1.Assessment of baseline CCL19+dendritic cell infiltration for predicting responses to immunotherapy in lung adenocarcinoma patients
Mingyang ZHU ; Bokang WANG ; Xiusen ZHANG ; Kexu ZHOU ; Zeyu MIAO ; Jiangtao SUN
Journal of Southern Medical University 2024;44(8):1529-1536
Objective To explore the correlation of baseline CCL19+dendritic cell(CCL19+DC)infiltration in lung adenocarcinoma microenvironment with immunotherapy efficacy and CD8+T cell infiltration.Methods We retrospectively analyzed the data of patients with lung adenocarcinoma hospitalized at First Affiliated Hospital of Henan University of Science and Technology from January,2020 to December,2023,and collected tissue samples from 96 patients undergoing immunotherapy for assessing CCL19+DC and CD8+T cell infiltration using immunofluorescence assay.We evaluated the predictive value of baseline CCL19+DCs for patient responses to immunotherapy using receiver-operating characteristics(ROC)curves and analyzed the correlations of baseline CCL19+DC expression with immunotherapy efficacy and CD8+T cell and cytotoxic T lymphocyte(CTL)infiltrations.In co-culture systems of lung adenocarcinoma PC9 cells,CD8+T cells and DCs(overexpressing CCL19 with or without anti PD-1 antibody treatment),the expressions of granzyme B,perforin,IFN-γ,and Ki-67 in T cells were analyzed using flow cytometry.Results The patients with partial or complete remission following immunotherapy had a significantly higher baseline CCL19+DC infiltration level in lung adenocarcinoma tissues than those with poor responses.CCL19+DC infiltration had an area under ROC curve of 0.785,a sensitivity of 75.6%,and a specificity of 62.8%for predicting immunotherapy efficacy.The expression of CD8+T cell surface molecules Granzyme B(P<0.01),Perforin(P<0.01),IFN-γ(P<0.01)and Ki-67(P<0.001)in patients with high expression of CCL19+DC were higher than those in patients with low expression of CCL19+DC.The baseline CCL19+DC infiltration level was positively correlated with immunotherapy efficacy(P=0.003),CTL infiltration of(r=0.6657,P<0.001)and CD8+T cell infiltration(P=0.007).In the co-cultured cells,CCL19 overexpression combined with anti-PD1 treatment of the DCs more strongly enhanced cytotoxicity and proliferation of CD8+T lymphocytes than either of the single treatments(P<0.01 or 0.001).Conclusion The baseline CCL19+DC infiltration level in lung adenocarcinoma microenvironment is positively correlated with immunotherapy efficacy and CTL infiltration and can thus predict the response to immunotherapy.
2.Assessment of baseline CCL19+dendritic cell infiltration for predicting responses to immunotherapy in lung adenocarcinoma patients
Mingyang ZHU ; Bokang WANG ; Xiusen ZHANG ; Kexu ZHOU ; Zeyu MIAO ; Jiangtao SUN
Journal of Southern Medical University 2024;44(8):1529-1536
Objective To explore the correlation of baseline CCL19+dendritic cell(CCL19+DC)infiltration in lung adenocarcinoma microenvironment with immunotherapy efficacy and CD8+T cell infiltration.Methods We retrospectively analyzed the data of patients with lung adenocarcinoma hospitalized at First Affiliated Hospital of Henan University of Science and Technology from January,2020 to December,2023,and collected tissue samples from 96 patients undergoing immunotherapy for assessing CCL19+DC and CD8+T cell infiltration using immunofluorescence assay.We evaluated the predictive value of baseline CCL19+DCs for patient responses to immunotherapy using receiver-operating characteristics(ROC)curves and analyzed the correlations of baseline CCL19+DC expression with immunotherapy efficacy and CD8+T cell and cytotoxic T lymphocyte(CTL)infiltrations.In co-culture systems of lung adenocarcinoma PC9 cells,CD8+T cells and DCs(overexpressing CCL19 with or without anti PD-1 antibody treatment),the expressions of granzyme B,perforin,IFN-γ,and Ki-67 in T cells were analyzed using flow cytometry.Results The patients with partial or complete remission following immunotherapy had a significantly higher baseline CCL19+DC infiltration level in lung adenocarcinoma tissues than those with poor responses.CCL19+DC infiltration had an area under ROC curve of 0.785,a sensitivity of 75.6%,and a specificity of 62.8%for predicting immunotherapy efficacy.The expression of CD8+T cell surface molecules Granzyme B(P<0.01),Perforin(P<0.01),IFN-γ(P<0.01)and Ki-67(P<0.001)in patients with high expression of CCL19+DC were higher than those in patients with low expression of CCL19+DC.The baseline CCL19+DC infiltration level was positively correlated with immunotherapy efficacy(P=0.003),CTL infiltration of(r=0.6657,P<0.001)and CD8+T cell infiltration(P=0.007).In the co-cultured cells,CCL19 overexpression combined with anti-PD1 treatment of the DCs more strongly enhanced cytotoxicity and proliferation of CD8+T lymphocytes than either of the single treatments(P<0.01 or 0.001).Conclusion The baseline CCL19+DC infiltration level in lung adenocarcinoma microenvironment is positively correlated with immunotherapy efficacy and CTL infiltration and can thus predict the response to immunotherapy.
