趋化因子受体6表达在肿瘤免疫微环境中作用的生物信息学分析及其对调节性T淋巴细胞功能的影响

doi:10.13481/j.1671-587X.20260401

摘要/Abstract

摘要：

目的探讨肿瘤组织中CXC基序趋化因子受体6（CXCR6）与免疫检查点分子表达及CD4?T淋巴细胞浸润丰度的关系，阐明其对调节性T淋巴细胞（Treg）中功能的影响及其潜在作用机制。方法生物信息学分析采用肿瘤免疫估计资源（TIMER）数据库，收集10种实体瘤的转录组及免疫浸润数据，通过Pearson相关性分析法分析CXCR6与免疫检查点分子及CD4⁺T淋巴细胞浸润丰度的相关性。采用健康人外周血单个核细胞（PBMCs），分离扩增CD4?T淋巴细胞并诱导为Treg细胞，构建CXCR6 shRNA1~4#重组慢病毒载体。实验分为空白组（不加Treg）、对照组和CXCR6敲低组，经慢病毒转染后，将Treg与CD8?细胞毒性T淋巴细胞（CTLs）、T2肿瘤靶细胞共培养。采用流式细胞术检测Treg的纯度，荧光计数法测定靶细胞裂解率，酶联免疫吸附试验（ELISA）检测各类细胞中细胞因子水平，RNA测序技术检测细胞中差异基因的表达水平，实时荧光定量PCR（RT-qPCR）法检测CXCR6的敲低效率和细胞中肽酶抑制因子6（PI16）mRNA表达水平。结果生物信息学分析，CXCR6在10种实体瘤中与免疫检查点分子及CD4?T淋巴细胞浸润丰度均呈高度正相关关系（P<0.01），且与肿瘤纯度呈负相关关系（胶质母细胞瘤除外）（P<0.01）。实验成功获得高纯度CD4?T淋巴细胞（90.13%±3.7%）及CD25?Foxp3?Treg细胞（69.6%±3.4%），CXCR6-KD3#敲低效率最高（P<0.05）。与对照组比较，CXCR6敲低组靶细胞裂解率升高（P<0.01），Treg细胞中γ干扰素（IFN-γ）及肿瘤坏死因子α（TNF-α）水平升高（P<0.01），Treg细胞中PI16 mRNA表达水平升高（P<0.01），差异基因富集于胞外基质空间、细胞迁移调控及磷脂酰肌醇3-激酶（PI3K）-蛋白激酶B（Akt）通路。结论 CXCR6与10种实体瘤的免疫检查点分子表达及CD4?T淋巴细胞浸润高度相关，主要定位于肿瘤免疫微环境。敲低Treg细胞中CXCR6表达可减弱其免疫抑制功能并促进CTLs分泌相关细胞因子，其机制可能与上调Treg细胞中PI16 mRNA表达水平有关。

关键词: CXC 基序趋化因子受体6, 免疫检查点, 调节性T淋巴细胞, 细胞毒性T淋巴细胞, 免疫逃逸, 肿瘤微环境, 免疫治疗靶点

Abstract:

Objective To discuss the relationship between CXC motif chemokine receptor 6 （CXCR6） expression in tumor tissue and the expression of immune checkpoint molecules as well as the infiltration abundance of CD4? T lymphocytes， and to clarify its impact in the function of regulatory T lymphocytes （Tregs） and underlying mechanism. Methods Bioinformatics analysis was performed using Tumor Immune Estimation Resource （TIMER） database. Transcriptomic and immune infiltration data of 10 solid tumors were collected. Pearson correlation analysis was used to analyze the correlations of CXCR6 with immune checkpoint molecules and the infiltration abundance of CD4? T lymphocytes. In the experiments， the human peripheral blood mononuclear cells （PBMCs） from healthy individuals were used. The CD4? T lymphocytes were isolated and expanded， and then induced into the Treg cells. Recombinant lentiviral vectors of CXCR6 shRNA1-4# were constructed. The experiment was divided into blank group （without Treg）， empty vector Treg group（control group）， and CXCR6 knockdown group. After lentiviral transfection， the Treg cells were co-cultured with CD8? cytotoxic T lymphocytes （CTLs） and T2 tumor target cells. Flow cytometry was used to detect the purity of Treg cells； fluorometric counting method was used to detect the lysis rate of target cells； enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of cytokines in the cells； RNA sequencing was used to detect the expression levels of differential genes in the cells； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the knockdown efficiency of CXCR6 and the expression level of peptidase inhibitor 16（PI16） mRNA. Results The bioinformatics analysis results showed that CXCR6 was highly positively correlated with immune checkpoint molecules and the infiltration abundance of CD4? T lymphocytes in 10 solid tumors （P<0.01）， and was negatively correlated with tumor purity （except for glioblastoma）（P<0.01）. The flow cytometry results showed that high-purity CD4? T lymphocytes （90.13%±3.7%） and CD25?Foxp3? Treg cells （69.6%±3.4%） were successfully obtained. The RT-qPCR results showed that CXCR6-KD3# had the highest knockdown efficiency （P<0.05）. The fluorometric counting method results showed that compared with control group， the lysis rate of target cells in CXCR6 knockdown group was increased （P<0.01）. The ELISA results showed that compared with empty control group， the levels of interferon-γ （IFN-γ） and tumor necrosis factor-α（TNF-α） in CXCR6 knockdown group were increased （P<0.01）. The RT-qPCR results showed that compared with control group， the expression level of PI16 mRNA in Treg cells in CXCR6 knockdown group was increased （P<0.01）. The RNA sequencing results showed that the differential genes were enriched in the extracellular matrix space， regulation of cell migration， and phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt） pathway. Conclusion CXCR6 is highly correlated with the expression of immune checkpoint molecules and CD4? T lymphocyte infiltration in 10 solid tumors， and is mainly localized in the tumor immune microenvironment. Knockdown of CXCR6 expression in Treg cells can attenuate their immunosuppressive function and promote the secretion of related cytokines by CTLs， and the mechanism may be related to the upregulation of PI16 mRNA expression level in the Treg cells.

