Single-cell Sequencing Reveals Neoadjuvant Immune Landscape In Non-small Cell Lung Cancer

Objective:Non-small cell lung cancer(NSCLC)is one of the most common malignancies.Some patients have been diagnosed at advanced stage and have lost the opportunity of radical surgery.Neoadjuvant therapy is used to shrink tumors to regain the opportunity of radical surgery.However,the effect of platinum-based chemotherapy alone is limited.Currently,immunotherapy,especially immune checkpoint inhibitors(ICIs)alone or in combination with chemotherapy,has become one of the important therapeutic strategies for advanced NSCLC.The application of neoadjuvant ICIs combined with chemotherapy in non-small cell lung cancer has also attracted more attention.Several clinical studies demonstrated that neoadjuvant ICIs and chemotherapy have achieved favorable pathological response and clinical benefits,but the underlying immune mechanism of tumor regression and biomarkers of response remain unclear.Methods:To identify factors associated with the clinical outcome,we generated single-cell RNA sequencing profiles by 10x Genomics technology on CD45~+immune cells isolated from lung cancer tumor tissues(T)and matched five immune-relevant sites,including peripheral blood mononuclear cells before and after neoadjuvant therapy(hereafter we called P0 and P1),distal normal tissues(D),nearest non-cancer tissues(N),regional draining lymph nodes(LNs)of 12ⅢA(N2)NSCLC patients,including4 treatment-na?ve and 8 neoadjuvant pembrolizumab and chemotherapy patients.Bioinformatics analysis was used to screen immune cell factors relevant to therapy response in single-cell sequencing profiles,and results were verified at protein level by flow cytometry,multiplex fluorescent immunohistochemistry(m IHC)and ELISA.Finally,we collected surgical tumor tissue from 26 treatment-na?veⅢA NSCLC tumor tissues and 30 surgical tumor specimens from patients who diagnosed withⅢA/ⅢB NSCLC and received 2 cycles of neoadjuvant pembrolizumab and chemotherapy before surgery.MIHC of CD4,IL-21,CD20,Ig G1,Ig G3 and Ig A antibodies was performed to further verify the possibility that these indicators could be used as biomarkers for predicting clinical response to neoadjuvant ICIs and chemotherapy.Results:1.After initial filters and quality control,we acquired single-cell transcriptomes on a total of 186,477 immune cells and identified 29 main clusters,including 2clusters enriched for NK/NKT cells(C0-NK/NKT-GNLY/NKG7,C20-NK-XCL1),3clusters for CD4~+T cells(C2-CD4-IL7R,C4-CD4-TCF7,C11-Treg-Fox P3),6clusters for CD8~+T cells(C3-CD8-KLRG1,C5-CD8-IL7R,C7-CD8-PDCD1,C8-CD8-GZMK,C21-CD8-MKI67,C24-CD8-TCF7),3 B cell clusters(C6-B cells-CD79A,C17-Plasma cells-IGHG1,C22-B cells-MS4A1),9 myeloid clusters(C1-TAM-CD81,C9-MDSC-like macrophage-S100A8/A9,C10-Neotrophil-CXCL8,C15-Macrophage-CCL18,C16-DC-HLA II molecules,C18-Mast cells-TPSAB1,C19-Monocyte-CD16,C25-p DC-IL3RA,C26-Megakaryocyte-PPBP),as well as C23-Endothelial cells-VWF,C27-Epithelial cells-KRT17 and cycling cell clusters such as C14 and C28.2.We identified significant changes of lymphocytes and myeloid cells in neoadjuvant tumor lesions,with lymphocytes striking increased,while myeloid cells deceased.We uncovered that tertiary lymphoid structures(TLSs),B cells and CD4~+T cells were significantly enriched in neoadjuvant tumor tissues,and IL-21 secreted by Tfh cells promoted B cells class switched to Ig G(mainly Ig G1 and Ig G3),which was associated with the anti-tumor immune response of neoadjuvant pembrolizumab and chemotherapy.3.Cellular connection analysis identified the possibility of cell migration between tumor tissue and peripheral blood.TCR clonotype analysis confirmed the locally activation and expansion of intra-tissue CD4~+T clones,the dynamic exchange and expansion of CD8~+T clones between tumor and peripheral blood contributed to anti-PD-1 therapy response,and clonal replenishment of peripherally-expanded CD8~+T clones improved the exhaustion and dysfunctional status of intratumoral CD8~+T cells in tumor lesions,suggesting that the peripheral blood may be a valuable compartment for immune checkpoint blockage therapy.4.The diminished TNFRSF4~+regulatory T cells(Tregs)in tumor is a favorable predictor of neoadjuvant pembrolizumab and chemotherapy.5.Single-cell sequencing and flow cytometry confirmed that LAMP3~+DCs was agrregated in tumor tissue after neoadjuvant pembrolizumab and chemotherapy,which was significantly correlated with therapy response.LAMP3~+DCs was predicted to interact with CD4~+T cells,CD8~+T cells and B cells via the CCL22/CCR4,CCL17/CCR4 and CCL19/CCR7 axes,indicating a potential role of LAMP3~+DCs for lymphocytes recruitment during neoadjuvant chemoimmunotherapy.Moreover,LAMP3~+DCs exhibit the regulation activity towards B cells,Tregs,CD4~+and CD8~+T lymphocytes through PD1/PD-L1 and PD1/PD-L2 signaling pathways,as well as NK/NKT cells via CD226/NECTIN2 and TIGIT/NECTIN2 pathways.6.MIHC of tumor tissues from validation cohort confirmed that CD20~+B cells,Ig G1,Ig G3,Ig A,CD4~+T cells and IL-21 in tumor tissues were significantly correlated with the therapeutic response of neoadjuvant pembrolizumab and chemotherapy.Moreover,plasma IL-21 concentration was significantly related to the therapuetic response of neoadjuvant pembrolizumab and chemotherapy.Conclusion:The co-increase of TLSs,B cells and CD4~+T cells are associated with positive therapeutic response of neoadjuvant pembrolizumab and chemotherapy.B cell Ig G subclasses Ig G1 and Ig G3 play a critical role in anti-tumor immune response in tumor lesion,and this process was driven by increased IL-21 protein secreted by infiltrated T follicular helper(Tfh)cells after neoadjuvant ICIs and chemotherapy.Pre-therapy peripheral blood T cell diversity is also a good predictor of clinical response to neoadjuvant ICIs and chemotherapy,and detection of TCR diversity before treatment can help identify patients who will respond to treatment.The diminished TNFRSF4~+Tregs in tumor is a favorable predictor of neoadjuvant ICIs and chemotherapy.LAMP3~+DCs are involved in the recruitment and regulation of multiple lymphocyte subsets via ligand-recepror interactions,and are more likely associated with the abundant intratumoral lymphocyte infiltration and the clinical response to neoadjuvant pembrolizumab and chemotherapy.Plasma IL-21 may serve as a potential biomarker for predicting and detecting the efficacy of neoadjuvant ICIs and chemotherapy,and may assist in screening patients who are more likely to benefit from neoadjuvant therapy.Taken together,our extensive immune single-cell RNA-seq and TCR-seq landscape provide novel insights into the cellular mechanisms of underlying the synergistic interaction in clinical responses to neoadjuvant pembrolizumab and chemotherapy and provide potential predictive biomarkers and therapeutic targets to improve the positive clinical outcome.