

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Induction of maturation is stimulated by viable canarypox virus. (A) Immature human DCs were infected with an MOI of 10 of either vCP180 or the parental strain (ALVAC) or left uninfected (medium). CD25 surface expression by large HLA-DR-positive cells was assessed 4 days after infection (CD25-PE, y axes; HLA-DR–FITC, x axes). The percentage of CD25-positive cells (above isotype control) are indicated in each panel (highlighted by arrowheads). (B) Immature human DCs that had been infected with vCP180 3 to 4 days earlier were sorted into CD25-negative (CD25 neg.) and CD25-positive (CD25 pos.) fractions. Each fraction was then immunostained for intracellular expression of p27 and analyzed by FACS. The percentage of SIV p27-positive cells, above the immunoglobulin control, is shown for each subset. (C) Immature DCs (human) were infected with live (ALVAC) or heat-inactivated (H.I.) ALVAC or left untreated (medium). After 4 days the DCs were examined for CD25 expression by FACS. The percentages of CD25-positive large cells (compared to the isotype control) are shown in each panel. The CD25-positive subset is highlighted by an arrowhead.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Expressing

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Inhibition of maturation of ALVAC-infected DCs by the addition of a caspase 3 inhibitor. A total of 250 or 500 μM Z-DEVD-FMK, or the equivalent dilution of DMSO diluent for the high dose (DMSO), were added to immature DCs 30 min before infection with ALVAC. Inhibition of maturation was assessed by the lack of CD25 expression 4 days after infection using the DMSO-treated cells as the 100% matured population. The data represent the mean percentages of CD25-expression (% CD25 pos.) of three experiments.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Inhibition, Infection, Expressing

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Maturation of uninfected DCs by ALVAC-infected immature DCs. Immature DCs were infected with ALVAC, and free virus was washed out. Uninfected immature DCs that had been stained with the green fluorescent dye CMFDA were added to the infected (unstained) DCs at a ratio of 1:1 (Inf. DCs). As controls, the supernatant from infected cells (collected directly after washing off the virus) was added to CMFDA-stained cells (Sup't) or the green-uninfected cells were kept in medium (Medium). Maturation (highlighted by arrowheads) was assessed by CD25 expression 4 days later. The log PE is expressed on the y axes (CD25 versus the IgG control), and the CMFDA fluorescence intensity is expressed on the x axes. The results from one of two similar experiments are provided.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Staining, Expressing, Fluorescence

Journal: Journal of Inflammation Research

Article Title: Cerium Oxide-Loaded Exosomes Derived From Regulatory T Cells Ameliorate Inflammatory Bowel Disease by Scavenging Reactive Oxygen Species and Modulating the Inflammatory Response

doi: 10.2147/JIR.S502388

Figure Lengend Snippet: Synthesis and characterization of Treg-exo@nCeO. ( A ) TEM images of nCeO, scale bars: 10 nm. ( B ) EDS spectrum of nCeO. ( C ) Appearance images of nCeO. ( D – F ) hydrogen peroxide and free radical scavenging assay, n=3; *p < 0.05 vs 0 μg/mL, **p < 0.01 vs 0 μg/mL, ns, no significance vs 0 μg/mL. ( G ) Flow chart of the isolation of CD4+CD25+ T cells. ( H ) Relative proportion of living cells, n=3; **p < 0.01 vs 0 μg/mL, ns, no significance vs 0 μg/mL. ( I ) Uptaken Cy5-labeled nCeO and fluorescence imaging, scale bars: 2 μm. ( J ) NTA of exo and exo@nCeO. ( K ) Western blot assay of the expression of Calnexin, Tsg101 and CD63 in Tregs and exo. ( L ) TEM images and average size of exo and exo@nCeO, n=3, scale bars: 100 nm. ( M ) Uptaken Dio-exo@Cy5-nCeO and fluorescence imaging, scale bars: 5 μm.

