Single-cell RNA and TCR sequencing reveals distinct systematic immune response induced by SABR with or without prior anti-PD-1 therapy. Single-cell RNA and TCR sequencing reveals distinct systematic immune response induced by SABR with or without prior anti-PD-1 therapy

Previous studies have demonstrated that stereotactic body radiation therapy (SBRT) could activate systemic immune response, however, these researches showed limitations in comprehensively characterizing changes of T cell profile and T cell receptor (TCR). In the present study, we applied scRNA-seq and scTCR-seq to observe the dynamics of T cell and TCR repertoire from peripheral blood mononuclear cells (PBMCs) in early stage non-small-cell lung cancer (NSCLC) patients receiving SBRT with or without prior anti-PD-1 therapy, demonstrating the distinct systemic immune response between SBRT and ISBRT group. An enrichment of CD8-TE, CD8-EM, and CD4-TE clusters with increased cytotoxic and exhausted score were observed after treatment in SBRT group, while a decrease of those were presented in ISBRT group. Moreover, gene expression analysis revealed T cell mediated cytotoxicity signaling and T cell proliferation signaling were enriched in SBRT group while decreased in SBRT group. Analysis of TCR repertoire indicated a substantial alteration of TCR repertoire after treatment. The proportions of the large clone of TCR were increased after treatment in SBRT group while an opposite alteration existed in ISBRT group. After treatment, both SBRT and ISBRT group generated numerous new TCR clones which were mainly enriched in CD8 TE. Single clones and large clones were the mainly types of new TCRs in SBRT group, whereas in ISBRT group single clones accounted for the majority of new TCRs. These findings suggested that systemic immunity was activated in distinct pattern between the SBRT and ISBRT group, and provide evidence for future development of new treatment regimen. Overall design: Single cell RNA-seq and T-cell receptor repertoire sequencing with 43,051 T cells sorted from the peripheral blood of patients with metastatic non-small cell lung cancer (NSCLC) were performed on the 10x Genomics platform. Sampling grouping included pre- and post-therapy of stereotactic ablative radiation therapy (SABR) with or without prior anti-PD-1 therapy.

既往研究已证实，体部立体定向放射治疗（stereotactic body radiation therapy, SBRT）可激活系统性免疫应答，但此类研究在全面刻画T细胞谱系与T细胞受体（T cell receptor, TCR）的变化层面存在局限性。本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与单细胞T细胞受体测序（single-cell T cell receptor sequencing, scTCR-seq），对接受体部立体定向放射治疗（SBRT）联合或未联合前期抗PD-1治疗的早期非小细胞肺癌（non-small-cell lung cancer, NSCLC）患者外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中的T细胞及TCR库动态变化进行观测，结果显示SBRT组与ISBRT组的系统性免疫应答模式存在显著差异。治疗后，SBRT组中CD8-TE、CD8-EM及CD4-TE细胞簇的丰度显著升高，且其细胞毒性评分与耗竭评分均有所增加，而ISBRT组中此类细胞簇的丰度则呈下降趋势。此外，基因表达分析结果显示，T细胞介导的细胞毒性信号通路与T细胞增殖信号通路在SBRT组中显著富集，而在ISBRT组中则呈下调趋势。TCR库分析表明，治疗后患者的TCR库发生了显著改变。SBRT组中TCR大克隆的占比在治疗后有所升高，而ISBRT组则呈现出相反的变化趋势。治疗后，SBRT组与ISBRT组均产生了大量新的TCR克隆，且此类克隆主要富集于CD8 TE细胞亚群中。SBRT组中新产生的TCR克隆主要以单一克隆与大克隆为主，而ISBRT组中新产生的TCR克隆则绝大多数为单一克隆。上述研究结果表明，SBRT组与ISBRT组的系统性免疫激活模式存在显著差异，本研究为新型治疗方案的后续开发提供了理论依据。实验整体设计：本研究依托10x Genomics平台，对从转移性非小细胞肺癌（NSCLC）患者外周血中分选得到的43051个T细胞开展单细胞RNA测序与T细胞受体库测序。采样分组涵盖接受立体定向消融放疗（stereotactic ablative radiation therapy, SABR）联合或未联合前期抗PD-1治疗的患者的治疗前与治疗后样本。