3.RgpB contributes to chemoresistance in esophageal squamous cell carcinoma by preventing Cx43 degradation via inhibiting autophagosome-lysosome fusion
Yue DU ; Xiusen ZHANG ; Kexu ZHOU ; Xing JIN ; Xiang YUAN ; Shegan GAO
Journal of Southern Medical University 2024;44(9):1670-1676
Objective To investigate the mechanism through which RgpB,a virulence factor of Porphyromonas gingivalis(Pg),induces chemoresistance in esophageal squamous carcinoma.Methods The autophagy-regulating factors that interact with RgpB were screened by immunoprecipitation-mass spectrometry.The interaction between RgpB and the autophagy regulator TBC1D5 was investigated using co-immunoprecipitation.The impact of Pg infection on the expression of esophageal cancer cell membrane receptor molecule Cx43 was assessed using Western blotting.Immunofluorescence assay was used to analyze the relationship among Lamp1,Cx43 and TBC1D5.The effect of Pg infection on autophagosome-lysosome fusion was evaluated using autophagy double fluorescence technique.The effects of Pg infection and a Cx43 inhibitor on proliferation of esophageal cancer cells after chemotherapy were examined with plate cloning assay and CCK-8 method.Results Immunoprecipitation-mass spectrometry identified TBC1D5 as an autophagy regulator interacting with RgpB,and co-immunoprecipitation suggested that RgpB could directly bind to TBC1D5.In Pg-infected esophageal cancer cells,the expression of Cx43 on the cell membrane was significantly higher than that in non-infected cells.Immunofluorescence assay showed that the expression of Cx43 on the membrane of esophageal cancer cells increased significantly after Pg infection,which blocked autophagosome-lysosome fusion as shown by stubRFP-sensGFP-LC3 lentivirus study.Plate cloning assay and CCK-8 assay showed that the Cx43 inhibitor significantly attenuated the effect of Pg infection for promoting proliferation of esophageal cancer cells after chemotherapy.Conclusion Pg infection in esophageal cancer blocked autophagosome-lysosome fusion in the tumor cells,thereby preventing Cx43 from lysosomal degradation and leading to chemoresistance of esophageal cancer.
4.RgpB contributes to chemoresistance in esophageal squamous cell carcinoma by preventing Cx43 degradation via inhibiting autophagosome-lysosome fusion
Yue DU ; Xiusen ZHANG ; Kexu ZHOU ; Xing JIN ; Xiang YUAN ; Shegan GAO
Journal of Southern Medical University 2024;44(9):1670-1676
Objective To investigate the mechanism through which RgpB,a virulence factor of Porphyromonas gingivalis(Pg),induces chemoresistance in esophageal squamous carcinoma.Methods The autophagy-regulating factors that interact with RgpB were screened by immunoprecipitation-mass spectrometry.The interaction between RgpB and the autophagy regulator TBC1D5 was investigated using co-immunoprecipitation.The impact of Pg infection on the expression of esophageal cancer cell membrane receptor molecule Cx43 was assessed using Western blotting.Immunofluorescence assay was used to analyze the relationship among Lamp1,Cx43 and TBC1D5.The effect of Pg infection on autophagosome-lysosome fusion was evaluated using autophagy double fluorescence technique.The effects of Pg infection and a Cx43 inhibitor on proliferation of esophageal cancer cells after chemotherapy were examined with plate cloning assay and CCK-8 method.Results Immunoprecipitation-mass spectrometry identified TBC1D5 as an autophagy regulator interacting with RgpB,and co-immunoprecipitation suggested that RgpB could directly bind to TBC1D5.In Pg-infected esophageal cancer cells,the expression of Cx43 on the cell membrane was significantly higher than that in non-infected cells.Immunofluorescence assay showed that the expression of Cx43 on the membrane of esophageal cancer cells increased significantly after Pg infection,which blocked autophagosome-lysosome fusion as shown by stubRFP-sensGFP-LC3 lentivirus study.Plate cloning assay and CCK-8 assay showed that the Cx43 inhibitor significantly attenuated the effect of Pg infection for promoting proliferation of esophageal cancer cells after chemotherapy.Conclusion Pg infection in esophageal cancer blocked autophagosome-lysosome fusion in the tumor cells,thereby preventing Cx43 from lysosomal degradation and leading to chemoresistance of esophageal cancer.