基线水平CCL19+树突状细胞可有效预测肺腺癌患者免疫治疗的敏感性

doi:10.12122/j.issn.1673-4254.2024.08.11

摘要/Abstract

摘要：

目的探讨肺腺癌微环境基线水平CCL19⁺树突状细胞（CCL19⁺DC）与肺腺癌患者接受免疫治疗疗效及肺腺癌患者CD8⁺T细胞浸润的相关性。方法回顾性分析2020年1月~2023年12月在河南科技大学第一附属医院住院的肺腺癌患者资料，并收集行免疫治疗的肺腺癌患者组织标本96例。应用免疫荧光法检测96例肺腺癌组织中CCL19⁺DC及CD8⁺T细胞的浸润状况。受试者工作特征（ROC）曲线评估基线水平CCL19⁺DC对肺腺癌免疫治疗疗效预测价值，并进行敏感性分析。采用卡方检验分析肺腺癌组织中基线水平CCL19⁺DC与免疫疗效及CD8⁺T细胞浸润的相关性。采用Pearson相关性分析评价基线水平CCL19⁺DC表达与细胞毒性T细胞（CTL）浸润相关性。将PC9肺腺癌细胞、CD8⁺T细胞及DC（过表达CCL19和/或抗PD-1处理）进行分组体外共培养，采用流式细胞学方法检测T细胞各表面分子（GranzymeB、Perforin、IFN-γ、Ki-67）表达情况。结果免疫荧光结果显示，PR/CR组（Respond）的患者肺腺癌组织中CCL19⁺ DC浸润高于PD/SD组（Unrespond）患者，表现出更好的免疫治疗疗效，ROC曲线下面积为0.785，敏感度为75.6%，特异度为62.8%。CCL19⁺ DC高表达患者肺腺癌组CD8⁺ T细胞表面分子Granzyme B（P<0.01）、Perforin（P<0.01）、IFN-γ（P<0.01）及 Ki-67（P<0.001）表达均高于CCL19⁺ DC低表达患者。肺腺癌组织基线水平CCL19⁺DC的表达与免疫治疗疗效具有显著的相关性（P=0.003），与CTL细胞浸润呈正相关（r=0.6657，P<0.001），CCL19⁺DC丰富度与CD8⁺T细胞的浸润具有显著的相关性（P=0.007）。与对照组相比，转染过表达CCL19质粒及单独使用anti-PD1处理组中CD8⁺T淋巴细胞杀伤及增殖功能标志物增加（P<0.01）。使用anti-PD1联合转染过表达CCL19质粒处理组中CD8⁺T淋巴细胞杀伤及增殖功能标志物水平显著增加（P<0.001）。结论在免疫治疗背景下，肺腺癌微环境中基线水平CCL19⁺DC的表达与肺腺癌患者免疫治疗疗效及CTL细胞浸润呈正相关，并且肺腺癌微环境中CCL19⁺DC可以提升CD8⁺T细胞杀伤及增殖功能标志物水平，发挥潜在抗肿瘤免疫作用。因此，肺腺癌微环境中基线水平CCL19⁺DC可以有效预测肺腺癌免疫治疗的敏感性。

关键词: 肺腺癌, CCL19⁺树突状细胞, 免疫治疗, CD8⁺T细胞

Abstract:

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.

Key words: lung adenocarcinoma, CCL19⁺ dendritic cells, immunotherapy, CD8⁺ T cells

朱名扬, 王博康, 张秀森, 周克旭, 苗泽宇, 孙江涛. 基线水平CCL19⁺树突状细胞可有效预测肺腺癌患者免疫治疗的敏感性[J]. 南方医科大学学报, 2024, 44(8): 1529-1536.

Mingyang ZHU, Bokang WANG, Xiusen ZHANG, Kexu ZHOU, Zeyu MIAO, Jiangtao SUN. Assessment of baseline CCL19⁺ dendritic cell infiltration for predicting responses to immunotherapy in lung adenocarcinoma patients[J]. Journal of Southern Medical University, 2024, 44(8): 1529-1536.

图/表 6

表1 基线水平CCL19+ DC的表达与肺腺癌患者免疫治疗疗效之间相关性

Tab.1 Correlation between baseline expression of CCL19+ DCs and immunotherapy efficacy in patients with lung adenocarcinoma

Patient type

CCL19⁺DC Low expression

CCL19⁺DC High expression

8.663

图 2 CCL19+ DC高表达及CCL19+ DC低表达组患者肺癌组织中CD8+ T细胞表面分子 GranzymeB、Perforin、IFN-γ 及Ki-67的表达情况

Fig. 2 Expression of CD8+ T cell surface molecules granzyme B, perforin, IFN-γ, and Ki-67 in lung adenocarcinoma tissues of patients with high and low levels of CCL19+ DC infiltration. A: Immunofluorescence images of CD8+ T surface molecule expressions in each group (scale bar=10 μm, 5 μm). B: Statistical graph of CD8+ T surface molecule expressions. **P<0.01, ***P<0.001.

表2 基线水平CCL19+ DC的表达与CD8+ T细胞浸润之间相关性。

Tab.2 Correlation between baseline expression of CCL19+ DCs and CD8+ T cell infiltration

Patient	n	CD8⁺ T cell desert type	CD8⁺ T cell rich type	χ²	P
CCL19⁺DC high expression	53	21	32	7.362	0.007
CCL19⁺DC low expression	43	29	14	7.362	0.007

图3 CTL在不同肺腺癌组织中的浸润情况

Fig.3 Infiltration of CTL in different lung adenocarcinoma tissues.

图4 CCL19免疫印记分析

Fig.4 Western blotting of CCL19. A: Expressions of CCL19 protein in different groups detected by Western blotting. B: Statistical analysis of CCL19 expressions. ***P<0.001.

图5 不同处理组对共培养体系中CD8+ T细胞表面分子表达情况的影响

Fig.5 Effect of different treatments on the expression of surface molecules of CD8+ T cells in the co-culture systems. A: Flow cytometry of surface molecule expressions on CD8+ T cells. B: Statistical analysis of surface molecule expression on CD8+ T cells. **P<0.01, ***P<0.001.

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