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6P Expression of PD-L1 in lung cancer cells regulates Treg cell differentiation
April 2016
Abstracts, ELCC 2016 – Tumour biology and pathology
results were statistically analyzed for two preplanned cut-off values, namely 5% and 10% of PD-L1 positivity in either TCs or ICs, regardless of the intensity. Baseline patients’ characteristics were obtained from the hospital registry database. Results: There were 29 (54%) AC and 25 (46%) SCC; the mean age was 62.4 years, 34 (63%) were male, most of them current or ex-smokers 46 (85%). TC PD-L1 positivity was significantly higher in SCC vs. AC, at both 5% and 10% cut-offs (52% vs. 17%, p = 0.016 and 52% vs. 14%, p = 0.007, respectively) while IC PD-L1 positivity was similar in SCC and AC, at both cut-offs (76% vs. 72%, p = 1 and 64% vs. 41%, p = 0.166, respectively). PD-L1 positivity was significantly higher in IC compared to TC in AD but not in SCC, regardless the cut-off value.
Treg cells in the absence of TGF-beta. However, in the presence of TGF-beta, when adding anti-PD-L1 monoclonal antibody, the differentiation of T lymphocytes into Treg cells was reduced. When TGF-beta and PD-L1 existed at the same time, the highest degree of T lymphocyte differentiation into Treg cells were observed. Conclusions: Expression of PD-L1 in lung cancer cells can coordinate TGF-beta to induce human peripheral blood T lymphocyte differentiation to Treg cells. Its role in immunosuppression may promote the occurrence and development of tumors. Legal entity responsible for the study: The Ethics Committee of The Second Affiliated Hospital of Soochow University Funding: The National Natural Science Foundation of China (No. 81272610) Disclosure: All authors have declared no conflicts of interest.
Table: PD-L1 positivity according to histology and different cutoffs N
Total AC SCC p (histology)
54 29 25
PD-L1 > 5%
PD-L1 > 10%
TC or IC N (%)
TC N (%)
IC N (%)
p (TC vs IC)
TC or IC N (%)
TC N (%)
IC N (%)
p (TC vs IC)
43 (80) 21 (72) 22 (88)
18 (33) 5 (17) 13 (52) 0.016
40 (74) 21 (72) 19 (76) 0.764
< 0.001 0.146
31 (57) 12 (41) 19 (76)
17 (31) 4 (14) 13 (52) 0.007
28 (52) 12 (41) 16 (64) 0.166
0.008 0.508
Conclusions: PD-L1 positivity is significantly higher in SCC compared to AC when looking only at TC and not at IC or combination. PD-L1 positivity in IC, which is highly positive in both histologic subtypes, adds to the overall positivity of AC but not SCC. There is no significant difference in IC PD-L1 positivity between SCC and AC, however by lowering the cut-off value the share of PD-L1 positive AC samples increases substantially, mostly due to IC positivity. Legal entity responsible for the study: University Clinic Golnik Funding: University Clinic Golnik Disclosure: All authors have declared no conflicts of interest. 6P Expression of PD-L1 in lung cancer cells regulates Treg cell differentiation M. Shi. Respiratory Diseases, 2nd Affiliated Hospital Suzhou (Soochow) University, Suzhou, China Background: Both regulatory T cells (Tregs) and the programmed cell death-1 (PD-1)/PD-L1 pathway play important roles in lung cancer pathogenesis; however, the association between these two factors remains poorly understood. Hereby, we examined PD-L1 expression in lung cancer cells, and analyzed its effect on induced Treg cells. Methods: PD-L1 expression was studied on lung cancer cells by flow cytometry. Peripheral blood mononuclear cells (PBMC) were isolated from a healthy volunteer, and then immune magnetic beads were used to sort out human peripheral blood T lymphocyte and detect separation purity. Plant blood agglutinin (PHA) was used to stimulate the peripheral blood T lymphocyte. Activated T cells were co-cultured with lung cancer cells which expressed the highest level of PD-L1, to which transforming growth factor-b (TGF-beta) was added to block PD-L1 type antibody. Then the degree of Treg cell differentiation was detected. Results: The expression of B7-H1 was detected in four human lung cancer cell lines using flow cytometry. We found that the membrane expression level of PD-L1 was the highest on the A549 cells. After PHA stimulation, T lymphocyte proliferation was activated. Then the activated T lymphocytes and A549 cells were co-cultured. Only a small part of T lymphocytes differentiated into
