T-cell CX3CR1 expression as a dynamic blood-based biomarker of response to immune checkpoint inhibitors

We investigated the frequency of CX3CR1+CD8+ T cells in peripheral blood (PB) before and during immune checkpoint inhibitor (ICI) therapy, and delineated the TCR repertoire in peripheral CX3CR1+CD8+ T-cell subsets and CD8+ tumor-infiltrating lymphocytes (TILs) using preclinical models. To understand the clinical utility of CX3CR1 as a circulating T-cell biomarker, we analyzed longitudinal PB samples from non-small cell lung cancer (NSCLC) patients undergoing anti-PD-1 therapy, and evaluated predictive and prognostic value of changes in the frequency of PB CX3CR1+ CD8+ T cells Methods: Female C57BL/6 mice were inoculated subcutaneously with 5 x 10^5 MC38 tumor cells per mouse on the right flank on day 0. When tumor volume reached approximately 50 mm^3, 200 µg of anti-PD-L1 Ab (clone 10F.9G2, BioXcell) and 100 µg of anti-CTLA-4 Ab (clone 9H10, BioXcell) were administered intraperitoneally every 3 days and every other day, respectively. MC38 tumor-bearing mice were treated for 14 days, and spleens and tumors were harvested. [Tumor cells] Tumors were cut into small pieces of 2-4 mm. Single-cell suspensions were obtained by mechanical dispersion consisting of two 30-min incubations at 37°C, 5% CO2 in 5 ml RPMI 1640 (Gibco) and tumor dissociation kit (Miltenyi Biotec) in C Tubes (Miltenyi Biotec) interspersed with three mechanical dispersions on a GentleMACS dissociator (Miltenyi Biotec). The tumor cell suspensions were then filtered through a cell strainer (70 μm; BD Biosciences). An EasySep Mouse CD8a Positive Selection Kit II (STEMCELL Technologies) was used to isolate murine CD8+ tumor-infiltrating lymphocytes (TILs) for TCR sequencing. [Splenocytes] Spleens were homogenized by forcing the tissue through a cell strainer (70 μm; BD Biosciences). Red blood cells in blood and spleen were lysed using ACK Lysis Buffer (Gibco). For TCR sequencing of murine splenic CD8+ T cells, single cell suspensions from freshly isolated splenocytes were stained. CD45+ CD3+ CD8+ T cells were gated, and CD27lo CX3CR1-, CD27hi CX3CR1-, and CX3CR1+ CD8+ T cells were sorted using BD FACSAria II Cell Sorter. DNA from flow-isolated murine splenic CD8+ T cells and CD8+ TILs, and PB CD8+ T cells was extracted using QIAamp DNA Micro Kit (QIAGEN). DNA was quantified using Qubit dsDNA BR Assay (Invitrogen). Amplification and sequencing of TCRβ CDR3 regions was performed using ImmunoSEQ immune profiling system at the survey level (Adaptive