Article Snippet: 1×10 6 Tregs were collected and stained with phycoerythrin (PE) monoclonal antibodies against mouse CD25 (E-AB-F1102D, Elabscience, China) and fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies against mouse CD4 (E-AB-F1097C, Elabscience, China) for 30 min, followed by data collection on a flow cytometer (Beckman Coulter, USA).

Techniques: Isolation, Labeling, Fluorescence, Imaging, Western Blot, Expressing

Journal: Frontiers in Physiology

Article Title: Regulatory T Cells as a Novel Candidate for Cell-Based Therapy in Kidney Disease

doi: 10.3389/fphys.2020.00621

Figure Lengend Snippet: (A) Histology of kidney with Adriamycin-induced nephropathy showed severe structural injury, in contrast to healthy control kidney. (B–D) Serum creatinine, urine protein, and creatinine clearance were assessed in control mice and mice with Adriamycin-induced nephropathy. (E) Dot plots from flow cytometry showing numbers of CD4+ CD25+ Tregs isolated from control kidney and kidney with Adriamycin-induced nephropathy. Percentages of cells are indicated within each quadrant. LL, lower left; LR, lower right; Q, quadrant; UL, upper left; UR, upper right.

Article Snippet: The surface of CD3+ cells was labeled with antibodies against CD25 and FOXP3 (BD Accuri C6, United States).

Techniques: Flow Cytometry, Isolation

Journal: Frontiers in Physiology

Article Title: Regulatory T Cells as a Novel Candidate for Cell-Based Therapy in Kidney Disease

doi: 10.3389/fphys.2020.00621

Figure Lengend Snippet: (A) Dot plots from flow cytometry showing numbers of CD4+ CD25+ Tregs isolated from spleens of healthy mice. (B) Increase in Treg numbers after in vitro amplification. (C) Dot plots from flow cytometry showing purity of CD4+ CD25+ Tregs after amplification. (D) Flow cytometry histogram depicting Treg numbers isolated from mice with Adriamycin-induced nephropathy in which Tregs from healthy donors had been adoptively transferred. (E) Fluorescence images of kidneys from control mice, mice with Adriamycin-induced nephropathy, and mice with nephropathy after treatment with Tregs from healthy donors. (F) Histological examination suggested that kidney with AN treated with Tregs show less damage than AN. AN, Adriamycin-induced nephropathy.

Article Snippet: The surface of CD3+ cells was labeled with antibodies against CD25 and FOXP3 (BD Accuri C6, United States).

Techniques: Flow Cytometry, Isolation, In Vitro, Amplification, Fluorescence

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: International Journal of Cell Biology

Article Title: Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

doi: 10.1155/2015/614297

Figure Lengend Snippet: CD44 cross-linking and IL-2 both promote Foxp3 expression and Treg persistence despite CSA treatment. (a) Representative flow cytometric analysis of CD25 labeled and GFP/Foxp3+ cells after 3 days in culture in the presence of anti-CD3 and anti-CD28 alone or with the addition of anti-CD44, and with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). (b) Fold increase (FI) in GFP/Foxp3 MFI after 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or in conjunction with anti-CD44 Ab, with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 independent experiments, among these are included . (c) Fold Increase in GFP/FoxP3 MFI in the presence of anti-CD3 and anti-CD28 alone, or in conjunction with anti-CD44 and increasing concentrations of CSA. Data are representative of two experiments. (d) Fold increase in the fraction of viable GFP/FoxP3+ cells (Annexin V-, 7AAD-) upon culture with aCD3/28 or aCD3/28/44 with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 experiments among these are included in and the other experiments are in .

Article Snippet: Where indicated, the following reagents were incubated at 37°C in a CO 2 incubator with the cells 30 minutes prior to cross-linking CD44: CSA (50 ng/mL or at concentrations indicated) and neutralizing Ab against CD25 (100 μ g/mL; R&D Systems, Cat number AB-223-NA).