Key words: CXC Motif chemokine receptor 6, Immune checkpoint, Regulatory T lymphocyte, Cytotoxic T lymphocyte, Immune escape, Tumor microenvironment, Immunotherapy target

中图分类号:

R392.1

李佳鑫,张欣阳. 趋化因子受体6表达在肿瘤免疫微环境中作用的生物信息学分析及其对调节性T淋巴细胞功能的影响[J]. 吉林大学学报(医学版), 2026, 52(4): 881-892.

Jiaxin LI,Xinyang ZHANG. Bioinformatics analysis on effect of chemokine receptor 6 expression on tumor immune microenvironment and its impact on regulatory T lymphocyte function[J]. Journal of Jilin University(Medicine Edition), 2026, 52(4): 881-892.

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参考文献 31

[1]	SAKAGUCHI S， MIYARA M， COSTANTINO C M， et al. FOXP3⁺ regulatory T cells in the human immune system［J］. Nat Rev Immunol， 2010， 10（7）：490-500.
[2]	DENG L， CHEN N Y， LI Y， et al. CXCR6/CXCL16 functions as a regulator in metastasis and progression of cancer［J］. Biochim Biophys Acta BBA Rev Cancer， 2010， 1806（1）： 42-49.
[3]	BAO N D， FU B， ZHONG X L， et al. Role of the CXCR6/CXCL16 axis in autoimmune diseases［J］. Int Immunopharmacol， 2023， 121： 110530.
[4]	MENG J， YANG Y L， LV J J， et al. CXCR6 expression correlates with radiotherapy response and immune context in triple-negative breast cancer-experimental studies［J］. Int J Surg， 2024， 110（8）： 4695-4707.
[5]	胡文露，张盼盼，贺玉杰，等. 血清γ-谷氨酰转移酶水平与抗黑色素瘤分化相关基因5抗体阳性皮肌炎早期死亡的关联性［J］. 郑州大学学报（医学版）， 2026， 61（3）：166-168.
[6]	FRIDMAN W H， PAGÈS F， SAUTÈS-FRIDMAN C， et al. The immune contexture in human tumours： impact on clinical outcome［J］. Nat Rev Cancer， 2012， 12（4）： 298-306.
[7]	MABROUK N， TRAN T， SAM I， et al. CXCR6 expressing T cells： Functions and role in the control of tumors［J］. Front Immunol， 2022， 13： 1022136.
[8]	LI L T， HUANG Y F， XUE R Z， et al. T cell-mediated mechanisms of immune-related adverse events induced by immune checkpoint inhibitors［J］. Crit Rev Oncol， 2025， 213： 104808.
[9]	LI T T， PAN J， CHEN H Q， et al. CXCR6-based immunotherapy in autoimmune， cancer and inflammatory infliction［J］. Acta Pharm Sin B， 2022， 12（8）： 3255-3262.
[10]	LIANG Z， TANG Z W， ZHU C J， et al. Intestinal CXCR6⁺ ILC3s migrate to the kidney and exacerbate renal fibrosis via IL-23 receptor signaling enhanced by PD-1 expression［J］. Immunity， 2024， 57（6）： 1306-1323.e8.
[11]	LESCH S， BLUMENBERG V， STOIBER S， et al. T cells armed with C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours［J］. Nat Biomed Eng， 2021， 5（11）： 1246-1260.
[12]	LI T W， FAN J Y， WANG B B， et al. TIMER： a web server for comprehensive analysis of tumor-infiltrating immune cells［J］. Cancer Res， 2017， 77（21）： e108-e110.
[13]	吴琴. PI16通过DOT1L影响CD4+CD25+Foxp3+调节性T细胞数量及功能参与类风湿关节炎发病的研究［D］. 南京：南京医科大学，2018.
[14]	PU J， LIU T， ZHOU Y， et al. T cells in cancer： mechanistic insights and therapeutic advances［J］. Biomark Res， 2025， 13： 97.
[15]	GAVIL N V， CHENG K， MASOPUST D. Resident memory T cells and cancer［J］. Immunity， 2024， 57（8）：1734-51.
