Correlation Between B Cells And The Efficacy Of Immunotherapy In Non-small Cell Lung Cancer

Objective: 1)To investigate the prognostic value of the CD20+B cells in non-small cell lung cancer(NSCLC)patients with immunotherapy.2)To explore the effect of B cell-related therapies on the efficacy of immunotherapy,and to explore the potential mechanism of combination therapy with regard to the B cells in Lewis lung carcinoma mouse.3)To construct a B cell-related gene pairs(BRGPs)signature for predicting the prognosis as well as the efficacy of immunotherapy in NSCLC patients.Methods: 1)TCGA-NSCLC and GEO-NSCLC-immunotherapy datasets were obtained from TCGA and GEO databases to analyze the correlation between MS4A1(CD20 m RNA)expression and the prognosis as well as the efficacy of immunotherapy in NSCLC patients.Tumor samples of immune checkpoint inhibitors(ICIs)-treated NSCLC patients in the Affiliated Tumor Hospital of Shandong First Medical University were collected to evaluate the CD20 expression by immunohistochemical staining.Corresponding clinical data and follow-up data for these patients were collected,and the correlation between immunotherapy efficacy and CD20+B cells was analyzed.2)To investigate the anti-tumor effect of B cell-related therapies in C57BL/6 mouse with Lewis lung carcinoma,anti-CD20 m Ab/CD40 agonist m Ab with or without anti-PD-1 treatment were given,and the tumor volume together with the survival time of mouse were collected.In addition,the function and differentiation related markers of B cells in TDLN and spleen samples,such as MHC-Ⅱ,CD5,CD1 d,CD138,Ig D,Ig M,Ig G,CD80 and PD-L2 were detected by flow cytometry method.3)Based on a public single-cell RNA sequencing data,the B cell-related genes from NSCLC samples were identified.TCGA training and GEO validation datasets were used to screen the prognostic B cell-related gene pairs in NSCLC patients,and Lasso-Cox proportional hazard model was used to construct BRGPs signature.Then,the ROC curve of the BRGPs signature was applied to divide NSCLC patients into high and low-risk groups,followed by evaluation of the patients’ clinical outcomes by Kaplan-Meier method and Log-rank test in the training and validation datasets,respectively.The relationship between BRGPs signature and the clinicopathological characteristics of NSCLC patients were analyzed by chi-square test and Wilcoxon test.Subsequently,TME conditions including immune scores,stromal scores,tumor purity and 22 immune cell types in NSCLC samples were evaluated by ESTIMATE and CIBERSORT methods.In addition,functional annotation and analyses of the BRGPs signature were performed based on GSEA and ss GSEA.Finally,the correlation between the risk score of the BRGPs signature and CD274(PD-L1 m RNA)expression as well as TMB score were explored,and the potential immunotherapeutic responses of NSCLC patients were predicted based on TIDE algorithm.Results: 1)The expression level of MS4A1 in NSCLC tumor tissues was significantly higher than adjacent normal tissues(P<0.05).The expression of MS4A1 was negatively correlated with the clinical stage of NSCLC patients(P < 0.05).NSCLC patients with high expression of MS4A1 were significantly correlated with longer overall survival(OS)time compared with their counterparts(P=0.014).In the ICIs-treated NSCLC patients,there were no significant correlation between the density of tumor infiltrating CD20+B cells and ORR or DCR(P>0.05).However,the OS was significantly longer in NSCLC patients with high CD20+B cells infiltration(P=0.025),but the PFS was only numerically increased(P > 0.05).2)Anti-CD20 m Ab was given intravenously resulting in the completely depletion of B cells in the blood,lymph nodes,spleen and tumor tissues of Lewis lung carcinoma mouse,and the depletion efficacy lasted more than 20 days.In Lewis lung carcinoma mouse,the tumor volume was significantly increased after B cell-depletion regardless of anti-PD-1 treatment.Otherwise,CD40 agonist m Ab administration induced the formation of CD20+B cell clusters and increased the anti-tumor efficacy of anti-PD-1 m Ab.The combination therapy significantly inhibited tumor growth and prolonged the survival time of the Lewis lung carcinoma mouse(median OS was 26,28,28,40 days for the mouse in control group,anti-PD-1 group,CD40 agonist group and combination group,respectively).CD40 agonist m Ab combined with anti-PD-1 m Ab showed a synergistic effect on tumor control in Lewis lung carcinoma mouse,through improving the expression of MHC-Ⅱ on the surface of B cells in TDLN and spleen,and promoting the differentiation of memory B cell subtypes and plasma cells.3)We successfully constructed the BRGPs signature consisting of 23 B cell-related gene pairs in NSCLC patients.In the training and validation datasets,NSCLC patients with low-risk score had significantly better OS than patients with high-risk score,respectively(P<0.001,P=0.001,respectively).Univariate and multivariate Cox analysis demonstrated that the risk score of BRGPs signature was a independent prognostic factor in NSCLC patients.In addition,the risk score was significantly correlated with the tumor TNM stage and NSCLC patients with high-risk score often had advanced clinical stage.TME analysis showed that patients with low-risk score had significantly higher ESTIMATE scores,immune scores,stroma scores(all P<0.05),and lower tumor purity(P<0.05)than patients with high-risk score.Immune cells analysis showed that the proportion of CD8+T cells,resting mast cells,plasma cells,resting dendritic cells,memory B cells,Treg and gamma delta T cells were significantly lower in low-risk group compared with high-risk group(all P<0.05).GSEA analysis indicated that patients with low-risk score enriched with several immune-related activation pathways.There was no significant differences in CD274 expression of NSCLC patients between the high and low-risk groups(P=0.940),but the TMB score in the high-risk group was significantly higher compared with low-risk group(P<0.001).NSCLC patients with high-risk score had significantly lower TIDE scores compared with patients with low-risk score(P<0.01),which indicates that high-risk patients are more likely to benefit from immunotherapy.Conclusion: 1)The density of CD20+B cells is correlated with the efficacy of immunotherapy in patients with advanced NSCLC.ICIs-treated NSCLC patients with high expression of CD20 have better OS,and CD20 can be used as a potential biomarker to predict the prognosis of NSCLC patients with immunotherapy.2)In Lewis lung carcinoma mouse,B cell depletion can significantly promote tumor growth,while CD40 agonist m Ab can increase the efficacy of anti-PD-1 m Ab.CD40 agonist m Ab combined with anti-PD-1 immunotherapy exhibits a synergistic effect on tumor control,partially due to the improvement of anti-tumor B cell functions and subsets.The strategy of CD40 agonist m Ab in combination with ICIs therapy may be a promising option to manage NSCLC patients in the future.3)The BRGPs signature is significantly correlated with the clinical stage,TME and TMB scores of NSCLC patients,and BRGPs signature might be a potential biomarker to predict prognosis and immunotherapeutic effect in NSCLC patients.