Techniques: Expressing, Labeling

Journal: International Journal of Cell Biology

Article Title: Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

doi: 10.1155/2015/614297

Figure Lengend Snippet: CD44 cross-linking promotes Foxp3 expression in an IL-2-independent manner . (a) Fold Increase (FI) in GFP/FoxP3 MFI for Treg activated with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab with or without anti-IL-2 Ab (Anti-IL-2), recombinant CD25 (rCD25), or IL-2 ( n = 7). (b) Representative histograms demonstrating GFP/Foxp3 expression by Treg isolated from GFP/Foxp3 knock-in mice on a conventional B6 background mice or on a CD25 −/− background (B6 GFP/Foxp3.CD25 −/− mice) following 3 days of culture with anti-CD3 and anti-28 alone or in conjunction with plate-bound anti-CD44. (c) Representative histograms illustrating GFP/FoxP3 expression of Treg following 3 days of culture with anti-CD3 and anti-CD28 alone, or in conjunction with plate-bound CD44 Ab, and with or without varying doses of IL-2. Data are representative of two experiments.

Article Snippet: Where indicated, the following reagents were incubated at 37°C in a CO 2 incubator with the cells 30 minutes prior to cross-linking CD44: CSA (50 ng/mL or at concentrations indicated) and neutralizing Ab against CD25 (100 μ g/mL; R&D Systems, Cat number AB-223-NA).

Techniques: Expressing, Recombinant, Isolation, Knock-In

Journal: International Journal of Cell Biology

Article Title: Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

doi: 10.1155/2015/614297

Figure Lengend Snippet: Inhibition of HA synthesis impairs Treg homeostasis which can be overcome with exogenous IL-2 or CD44-cross-linking . (a) Representative histograms of GFP/FoxP3 expression of Treg following 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or with IL-2, CD44 cross-linking, or exogenous plate-bound HA. N = 3 independent experiments. (b) Representative FACS plots illustrating GFP/Foxp3 and CD25 expression on Day 0 immediately following isolation of CD4+GFP/Foxp3+ Treg from murine splenocytes and following 3 days of culture with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab, the HA synthesis inhibitor 4-MU, and/or IL-2. (c) Fold change in GFP/Foxp3 MFI for the same conditions as in (b), here for N = 3 independent experiments. (d) Viability (the percentage of GFP/Foxp3+ cells negative for 7AAD and Annexin V) for Treg cultured in the setting of either DMSO or 4MU.

Article Snippet: Where indicated, the following reagents were incubated at 37°C in a CO 2 incubator with the cells 30 minutes prior to cross-linking CD44: CSA (50 ng/mL or at concentrations indicated) and neutralizing Ab against CD25 (100 μ g/mL; R&D Systems, Cat number AB-223-NA).

Techniques: Inhibition, Expressing, Isolation, Cell Culture

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: The antibodies against mouse CD25 (clone PC61 and clone 7D4) were produced at Evitria and Absolute Antibody as mIgG2a.

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: The antibodies against mouse CD25 (clone PC61 and clone 7D4) were produced at Evitria and Absolute Antibody as mIgG2a.

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: The antibodies against mouse CD25 (clone PC61 and clone 7D4) were produced at Evitria and Absolute Antibody as mIgG2a.

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: The antibodies against mouse CD25 (clone PC61 and clone 7D4) were produced at Evitria and Absolute Antibody as mIgG2a.

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: 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: The antibodies against mouse CD25 (clone PC61 and clone 7D4) were produced at Evitria and Absolute Antibody as mIgG2a.

