Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: PBF-509 is specific for the A2aR. (A) Dose response curves of PBF-509 versus 4 adenosine receptors in transfected CHO (hA1), HeLa (hA2a and hA3), and HEK-293 (hA2b) cells. (B) Representative dose–response curves of PBF-509 in a binding assay against the human A2aR in buffer (median Ki = 12 nM) and in human plasma (median Ki = 32 nM).

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Transfection, Binding Assay, Clinical Proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: PBF-509 inhibits accumulation of cAMP. CHO cells transfected with the A2aR were used to determine the concentration of PBF-509 to reduce the concentration of cAMP by 50%.

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Transfection, Concentration Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: Human primary tumor, CAF, and TIL cell lines express CD73 and A2aR. Tumor (Tm), cancer-associated fibroblast (CAF), and tumor infiltrating lymphocyte (TIL) cell lines were established from 12 resected NSCLC tumors. (A) Tumor cells or CAFs were stained with anti-CD73 and subjected to flow cytometry. Most cells from nearly all of the cell lines (both tumor cells and CAFs) expressed high levels of CD73. (B) Representative experiment of tumor cells stained with anti-CD73 (blue) or isotype control (green). (C) CD4+ or CD8+ TIL cells were stained with anti-A2aR and subjected to flow cytometric analysis showing that high levels of A2aR were being expressed in CD4+ cells, with variable expression in CD8+ cells.

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Staining, Flow Cytometry, Control, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: Anti-PD-L1 and PBF-509 restore immune responsiveness of TILs. Four different resected NSCLC tumors were disaggregated and cultured with or without anti-PD-L1 (aPDL1; 10 mg/ml), anti-PD-1 (aPD1; 10 μg/ml), or the A2aR antagonist PBF-509 (1 μM). After 3 days in culture, the supernatants were assayed for the presence of γ-interferon using an ELISA.

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Baseline expression level of inhibitory receptors in T cells of patients with EOC and healthy controls. The baseline percentage of TIM3+ (A) , PD1+ (B) , and A2aR+ (C) cells are significantly higher in EOC patient-derived T cells post thawing. The frequency of T tex cells is significantly higher in patients’ samples compared to normal controls (D) . However, the frequency of T pex cells is not significant different between patients and healthy samples (D). Representative overlay histogram plots representing expression level of TIM3 (A), PD1 (B), and A2aR (C) in EOC patient-derived T cells sample (Red color) vs. unstained sample (Blue color). Representative overlay dot plot (D) representing the frequency of terminally exhausted and progenitor exhausted T cells in an EOC patient sample (Red color) vs. unstained sample (Blue color). Data from healthy donors (n = 30) and EOC patients (n = 30) were analyzed using Welch’s t-test (A and B) Mann-Whitney U test (C and D). * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Derivative Assay, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Expression pattern of inhibitory receptors during CAR T manufacturing process. CAR T cell population positive for TIM3 (A) , PD1 (B) , and A2aR (C) in EOC patient-derived T cells during CAR T cell manufacturing steps. The frequency of T pex (D) and T tex (E) cells during manufacturing steps of CAR T cells derived from EOC patient T cells. Median fluorescence intensity (MFI) of A2aR (F) , TIM3 (G) , and PD1 (H) in EOC patient-derived T cells during CAR T cells manufacturing process. Healthy donors (n = 30) and EOC patients (n = 30) data were analyzed using RM one-way ANOVA and Tukey multiple comparison test. * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Derivative Assay, Fluorescence, Comparison

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Expression pattern of inhibitory receptors in patient-derived CAR T cells. CAR T cell population positive for TIM3 (A) , PD1 (B) , and A2aR (C) in EOC patient-derived T cells after activation and transduction vs. healthy controls. The frequency of T tex (D) and T pex (E) cells after activation and transduction of EOC patient-derived T cells vs. healthy controls. Data from healthy donors (n = 30) and EOC patients (n = 30) were analyzed using Mann-Whitney U test (A, B, and D) and Welch’s t-test (C and E). * P < 0.05; **P < 0.01; ***P < 0.001. Data are represented by mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Derivative Assay, Activation Assay, Transduction, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Differential expression of immune inhibitory receptors in different cancer stages during CAR T cell manufacturing. Cell surface expression levels of TIM3 (A) , PD1 (B) , A2aR (C) , and the frequency of T pex (D) and T tex (E) cells at different steps of CAR T cell manufacturing process for different stages of EOC. Data from stage III patients (n = 18) and stage IV patients (n = 12) were analyzed using Welch’s t-test. * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented by mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Quantitative Proteomics, Expressing