[16]	MA K， XU Y， CHENG H， et al. T cell-based cancer immunotherapy： opportunities and challenges［J］. Sci Bull， 2025， 70（11）：1872-1890.
[17]	OLIVEIRA G， WU C J. Dynamics and specificities of T cells in cancer immunotherapy［J］. Nat Rev Cancer， 2023， 23：295-316.
[18]	STEIER Z， KIM EJY， AYLARD DA， et al. The CD4 versus CD8 T cell fate decision： A Multiomics-Informed perspective［J］. Annu Rev Immunol， 2024， 42（1）：235-58.
[19]	GALASSI C， CHAN TA， VITALE I， et al. The hallmarks of cancer immune evasion［J］. Cancer Cell， 2024， 42（11）：1825-63.
[20]	MADDEN M Z， RATHMELL J C. The complex integration of T-cell metabolism and immunotherapy［J］. Cancer Discov， 2021， 11（7）：1636-43.
[21]	ZONG Y， DENG K， CHONG WP. Regulation of Treg cells by cytokine signaling and co-stimulatory molecules［J］. Front Immunol， 2024， 15：1387975.
[22]	CAO X， CAI S F， FEHNIGER T A， et al. Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance ［J］. Immunity， 2007 Oct.27（4）： 635-646.
[23]	MARANGONI F， et al. Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop［J］. Cell， 184（15）：3998-4015.e19.
[24]	ZAPPASODI R， SERGANOVA I， COHEN IJ， et al. CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours［J］. Nature， 2021， 591：652-658.
[25]	MOTOHASHI S， NISHIKAWA H. Regulatory T cells in the tumour microenvironment and their therapeutic targeting［J］. Nat Rev Clin Oncol， 2018， 15（5）：329-346.
[26]	DI PILATO M， KFURI-RUBENS R， PRUESSMANN J N， et al.CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment［J］. Cell， 2021， 184（17）：4512-4530.
[27]	KIM M J， SUN H J， SONG Y S， et al.CXCL16 positively correlated with M2-macrophage infiltration， enhanced angiogenesis， and poor prognosis in thyroid cancer［J］. Sci Rep， 2019；（9）：13288
[28]	MIR H， KAPUR N， GALES D N， et al. CXCR6-CXCL16 axis promotes breast cancer by inducing oncogenic signaling［J］. Cancers， 2021， 13（14）： 3568.
[29]	SUN Y K， LIN S Y， WANG H， et al. Regulatory role of PI16 in autoimmune arthritis and intestinal inflammation： implications for Treg cell differentiation and function［J］. J Transl Med， 2024， 22（1）： 327.
[30]	NICHOLSON I C， MAVRANGELOS C， BIRD D R， et al. PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20［J］. Cell Immunol， 2012， 275（1/2）： 12-18.
[31]	BROQUET A， GOURAIN V， GORONFLOT T， et al. Sepsis-trained macrophages promote antitumoral tissue-resident T cells［J］. Nat Immunol， 2024， 25（5）： 802-819.

Sequence ID	Sense（5'-3'）	Anti-sense（5'-3'）
CXCR6-KD1#	CCGGGCGCATTCTCATGGAGAAGTTCTCGAGAACTTCTCCATGAGAATGCGCTTTTT	AAAAAGCGCATTCTCATGGAGAAGTTCTCGAGAACTTCTCCATGAGAATGCGCCCGG
CXCR6-KD2#	CCGGGCTTGCTCATCTGGGTGATATCTCGAGATATCACCCAGATGAGCAAGCTTTTT	AAAAAGCTTGCTCATCTGGGTGATATCTCGAGATATCACCCAGATGAGCAAGCCCGG
CXCR6-KD3#	CCGGCTCACCATGATTGTCTGCTATCTCGAGATAGCAGACAATCATGGTGAGTTTTT	AAAAACTCACCATGATTGTCTGCTATCTCGAGATAGCAGACAATCATGGTGAGCCGG
CXCR6-KD4#	CCGGCTGAGGACAATTCCAAGACTTCTCGAGAAGTCTTGGAATTGTCCTCAGTTTTT	AAAAACTGAGGACAATTCCAAGACTTCTCGAGAAGTCTTGGAATTGTCCTCAGCCGG