Biotechnologies). Sequencing was performed on an Illumina NextSeq system using 150 cycle mid-output kit (Illumina Inc.). Processed data were uploaded to the ImmunoSEQ Platform (Adaptive Biotechnologies) for bioinformatics analysis. Processed data were downloaded and frequencies/counts for TCR clonotypes and diversity were examined by nucleotide sequences after non-productive reads were filtered out. Results: Clonally expanded TCR repertoires of CD8+ TILs are enriched in the peripheral CX3CR1+ subset during ICI therapy. ICI therapy induces high degree of TCR sequence similarity and clonality between CD8+ TILs and the peripheral CX3CR1+ CD8+ T cells. 12 samples, splenic CD27loCX3CR1lo, CD27hiCX3CR1lo, and CX3CR1hi CD8 T cells, and CD8 TILs *** Comment from submitter: "Files are from Adaptive Biotech ImmunoSeq" ***

本研究探究了免疫检查点抑制剂（immune checkpoint inhibitor, ICI）治疗前后及治疗过程中外周血（peripheral blood, PB）中CX3CR1阳性CD8阳性T细胞（CX3CR1+CD8+ T cells）的频率，并通过临床前模型解析了外周血CX3CR1阳性CD8阳性T细胞亚群与CD8阳性肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes, TILs）的T细胞受体库（TCR repertoire）。为明确CX3CR1作为循环T细胞生物标志物的临床应用价值，本研究分析了接受抗PD-1治疗的非小细胞肺癌（non-small cell lung cancer, NSCLC）患者的纵向外周血样本，并评估了外周血CX3CR1阳性CD8阳性T细胞频率变化的预测价值与预后价值。
方法：于第0天，将每只小鼠5×10^5个MC38肿瘤细胞接种于雌性C57BL/6小鼠右侧背部皮下。当肿瘤体积达到约50 mm³时，分别每隔3天、每隔1天腹腔注射200 μg抗PD-L1抗体（克隆号10F.9G2，BioXcell）与100 μg抗CTLA-4抗体（克隆号9H10，BioXcell）。携带MC38肿瘤的小鼠接受14天治疗后，采集脾脏与肿瘤组织。
【肿瘤细胞】将肿瘤组织剪切成2~4 mm的小块。采用机械解离法制备单细胞悬液：将组织置于含5 mL RPMI 1640培养基（Gibco）与肿瘤解离试剂盒（Miltenyi Biotec）的C管（Miltenyi Biotec）中，于37℃、5% CO₂条件下孵育两次，每次30分钟，期间在GentleMACS组织解离仪（Miltenyi Biotec）上进行三次机械解离。随后将肿瘤细胞悬液通过70 μm细胞筛过滤（BD Biosciences）。使用EasySep小鼠CD8α阳性分选试剂盒II（STEMCELL Technologies）分离小鼠CD8阳性肿瘤浸润淋巴细胞（TILs），用于TCR测序。
【脾脏淋巴细胞】将脾脏组织通过70 μm细胞筛过滤以制备单细胞悬液（BD Biosciences）。使用ACK裂解缓冲液（Gibco）裂解血液与脾脏样本中的红细胞。为完成小鼠脾脏CD8阳性T细胞的TCR测序，对新鲜分离的脾脏淋巴细胞制备的单细胞悬液进行染色。设门圈选CD45阳性CD3阳性CD8阳性T细胞，随后采用BD FACSAria II细胞分选仪分选出CD27低表达CX3CR1阴性、CD27高表达CX3CR1阴性以及CX3CR1阳性CD8阳性T细胞。使用QIAamp DNA微量提取试剂盒（QIAGEN）提取流式分选得到的小鼠脾脏CD8阳性T细胞、CD8阳性TILs以及外周血CD8阳性T细胞的基因组DNA。采用Qubit dsDNA BR定量试剂盒（Invitrogen）对DNA进行定量。采用ImmunoSEQ免疫组库分析系统（Adaptive Biotechnologies）的全景级分析流程扩增并测序TCRβ链CDR3区域。测序在Illumina NextSeq测序平台上完成，使用150个循环中通量测序试剂盒（Illumina公司）。将处理后的测序数据上传至ImmunoSEQ平台（Adaptive Biotechnologies）进行生物信息学分析。过滤掉非功能性读段后，下载处理后的数据，基于核苷酸序列分析TCR克隆型的频率/计数以及多样性。
结果：免疫检查点抑制剂（ICI）治疗期间，外周血CX3CR1阳性CD8阳性T细胞亚群中富集了与CD8阳性肿瘤浸润淋巴细胞（TILs）克隆扩增一致的T细胞受体库。ICI治疗可诱导CD8阳性TILs与外周血CX3CR1阳性CD8阳性T细胞之间产生高度相似的TCR序列与克隆型。本次研究共纳入12份样本，包括脾脏CD27低表达CX3CR1低表达、CD27高表达CX3CR1低表达、CX3CR1高表达CD8阳性T细胞以及CD8阳性TILs。
*** 提交者备注："数据文件来源于Adaptive Biotechnologies公司的ImmunoSeq平台" ***