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Effect of CarboTaxol treatment on T cell expansion, frequency, and expression pattern of immune inhibitory receptors during CAR T cell manufacturing process. Fold increase of T cells from CarboTaxol-treated and untreated patients post T cell activation (A) . CD3 expression after T cell activation in CarboTaxol treated and newly diagnosed patients (B) . The percentage of A2aR (C) , TIM3 (D) , PD1 (E) positive cells, and the frequency of T tex (F) and T pex cells (G) during CAR T cell manufacturing process in CarboTaxol-treated and newly diagnosed patients. Data from CarboTaxol-treated patients (n = 20) and new patients (n = 10) were analyzed using Welch’s t-test. * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented by mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Activation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Preventing ATP Degradation by ASO-Mediated Knockdown of CD39 and CD73 Results in A2aR-Independent Rescue of T Cell Proliferation

doi: 10.1016/j.omtn.2020.06.020

Figure Lengend Snippet: Impact of A2aR Inhibition on T Cell Proliferation in the Presence or Absence of Extracellular ATP Human T cells were labeled with a proliferation dye, activated with anti-CD2/CD3/CD28 tetrameric antibody complexes, and treated with 5 μM of respective ASOs (or 2.5 μM in the case of the CD39 ASO + CD73 ASO condition) without using a transfection reagent. As mock control, cells were not treated with an ASO. On days 3 and 4, 10 μM of A2a receptor inhibitors (A2aRIs) AZD-4635 or CPI-444, dissolved in DMSO, or DMSO and different doses of ATP, was added to the cells. Proliferation of T cells was analyzed by flow cytometry on day 5 (A), and proliferation indices of CD8 + and CD4 + T cells were calculated (B). (A) Representative results from three technical replicates and two independent experiments are shown. (B) The mean + SD from three technical replicates is depicted. Representative results from two independent experiments are shown.

Article Snippet: Subsequently, T cells were spun down at 700 × g for 1 min and washed in fluorescence-activated cell sorting (FACS) buffer (1× PBS, 2% FBS; Thermo Fisher Scientific), followed by incubation for 30 min at 4°C in 50 μL FACS buffer per well in 96-well U-bottom plates containing the respective antibodies (anti-human CD8 allophycocyanin [APC]; Thermo Fisher Scientific; 17-0088-42, clone RPA-T81, 1:200 dilution), anti-human CD4 BV510 (BioLegend; 317444, clone OKT4, 1:200 dilution), anti-human CD39 phycoerythrin (PE)-cyanine 7 (Cy7) (BioLegend; 328212, clone A11:200 dilution), anti-human CD73 fluorescein isothiocyanate (FITC) (BioLegend; 344016, clone AD2, 1:200 dilution), anti-human adenosine A2aR PE (Santa Cruz Biotechnology; sc-32261 PE, clone 7F6-G5-A2, 1:10 dilution), anti-human CD8a BV785 (BioLegend; 301046, clone RPA-T8, 1:200 dilution), goat anti-human adenosine A2bR (Sigma-Aldrich; SAB2500030-100UG, polyclonal, 1:50 dilution), donkey anti-goat immunoglobulin G (IgG) Alexa Flour 647 (Invitrogen; A32849, polyclonal, 1:50 dilution), anti-human CD4 PE (BioLegend; 344605, clone SK3, 1:100 dilution), anti-human CD25 BV785 (BioLegend; 302638, clone BC96, 1:100 dilution), and 7-AAD viability staining solution (BioLegend; 420404, 1:50 dilution) or eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific; 65-0865-14, according to the manufacturer’s instructions).

Techniques: Inhibition, Labeling, Transfection, Control, Flow Cytometry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Preventing ATP Degradation by ASO-Mediated Knockdown of CD39 and CD73 Results in A2aR-Independent Rescue of T Cell Proliferation

