Journal: Cancer Immunology Research

Article Title: An IL6–Adenosine Positive Feedback Loop between CD73+ γδTregs and CAFs Promotes Tumor Progression in Human Breast Cancer

doi: 10.1158/2326-6066.cir-19-0923

Figure Lengend Snippet: Figure 3. CD73þ gdT cells are the predominant Tregs in breast cancer and exhibit direct immunosuppression via the adenosine-mediated pathway. A and B, Sorted CD73þ gdT cells, CD4þ Tregs (CD45þCD3þCD4þCD25þCD127low), CD4þCD73þ and CD8þCD73þ T cells, and CD73 gdT cells (1.0 104 cells, respectively) from tumor tissues and CD73þ gdT cells (1.0 104) from paired normal tissue were cocultured with CFSE-labeled allogeneic CD4þ T cells (5.0 104) in the presence of anti-CD3 and anti- CD28. n ¼ 5. A, CD4þ T-cell proliferation was evaluated on day 6 by flow cytometry. B, Bar diagram summarizes the percentages of proliferated CD4þ T cells (CFSElow). Data, mean SEM. n ¼ 5; , P < 0.01; , P < 0.001; paired Student t test for the analysis between CD73þ gdT cells from tumor and paired normal tissue and unpaired Student t test for the analysis among other cells from tumor tissue. C, Sorted CD73þ gdT, CD4þ Tregs, CD4þCD73þ and CD8þCD73þ T cells, and CD73 gdT cells (1.0 104 cells, respectively) from tumor tissues and CD73þ gdT cells (1.0 104) from paired normal tissue were cocultured with allogeneic CD8þ T cells (5.0 104) in the presence of anti-CD3 and anti-CD28. Concentrations of perforin in the supernatants were detected on day 6 by ELISA. Data, mean SEM. n ¼ 5; , P < 0.05; , P < 0.001; paired Student t test for the analysis between CD73þ gdT cells from tumor and paired normal tissue and unpaired Student t test for the analysis among other cells from tumor tissue. D and E, Sorted CD73þ gdT cells (1.0 104) from tumor tissues were cocultured with CFSE-labeled allogeneic CD4þ T cells (5.0 104) in the presence of anti-CD3 and anti-CD28 and pretreated with anti-CD73, an antibody cocktail, control antibody, or A2A (SCH58261) and A2B (PSB603) adenosine receptor antagonists. n ¼ 5. D, CD4þ T-cell proliferation was evaluated on day 6 by flow cytometry. n ¼ 5. E, Bar diagram summarizes the percentages of proliferated CD4þ T cells (CFSElow). Data, mean SEM. n ¼ 5; ns, no significance; , P <0.001;unpaired Student t test.mAb cocktail, the mixture of antibodies for LAG-3, CTLA-4, IL10, and TGFb; N, normal tissue; T, tumor tissue.

Article Snippet: A1 (KW3902, CAS No. 136199-02-5, 0.05 mmol/L, Tocris), A3 (MRS1220, CAS No. 183721-15-5, 0.05 mmol/L, Tocris), A2A (SCH58261, CAS No. 160098-96-4, 0.1 mmol/L, Tocris), and A2B (PSB603, CAS No. 1092351-10-4, 0.05 mmol/L, Tocris) adenosine receptor antagonists; p38 MAPK (SB203580, CAS No. 152121-47-6, 0.05 mmol/L, Abmole), ERK1/2 (SCH772984, CAS No. 942183-80-4, 0.01 mmol/L, Abmole); or JNK (SP600125, CAS No. 129-56-6, 0.05 mmol/L, Abmole) inhibitors; and anti-CD73 were added to some cultures.

Techniques: Labeling, Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Immunology Research

Article Title: An IL6–Adenosine Positive Feedback Loop between CD73+ γδTregs and CAFs Promotes Tumor Progression in Human Breast Cancer

doi: 10.1158/2326-6066.cir-19-0923

Figure Lengend Snippet: Figure 6. CD73þ gdTregs upregulate IL6 expression in CAFs via the adenosine/A2BR/p38MAPK pathway. A and B, Sorted CD73þ gdT cells from tumor tissue were cocultured with CAFs (5.0 104) in different E:T ratios (0:1, 0.1:1, 0.2:1, 0.5:1, or 1:1) in the presence of anti-CD3 and anti-CD28 for 6 days. n ¼ 5; unpaired Student t test. A, The relative mRNA expression of IL6 in CAFs was determined by RT-PCR and normalized to GAPDH. n ¼ 5; unpaired Student t test. B, Concentrations of IL6 in supernatants were detected by ELISAs. Data, mean SEM. n ¼ 5; ns, no significance; , P < 0.01; unpaired Student t test. C–E, Sorted CD73þ gdT cells from tumor tissues were cocultured with CAFs (5.0 104) in different E:T ratios (0:1, 0.2:1, or 0.5:1) in the presence of anti-CD3 and anti-CD28 for 6 days.The expression of p38 (C), ERK1/2 (D), and JNK (E) in CAFs was analyzed by flow cytometry. Bar diagrams summarize the percentages of p38, ERK1/2, and JNK in CAFs. Data, mean SEM. n ¼ 5; ns, no significance; , P < 0.01; unpaired Student t test. F and G, Sorted CD73þ gdT cells (2.5 104) from tumor tissues were cocultured with CAFs (5.0 104) in different conditions [medium, A1 (KW3902), A3 (MRS1220), A2A (SCH58261), and A2B (PSB603) adenosine receptor antagonists, p38 MAPK (SB203580), ERK1/2 (SCH772984), or JNK (SP600125) inhibitor, anti-CD73 or control antibody] in the presence of anti-CD3 and anti-CD28 for 6 days. F, The relative mRNA expression of IL6 in CAFs was determined by RT-PCR and normalized to GAPDH. G, Concentrations of IL6 in supernatants were detected by ELISAs. Data, mean SEM. n ¼ 5; ns, no significance; , P < 0.01.