S59
7P Proangiogenic effect of peritoneal macrophages from animals with Lewis lung carcinoma T. Nikolaienko, D. Shelest, L. Garmanchuk, O. Dzhus, V. Nikulina. Biochemistry, Educational and Scientific Centre «Institute of Biology» of Taras Shevchenko National University of Kyiv, Kyiv, Ukraine Background: Both inflammation and angiogenesis are exacerbated by increased production of chemokines/cytokines, growth factors, proteolytic enzymes and prostaglandins. Initiation and progression of cancer are also closely linked to angiogenesis. Infiltration of macrophages is a dramatic and common feature of inflammation, angiogenesis and cancer, and has been recently highlighted in an attempt to develop novel strategies for treating cancer. The recruitment and infiltration of tumor-associated macrophages in the tumor microenvironment activates them to support the malignant progression of cancer cells. The aim of our work is to study the angiogenic potential of macrophages at different stages of growth and metastasis of Lewis lung carcinoma. Methods: Mononuclear phagocytes obtained from peritoneal exudate of mice with Lewis lung carcinoma. Was carried out coculturing macrophages with endothelial cells and evaluated their effects on angiogenesis. Cell cycle parameters were assessed by cytoflourometry. The level of VEGFproduction by macrophages was determined by ELISA. Results: It was shown that cells of the control group passed to S+G2/M by 5.32%, while the number of cells stimulated by incubation medium of peritoneal macrophages on 19 and 25 days passed to S+G2/M increased by 52.82% and 49.72% respectively. It was found increased production of VEGF on 7 days to 2 times, 19 days – 4, 25 days – 4.7 as compared to intact group. Since 19 and 25 days characterized by intensive development of tumors and metestaz, in this time is formed of hypoxic areas. This process induces macrophages to increase production of VEGF for improving the access of oxygen and nutrients in these areas. Conclusions: The results suggest angiogenic potential of peritoneal macrophages because is observed active transition of endothelial cells to S+G2/M, increased production of VEGF and stimulate the formation of vessel-like structures by endothelial cells. Legal entity responsible for the study: Educational and Scientific Centre «Institute of Biology» of Taras Shevchenko National University of Kyiv Funding: Educational and Scientific Centre «Institute of Biology» of Taras Shevchenko National University of Kyiv Disclosure: All authors have declared no conflicts of interest.
Abstracts, ELCC 2016 – Tumour biology and pathology
results were statistically analyzed for two preplanned cut-off values, namely 5% and 10% of PD-L1 positivity in either TCs or ICs, regardless of the intensity. Baseline patients’ characteristics were obtained from the hospital registry database. Results: There were 29 (54%) AC and 25 (46%) SCC; the mean age was 62.4 years, 34 (63%) were male, most of them current or ex-smokers 46 (85%). TC PD-L1 positivity was significantly higher in SCC vs. AC, at both 5% and 10% cut-offs (52% vs. 17%, p = 0.016 and 52% vs. 14%, p = 0.007, respectively) while IC PD-L1 positivity was similar in SCC and AC, at both cut-offs (76% vs. 72%, p = 1 and 64% vs. 41%, p = 0.166, respectively). PD-L1 positivity was significantly higher in IC compared to TC in AD but not in SCC, regardless the cut-off value.
Treg cells in the absence of TGF-beta. However, in the presence of TGF-beta, when adding anti-PD-L1 monoclonal antibody, the differentiation of T lymphocytes into Treg cells was reduced. When TGF-beta and PD-L1 existed at the same time, the highest degree of T lymphocyte differentiation into Treg cells were observed. Conclusions: Expression of PD-L1 in lung cancer cells can coordinate TGF-beta to induce human peripheral blood T lymphocyte differentiation to Treg cells. Its role in immunosuppression may promote the occurrence and development of tumors. Legal entity responsible for the study: The Ethics Committee of The Second Affiliated Hospital of Soochow University Funding: The National Natural Science Foundation of China (No. 81272610) Disclosure: All authors have declared no conflicts of interest.