doi: 10.1016/j.omtn.2020.06.020

Figure Lengend Snippet: Influence of Inhibition of Different Components of the Purine Metabolism on T Cell Proliferation in the Presence of Extracellular ATP (A) Schematic illustration of the different components inhibited. (B–D) Human T cells were labeled with a proliferation dye and activated with anti-CD2/CD3/CD28 tetrameric antibody complexes. On day 3, (B) A2aR inhibitors AZD-4635 or CPI-444 (10 μM), A2bR inhibitor PSB-603 (10 μM), ENT inhibitor NBTI (20 μM), CNT inhibitor Phloridzin (20 μM), ADA inhibitor 2′-deoxycoformycin (dCF) (10 μM), PNP inhibitor 8-aminoguanosin (100 μM), AK inhibitor A-dAdo (10 μM), or (D) RNR inhibitor COH29 (at indicated concentrations) and (B–D) 400 μM ATP were added to the cells. Proliferation of T cells was analyzed by flow cytometry on day 5, and proliferation indices were calculated (B and D). (C) Proliferation of mock-treated cells and NBTI- or A-dAdo-treated cells in the absence or presence of ATP. (B and D) The mean + SD from three technical replicates is depicted. Representative results from three independent experiments are shown. (C) Representative results from three technical replicates and three independent experiments are shown.

Article Snippet: Subsequently, T cells were spun down at 700 × g for 1 min and washed in fluorescence-activated cell sorting (FACS) buffer (1× PBS, 2% FBS; Thermo Fisher Scientific), followed by incubation for 30 min at 4°C in 50 μL FACS buffer per well in 96-well U-bottom plates containing the respective antibodies (anti-human CD8 allophycocyanin [APC]; Thermo Fisher Scientific; 17-0088-42, clone RPA-T81, 1:200 dilution), anti-human CD4 BV510 (BioLegend; 317444, clone OKT4, 1:200 dilution), anti-human CD39 phycoerythrin (PE)-cyanine 7 (Cy7) (BioLegend; 328212, clone A11:200 dilution), anti-human CD73 fluorescein isothiocyanate (FITC) (BioLegend; 344016, clone AD2, 1:200 dilution), anti-human adenosine A2aR PE (Santa Cruz Biotechnology; sc-32261 PE, clone 7F6-G5-A2, 1:10 dilution), anti-human CD8a BV785 (BioLegend; 301046, clone RPA-T8, 1:200 dilution), goat anti-human adenosine A2bR (Sigma-Aldrich; SAB2500030-100UG, polyclonal, 1:50 dilution), donkey anti-goat immunoglobulin G (IgG) Alexa Flour 647 (Invitrogen; A32849, polyclonal, 1:50 dilution), anti-human CD4 PE (BioLegend; 344605, clone SK3, 1:100 dilution), anti-human CD25 BV785 (BioLegend; 302638, clone BC96, 1:100 dilution), and 7-AAD viability staining solution (BioLegend; 420404, 1:50 dilution) or eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific; 65-0865-14, according to the manufacturer’s instructions).

Techniques: Inhibition, Labeling, Flow Cytometry

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 1. Development of spontaneous and carcinogen-induced HCC is enhanced in A2AR-deficient mice (A) Experimental setting: WT and A2AR-deficient males were aged to 80 weeks old. Representative pictures of spontaneous HCC observed in WT and A2AR- deficient males are shown. Scale bar represents 2 cm. (B) HCC incidence (observation of macroscopic lesions) in 80-week-old WT and A2AKO males. (C) Hematoxylin and eosin staining of cancerous livers collected from A2AKO mice depicting the different stages of HCC: top panel, stages 1 and 2, and bottom panel, stages 3 and 4; scale bar represents 50 mm. An asterisk (*) indicates trabeculae that are present in stage II–IV mouse liver tumors. (D) Experimental setting: WT and A2AR-deficient pups were treated with DMBA, 3 to 4 days after birth, and aged until 40 weeks old.

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques: Staining

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 2. Loss of A2AR signaling triggers spontaneous obesity, NASH, and systemic inflammation (A) Weight gain of WT, A2A/, and A2A+/ mice (males) was monitored weekly starting from week 4 to 32 weeks old. (B) Body composition of 24-week-old WT and A2AKO males. (C) Representative picture of 32-week-old WT and A2AKO mice showing fat accumulation in A2AKO animals.

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques:

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 3. Myeloid- and hepatocyte-specific deletions of A2AR signaling promotes DMBA-induced HCC development (A) Experimental setting: lyzM-Cre+/ A2ARflox/flox

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques:

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 4. Exacerbated development of HCC in A2AR-deficient mice is TNF-a, IL-1b, and IL-17A dependent (A) Experimental setting: WT and A2AKO pups were treated with DMBA. At week 20, A2AKO mice were split into 5 groups receiving isotype control antibody or neutralizing monoclonal antibody (mAb) against TNF-a, IL-1b, IL-17A, and IL-6. Mice were sacrificed at 40 weeks old to evaluate tumor burden. (B) Representative pictures showing livers of 40-week-old A2AR-deficient mice treated with isotype control or anti-cytokine neutralizing mAbs. (C) Mice weights at 40 weeks old. (D) Total liver tumor burden. (E) Tumor incidence. (F) Number of macroscopic nodules. Results are representative of 1 biological replicate, and sample size is indicated for each panel. Data are presented as means ± SEM. Statistical tests: multiple t tests with Benjamin-Hochberg multiple comparison correction (C), Kruskal-Wallis with Dunn’s correction (D and F), or chi-squared test (E).