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

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) RG6292 did not block IL-2 signaling in a pSTAT5 assay using human PBMCs. Daclizumab was used as blocking control, human IgG1 isotype or untreated samples as reference control. Cells were incubated with 10 μg/ml antibody followed by 10 U/ml IL-2 for 10 min. pSTAT5 was analyzed on CD4 + , CD8 + and Treg cells. Shown is the mean of technical duplicates with SD. (B) RG6292 did not negatively impact Granzyme B induction and proliferation of αCD3/αCD28 activated Pan T cells, other than addition of antibodies either neutralizing IL-2 (αIL-2nAB) or blocking binding of IL2 to CD25 (Daclizumab). Antibodies were tested for 72 hours at 10 μg/ml. Shown is the mean of technical triplicates with SD. (C) The Fab fragment of RG6292 binds CD25 opposite of the IL2-CD25 interaction site. Shown is the superposition of the Fab RG6292 - CD25 structure with the IL2 quaternary signaling complex structure (PDB accession code 2B5I) . The overlay was prepared using all atoms of CD25. IL2Rβ and IL2Rγ of the quaternary complex are shown as white transparent surface whereas IL2 is highlighted in magenta and CD25 in yellow/ red. The Fab light and heavy chains of RG6292 are colored in cyan and blue, respectively. The close-up section provides a detailed view onto the epitope and paratope area with residues labeled contributing to the interface. Further details are provided in the and . (D) RG6292 mediated mostly depletion of CD25 high expressing regulatory T cells and not that of activated CD8 and CD4 effector T cells when present during polyclonal activation (αCD3 coated beads) of human PBMC. The counts of activated regulatory and non-regulatory CD4 + and CD8 + T cells within the PBMC sample were quantified on day 3 by flow cytometry and lysis by PBMC endogenous FcR+ cells was calculated. Shown is the mean of technical triplicates with SEM. (E) CD25 staining was performed to confirm target expression on Treg cells (CD4 + FoxP3 + CFSE high), activated CD4 (CD4 + FoxP3 - CD69 + ) and CD8 T cells (CD8 + CD69 + ) shown in (D) . (F and G) Graphs showing the killing of FoxP3 + CD25 + cells within human tumors (frozen dissociated tumor cells obtained from Conversant Bio) supplemented with allogeneic NK cells mediated by RG6292 (and the fully fucosylated (FF) or Fc silent version of RG6292). Shown is the mean of technical duplicates with SD. All quantification plots: 2-way ANOVA, Sidak’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: The antibodies against mouse CD25 (clone PC61 and clone 7D4) were produced at Evitria and Absolute Antibody as mIgG2a.

Techniques: Blocking Assay, Incubation, Binding Assay, Labeling, Expressing, Activation Assay, Flow Cytometry, Lysis, Staining

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: Stem cell humanized female NOG mice bearing an established s.c. BxPC-3 tumor were injected i.p. with vehicle, RG6292 [4 mg/kg] or Ipilimumab [10 mg/kg]. After 72 hrs, splenocytes, blood lymphocytes and tumor infiltrating lymphocytes were isolated and evaluated for counts of activated CD8 + T cells (huCD45 + , huCD3 + , huCD8 + huCTLA-4 + ) and Tregs (huCD45 + , huCD3 + , huCD4 + , huFoxP3 + ) as well as for markers of recent T cell activation. (A) Ipilimumab as well as RG6292 decreased the intratumoral Treg counts. An increase of intratumoral activated CD8 + T cell count was only evident after administration of RG6292. Normalized counts were plotted for the respective treatment groups. Each symbol represents one animal (n=5 mice), CD8 and Treg cells are connected for the same animals (B) Intratumoral CD8 + T cells after RG6292 treatment were highly activated and had increased levels of HLA-DR, PD-1 and CTLA-4 (MFI as well as % of positive cells). Each symbol represents one animal (n=5 mice). The box and whiskers plots show minima and maxima and the median. Statistical analysis of RG6292 and Ipilimumab treated groups against vehicle group is indicated. Data was analyzed using 2-way ANOVA, Dunnet’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) (p-value between RG6292 and Ipilimumab was 0.0001 for CTLA4 MFI on CD8 T cells and 0.0008 for HLA-DR on CD8 T cells. (C) Representative FACS plots showing CD25 expression versus FoxP3 expression in CD4 + T cells and PD-1 expression versus CTLA-4 expression in CD8 + T cells for vehicle, RG6292 and Ipilimumab treated animals.