Table: PD-L1 positivity according to histology and different cutoffs N
Total AC SCC p (histology)
54 29 25
PD-L1 > 5%
PD-L1 > 10%
TC or IC N (%)
TC N (%)
IC N (%)
p (TC vs IC)
TC or IC N (%)
TC N (%)
IC N (%)
p (TC vs IC)
43 (80) 21 (72) 22 (88)
18 (33) 5 (17) 13 (52) 0.016
40 (74) 21 (72) 19 (76) 0.764
< 0.001 0.146
31 (57) 12 (41) 19 (76)
17 (31) 4 (14) 13 (52) 0.007
28 (52) 12 (41) 16 (64) 0.166
0.008 0.508
Conclusions: PD-L1 positivity is significantly higher in SCC compared to AC when looking only at TC and not at IC or combination. PD-L1 positivity in IC, which is highly positive in both histologic subtypes, adds to the overall positivity of AC but not SCC. There is no significant difference in IC PD-L1 positivity between SCC and AC, however by lowering the cut-off value the share of PD-L1 positive AC samples increases substantially, mostly due to IC positivity. Legal entity responsible for the study: University Clinic Golnik Funding: University Clinic Golnik Disclosure: All authors have declared no conflicts of interest. 6P Expression of PD-L1 in lung cancer cells regulates Treg cell differentiation M. Shi. Respiratory Diseases, 2nd Affiliated Hospital Suzhou (Soochow) University, Suzhou, China Background: Both regulatory T cells (Tregs) and the programmed cell death-1 (PD-1)/PD-L1 pathway play important roles in lung cancer pathogenesis; however, the association between these two factors remains poorly understood. Hereby, we examined PD-L1 expression in lung cancer cells, and analyzed its effect on induced Treg cells. Methods: PD-L1 expression was studied on lung cancer cells by flow cytometry. Peripheral blood mononuclear cells (PBMC) were isolated from a healthy volunteer, and then immune magnetic beads were used to sort out human peripheral blood T lymphocyte and detect separation purity. Plant blood agglutinin (PHA) was used to stimulate the peripheral blood T lymphocyte. Activated T cells were co-cultured with lung cancer cells which expressed the highest level of PD-L1, to which transforming growth factor-b (TGF-beta) was added to block PD-L1 type antibody. Then the degree of Treg cell differentiation was detected. Results: The expression of B7-H1 was detected in four human lung cancer cell lines using flow cytometry. We found that the membrane expression level of PD-L1 was the highest on the A549 cells. After PHA stimulation, T lymphocyte proliferation was activated. Then the activated T lymphocytes and A549 cells were co-cultured. Only a small part of T lymphocytes differentiated into
S59
7P Proangiogenic effect of peritoneal macrophages from animals with Lewis lung carcinoma T. Nikolaienko, D. Shelest, L. Garmanchuk, O. Dzhus, V. Nikulina. Biochemistry, Educational and Scientific Centre «Institute of Biology» of Taras Shevchenko National University of Kyiv, Kyiv, Ukraine Background: Both inflammation and angiogenesis are exacerbated by increased production of chemokines/cytokines, growth factors, proteolytic enzymes and prostaglandins. Initiation and progression of cancer are also closely linked to angiogenesis. Infiltration of macrophages is a dramatic and common feature of inflammation, angiogenesis and cancer, and has been recently highlighted in an attempt to develop novel strategies for treating cancer. The recruitment and infiltration of tumor-associated macrophages in the tumor microenvironment activates them to support the malignant progression of cancer cells. The aim of our work is to study the angiogenic potential of macrophages at different stages of growth and metastasis of Lewis lung carcinoma. Methods: Mononuclear phagocytes obtained from peritoneal exudate of mice with Lewis lung carcinoma. Was carried out coculturing macrophages with endothelial cells and evaluated their effects on angiogenesis. Cell cycle parameters were assessed by cytoflourometry. The level of VEGFproduction by macrophages was determined by ELISA. Results: It was shown that cells of the control group passed to S+G2/M by 5.32%, while the number of cells stimulated by incubation medium of peritoneal macrophages on 19 and 25 days passed to S+G2/M increased by 52.82% and 49.72% respectively. It was found increased production of VEGF on 7 days to 2 times, 19 days – 4, 25 days – 4.7 as compared to intact group. Since 19 and 25 days characterized by intensive development of tumors and metestaz, in this time is formed of hypoxic areas. This process induces macrophages to increase production of VEGF for improving the access of oxygen and nutrients in these areas. Conclusions: The results suggest angiogenic potential of peritoneal macrophages because is observed active transition of endothelial cells to S+G2/M, increased production of VEGF and stimulate the formation of vessel-like structures by endothelial cells. Legal entity responsible for the study: Educational and Scientific Centre «Institute of Biology» of Taras Shevchenko National University of Kyiv Funding: Educational and Scientific Centre «Institute of Biology» of Taras Shevchenko National University of Kyiv Disclosure: All authors have declared no conflicts of interest.