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques: Control, Comparison

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 5. Pharmacological blockade of A2AR signaling promotes HFD-induced obesity and myeloid-derived liver inflammation (A) Growth curve. WT male mice were fed with a CD-HFD and treated daily with KW6002 (10 mg/kg p.o.), CGS21680 (0.5 mg/kg i.p.), or vehicle for a total of 8 weeks. Mice weight was monitored every week. (B) After 8 weeks of treatment, fat mass was measured using an EchoMRI. (C) Growth curve of male C57BL/6 mice fed with a CD-HFD and treated daily with NIR178 (10 mg/kg p.o.) or vehicle. (D) Fat mass measurement after 8 weeks of treatment with NIR178 using an EchoMRI.

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques: Derivative Assay, Mass Measurement

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 6. Diet-induced steatohepatitis im- pairs the anti-HCC activity of A2AR inhibi- tion (A) Experimental design of the hydrodynamic tail- vein HCC model. (B) Representative pictures of livers collected on mice treated with KW6002 or vehicle subjected to regular chow diet or CD-HFD feeding. (C) Tumor incidence as determined by visualization of macroscopic nodule on livers. (D) Total tumor burden on livers. (E) Liver-to-body weight ratio as a surrogate of total tumor burden. (F) Experimental design: male WT or A2AKO animals were exposed to DEN (50 mg/kg i.p.) at 3 weeks old and fed with a CD-HFD until sacrifice. From weeks 16 to 32, mice were treated daily with KW6002 (10 mg/kg p.o.) or vehicle as indicated. (G) Growth curve of the different group of mice. (H) Total liver tumor burden at sacrifice. (I) Number and size of macroscopic HCC nodules. Results are representative of 1 biological replicate, and sample size is indicated for each panel. Data are presented as means ± SEM. Statistical tests: chi-squared test (C), Mann-Whitney (D, E, H, and I), or Student’s t tests (D).

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques: Activity Assay, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: CD73 Ectonucleotidase Restrains CD8+ T Cell Metabolic Fitness and Anti-tumoral Activity

doi: 10.3389/fcell.2021.638037

Figure Lengend Snippet: Purinergic components are upregulated during in vitro activation. Naive CD8+ T cells from WT mice were isolated and activated with soluble α-CD3/CD28 antibodies for 4 days in vitro in the presence of IL-2 (10 ng/ml) for the generation of Tc1 lymphocytes. (A) Representative dot plots of CD39 and CD73 expression by in vitro -generated Tc1 cells at different time points. (B,C) Representative graph showing the concatenation and MFI of CD73 (B) and CD39 (C) on T cells at different days following activation. (D) Adenosine concentration obtained from naïve cells and in vitro Tc1 cultures at days 2 and 4 following activation. (E) Adenosine production by Tc1 cells cultured for 1 h in the presence of AMP (10 μM) and APCP (50 μM). (F–H) Representative dot plots and MFI graphs showing CD73 (F) , CD39 (G) , and A2AR (H) expression as a function of Tc1 proliferation cycles (at day 3) as assessed by cell trace violet dilution by FACS. Kruskal–Wallis Test, * p < 0.05, n = 3, Data is presented as the mean ± SEM.

Article Snippet: After fixation and permeabilization, cells were incubated with anti-A2AR antibody (clone 7F6-G5-A2) from Novus Biologicals for 30 min at 4°C.

Techniques: In Vitro, Activation Assay, Isolation, Expressing, Generated, Concentration Assay, Cell Culture

Journal: Cell stem cell

Article Title: CD150 high bone marrow Tregs maintain hematopoietic stem cell quiescence and immune privilege via adenosine

doi: 10.1016/j.stem.2018.01.017

Figure Lengend Snippet: KEY RESOURCE TABLES

Article Snippet: Anti adenosine A2aR (7F6-G5-A2) , Novus biologicals , NBP1–39474AF647.

Techniques: Recombinant, Reverse Transcription, Gene Expression, Activation Assay, Saline, Red Blood Cell Lysis, Blocking Assay, Software