Article Snippet: A1 (KW3902, CAS No. 136199-02-5, 0.05 mmol/L, Tocris), A3 (MRS1220, CAS No. 183721-15-5, 0.05 mmol/L, Tocris), A2A (SCH58261, CAS No. 160098-96-4, 0.1 mmol/L, Tocris), and A2B (PSB603, CAS No. 1092351-10-4, 0.05 mmol/L, Tocris) adenosine receptor antagonists; p38 MAPK (SB203580, CAS No. 152121-47-6, 0.05 mmol/L, Abmole), ERK1/2 (SCH772984, CAS No. 942183-80-4, 0.01 mmol/L, Abmole); or JNK (SP600125, CAS No. 129-56-6, 0.05 mmol/L, Abmole) inhibitors; and anti-CD73 were added to some cultures.

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

Journal: International Journal of Molecular Sciences

Article Title: Ivy Leaf Dry Extract EA 575 ® Has an Inhibitory Effect on the Signalling Cascade of Adenosine Receptor A 2B

doi: 10.3390/ijms241512373

Figure Lengend Snippet: Influence of EA 575 ® ( A , B ) or the antagonists PSB-603 and SCH 442416 ( C ) on the CRE activation in transiently transfected HEK cells mediated by stimulation with adenosine ( A , C ) or BAY 60-6583 ( B ). Pre-incubation with 40–240 µg/mL EA 575 ® was conducted for 16 h ( A , B ) or with 0.1–10 µM of the antagonists for 2 h ( C ) before cells were stimulated by adding 100 µM adenosine ( A , C ) or 10 µM BAY 60-6583 ( B ). The non-specifically mediated CRE activation was significantly inhibited by pre-incubation with 80–240 µg/mL EA 575 ® ( A ) or 1 µM PSB-603 ( C ), compared to stimulated control cells not pre-incubated with EA 575 ® (SC). SCH 442416 also significantly reduced the CRE activation at a concentration of 10 µM, whereas this effect could not be observed at lower concentrations of both antagonists. Instead, 0.1 µM SCH 442416 slightly increased the CRE activation ( C ). The inhibition of the specific A 2B AR-mediated CRE activation was achieved by pre-incubation with 80–240 µg/mL EA 575 ® ( B ). Influence of the A 2A AR agonist CGS 21680 and, in comparison, BAY 60-6583 and adenosine on the CRE activation in transiently transfected HEK cells ( D ). Stimulation was performed with 1–10 µM CGS 21680, 10 µM BAY 60-6583, or 100 µM adenosine. The A 2A AR-mediated CRE activation was significantly increased by 10 µM CGS 21680 compared with completely untreated control cells (UTC), but to a lesser extent than that mediated by A 2B AR or non-specifically using adenosine ( D ). Data are shown as AUC of the fold change (FC) after stimulation normalised to stimulated control cells not pre-incubated with EA 575 ® (SC) ( A – C ) or completely untreated control cells (UTC) ( D ). Results represent the mean and SEM ( n = 3 independent experiments performed in triplicate, * p < 0.05).

Article Snippet: PSB-603 and CGS 21680 were obtained from Biomol (Hamburg, Germany).

Techniques: Activation Assay, Transfection, Incubation, Concentration Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Ivy Leaf Dry Extract EA 575 ® Has an Inhibitory Effect on the Signalling Cascade of Adenosine Receptor A 2B

doi: 10.3390/ijms241512373

Figure Lengend Snippet: Influence of EA 575 ® ( A , B ) or the antagonists PSB-603 and SCH 442416 ( C ) on the IL-6 release of Calu-3 cells mediated by stimulation with adenosine ( A , C ) or BAY 60-6583 ( B ). Pre-incubation with 40–240 µg/mL EA 575 ® was conducted for 16 h ( A , B ) or with 0.1–10 µM of the antagonists for 1 h ( C ) before cells were stimulated by adding 100 µM adenosine ( A , C ) or 10 µM BAY 60-6583 ( B ) for another 24 h. The non-specifically mediated IL-6 release was significantly and dose-dependently inhibited by pre-incubation with 80–240 µg/mL EA 575 ® (A) or 1 µM PSB-603 ( C ), compared to stimulated control cells not pre-incubated with EA 575 ® (SC). SCH 442416, however, increased the concentration of IL-6 even further. ( C ). The inhibition of the specific A 2B AR-mediated IL-6 release was achieved by pre-incubation with 40–240 µg/mL EA 575 ® ( B ). Influence of the A 2A AR agonist CGS 21680 and, in comparison, BAY 60-6583 on the IL-6 release of Calu-3 cells ( D ). Stimulation was performed with 1–10 µM CGS 21680 or 10 µM BAY 60-6583 for 24 h. The A 2A AR-mediated IL-6 release was slightly, but neither significantly nor dose-dependently, increased compared to completely untreated control cells (UTC) ( D ). Results represent the mean normalised to stimulated control cells not pre-incubated with EA 575 ® (SC) ( A – C ) or completely untreated control cells (UTC) ( D ) and SEM ( n = 3 independent experiments performed in triplicate, * p < 0.05).

Article Snippet: PSB-603 and CGS 21680 were obtained from Biomol (Hamburg, Germany).

Techniques: Incubation, Concentration Assay, Inhibition