Article Snippet: The antibodies against mouse CD25 (clone PC61 and clone 7D4) were produced at Evitria and Absolute Antibody as mIgG2a.

Techniques: Injection, Isolation, Activation Assay, Cell Counting, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CD45RA - Foxp3 high but not CD45RA + Foxp3 low suppressive T regulatory cells increased in the peripheral circulation of patients with head and neck squamous cell carcinoma and correlated with tumor progression

doi: 10.1186/1756-9966-33-35

Figure Lengend Snippet: Percentage of Treg subsets in 112 HNSCC patients. (A) Gating strategy used is illustrated. (B) Flow dot plots of Foxp3 + CD25 + Tregs for one representative HD (left) and HNSCC patient (middle). Percentage (means ± SD) of Foxp3 + CD25 + Tregs in HNSCC patients or HD (right). (C) Flow dot plots of each Treg subset (I: CD45RA + Foxp3 low Tregs; II: CD45RA - Foxp3 high Tregs; III: CD45RA - Foxp3 low CD4 + T cells) for one representative HD (left) and HNSCC patient (right). (D) Percentage (means ± SD) of each Treg subset in HNSCC patients or HD. HNSCC: head and neck squamous cell carcinoma. HD: healthy donors. Statistical comparisons were performed using the Mann–Whitney U-test.

Article Snippet: Stained cells (mAbs against CD3, CD4, CD25, and CD45RA) at a concentration of 5 × 10 7 cells/100 μl were sorted using a FACS cell sorter (BD Influx, BD Biosciences).

Techniques: MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CD45RA - Foxp3 high but not CD45RA + Foxp3 low suppressive T regulatory cells increased in the peripheral circulation of patients with head and neck squamous cell carcinoma and correlated with tumor progression

doi: 10.1186/1756-9966-33-35

Figure Lengend Snippet: Percentage of Treg subsets in HNSCC patient subgroups. (A) Flow dot plots of Tregs (Foxp3 low and Foxp3 high Tregs) (top) and each Treg subset (I: CD45RA + Foxp3 low Tregs; II: CD45RA - Foxp3 high Tregs; III: CD45RA - Foxp3 low CD4 + T cells) (bottom) for one representative HD and patients with HPSCC, NPSCC, OPSCC, and LSCC. (B) Percentage (means ± SD) of Tregs and each Treg subset in HNSCC patient subgroups or HD. (C) Different proportions (means) of each Treg subset in HNSCC patient subgroups are presented. HD: healthy donors. OCSCC: oral squamous cell carcinoma. HPSCC: hypopharyngeal squamous cell carcinoma. NPSCC: nasopharyngeal squamous cell carcinoma. OPSCC: oropharyngeal squamous cell carcinoma. LSCC: laryngeal squanmous cell carcinoma. Statistical comparisons were performed using the Kruskal–Wallis test.

Article Snippet: Stained cells (mAbs against CD3, CD4, CD25, and CD45RA) at a concentration of 5 × 10 7 cells/100 μl were sorted using a FACS cell sorter (BD Influx, BD Biosciences).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CD45RA - Foxp3 high but not CD45RA + Foxp3 low suppressive T regulatory cells increased in the peripheral circulation of patients with head and neck squamous cell carcinoma and correlated with tumor progression

doi: 10.1186/1756-9966-33-35

Figure Lengend Snippet: Relationship between Treg subsets and tumor progression

Article Snippet: Stained cells (mAbs against CD3, CD4, CD25, and CD45RA) at a concentration of 5 × 10 7 cells/100 μl were sorted using a FACS cell sorter (BD Influx, BD Biosciences).

Techniques: