CD4+CD25+foxp3+调节性T细胞通过NKG2D增强急性髓性白血病中NK细胞毒性

doi:10.3877/cma.j.issn.2095-1221.2024.04.003

目的

CD4⁺CD25⁺foxp3⁺调节性T细胞在急性髓性白血病中调控NKG2D介导NK细胞毒性的研究。

方法

免疫磁珠法分离急性髓性白血病（AML）和健康对照外周血NK细胞和CD4⁺CD25⁺细胞，分析foxp3与NKG2D表达，CD4⁺CD25⁺foxp3⁺细胞的比例与NK细胞的细胞毒作用相关性。Spearman法分析CD4⁺CD25⁺foxp3⁺细胞比例与NK细胞的细胞毒性和NGK2D相关性。foxp3过表达处理AML外周血淋巴细胞后，流式细胞术检测NKG2D，IFN-γ和CD107a表达，LDH法检测NK细胞毒性。GEPIA2数据库分析AML患者NKG2D和foxp3基因表达相关性，TCGA数据库分析AML转录组数据中与NKG2D表达高相关性基因。IL-2 （10 μg/mL），IL-15 （10 μg/mL）处理NK细胞24 h后，流式细胞术检测NKG2D和CD107a表达，酶联免疫反应法检测NK细胞分泌的IFN-γ，LDH法检测NK细胞毒性。采用t检验等方法统计分析组间差异及变量相关性。

结果

与对照比较，AML组NK细胞中NKG2D表达（平均荧光强度MFI：942.25 ± 117.17比3245.28 ± 367.43）降低（P < 0.001）。foxp3表达与NKG2D呈正相关性（r = 0.590，P < 0.001），NKG2D表达百分数与外周血淋巴细胞中CD4⁺CD25⁺foxp3⁺细胞的比例呈正相关（r = 0.708，P = 0.002），AML患者外周血NK细胞活力也与CD4⁺CD25⁺foxp3⁺细胞的比例呈正相关（r = 0.655，P = 0.006）。与OE-NC比较，OE-foxp3组细胞中NKG2D的表达水平（MFI：2115.37 ± 269.53比914.28 ± 103.36）、杀伤能力（58.24 % ± 3.17%比24.18 % ± 2.35%）、NK细胞毒性评估指标CD107a （45.43% ± 1.28%比18.24% ± 1.49%）和IFN-γ水平（24.16 % ± 1.17%比16.28 % ± 0.95%）升高（P均< 0.001）。TCGA数据分析发现，NKG2D表达与CD3G、IL-3、IL-2、CD3E、IL-10、IL-17、IL-7R、IL-15、EZH2等基因相关性较高，流式细胞术检测发现，与对照比较，IL-2和IL-15处理后NK细胞的表面受体NKG2D和CD107a表达、IFN-γ水平、NK细胞毒性均升高。

结论

AML外周血中，NK细胞表面受体NKG2D低于正常对照，其中CD4⁺CD25⁺foxp3⁺调节性T细胞通过IL-2和IL-15影响NKG2D表达进而调控NK细胞毒性。

关键词: 急性髓性白血病, NK细胞, NKG2D, CD4⁺CD25⁺foxp3⁺, IL-2

Objective

Exploring the effects and mechanisms of CD4⁺CD25⁺foxp3⁺ regulatory T cells in regulating NK cytotoxicity through NKG2D in acute myeloid leukemia.

Methods

Immunomagnetic bead method was used to isolate peripheral blood NK cells and CD4⁺CD25⁺ cells from acute myeloid leukemia (AML) and healthy controls. The expression of foxp3 and NKG2D, and the relationship between the proportion of CD4⁺CD25⁺foxp3⁺ cells and the cytotoxicity of NK cells were analyzed. Spearman method was used to analyze the correlation between CD4⁺CD25⁺foxp3⁺ cell ratio and NK cell cytotoxicity as well as NGK2D. After overexpressed foxp3 in AML peripheral blood lymphocytes, flow cytometry was used to detect the expression of NKG2D, IFN-γ and CD107a, LDH assay was used to detect the toxicity of NK cell. The correlation between NKG2D and foxp3 gene expression in AML patients was analyzed by GEPIA2 database, while the genes with high correlation of NKG2D expression in AML transcriptome data was analyzed by TCGA database. After treating NK cells with IL-2 (10 μg/mL) and IL-15 (10 μg/mL) for 24 hours, 24 hours after treated with IL-2 (10 μg/mL) and IL-15 (10 μg/mL) in NK cells, flow cytometry was used to detect the expression of NKG2D and CD107a, enzyme-linked immunosorbent assay was used to detect the secretion of IFN-γ by NK cells, LDH assay to detect NK cell toxicity. Statistical analysis of inter group differences and variable correlations using t-tests and other methods.

Results

Compared to the control group, the expression of NKG2D in NK cells in the AML group was significantly reduced (MFI: 942.25 ± 117.17 vs 3245.28 ± 367.43, P < 0.001). The expression of foxp3 is positively correlated with NKG2D (r = 0.590, P < 0.001), and the percentage of NKG2D expression is positively correlated with the proportion of CD4⁺CD25⁺foxp3⁺ cells in peripheral blood lymphocytes (r = 0.708, P = 0.002). The activity of peripheral blood NK cells in AML patients is also positively correlated with the proportion of CD4⁺CD25⁺foxp3⁺ cells (r = 0.655, P = 0.006). Compared to the OE-NC group, the expression level of NKG2D (MFI: 2115.37 ± 269.53 vs 914.28 ± 103.36), the cell killing ability (58.24 % ± 3.17 %vs 24.18 % ± 2.35 %), the NK cell toxicity evaluation indicators CD107a and the level of IFN-γ in the OE-foxp3 group cells were significantly increased (45.43 % ± 1.28 %vs 18.24 % ± 1.49%), (24.16 % ± 1.17 %vs 16.28 % ± 0.95 %) (all P < 0.001). TCGA data analysis found that the expression of NKG2D was highly correlated with genes such as CD3G, IL-3, IL-2, CD3E, IL-10, IL-17, IL-7R, IL-15, EZH2, etc. Flow cytometry analysis showed that after treatment with IL-2 and IL-15, the expression of surface receptors NKG2D and CD107a on NK cells was significantly increased, and IFN-γ level was increases, NK cell toxicity is enhanced.

Conclusion

In peripheral blood of AML, the surface receptor NKG2D of NK cells is lower than that of control, and CD4⁺CD25⁺foxp3⁺ regulatory T cells regulate NK cell toxicity through the expression of NKG2D effecting by IL-2 and IL-15.

Key words: AML, NK cell, NKG2D, CD4⁺CD25⁺foxp3⁺, IL-2

图1 AML患者外周血NK细胞表面受体NKG2D低表达注：a图为流式细胞术检测AML患者及健康对照外周血NK细胞中NKG2D的表达；b图为AML组和对照NKG2D表达分析，与对照比较，^aP < 0.0001

图2 外周血淋巴细胞中CD4⁺ CD25⁺ foxp3⁺细胞比例与NK活性呈正相关注：a图为GEPIA2数据库分析foxp3与NKG2D在AML患者中表达相关性；b图为分析AML患者（AML）外周血淋巴细胞中NKG2D表达与CD4⁺CD25⁺foxp3⁺细胞群比例；c图为分析AML患者外周血淋巴细胞中NK细胞活性与CD4⁺CD25⁺foxp3⁺细胞群比例

图3 过表达foxp3促进外周血NK细胞NKG2D表达及细胞毒性注：a图为流式细胞术检测OE-NC组及OE-foxp3组NK细胞中NKG2D表达；b、c图为OE-NC组及OE-foxp3组NK细胞中NKG2D表达、NK细胞活性分析，与OE-NC比较，^aP < 0.001

图4 过表达foxp3促进外周血NK细胞中CD107a和IFN-γ的表达注：a ~ c图为流式细胞术检测OE-NC组及OE-foxp3组NK细胞中CD107a和IFN-γ的表达水平；d图为OE-NC组及OE-foxp3组NK细胞中CD107a和IFN-γ的表达水平分析，与OE-NC比较，^aP < 0.001

图5 NKG2D与CD3G，IL3等基因的相关性分析

表1 IL2，IL15处理NK细胞后NKG2D和CD107a表达（%，

± s）

分组	实验次数	NKG2D	CD107a
对照	3	13.29 ± 0.32	4.87 ± 0.42
IL2	3	18.09 ± 0.28^a	7.18 ± 0.26^a
IL15	3	21.23 ± 0.16^b	13.27 ± 0.24^b
F值		104.026	128.083
P值		< 0.001	< 0.001

表1 IL2，IL15处理NK细胞后NKG2D和CD107a表达（%，

± s）

分组	实验次数	NKG2D	CD107a
对照	3	13.29 ± 0.32	4.87 ± 0.42
IL2	3	18.09 ± 0.28^a	7.18 ± 0.26^a
IL15	3	21.23 ± 0.16^b	13.27 ± 0.24^b
F值		104.026	128.083
P值		< 0.001	< 0.001

图6 foxp3调控IL15、IL2增强NK细胞毒性注：a图为IL2，IL15处理后NK细胞中IFN-γ含量检测；b图为IL2，IL15处理后NK细胞毒性检测；c图为OE-NC组及OE-foxp3组细胞中IL2，IL15水平检测；与对照比较，^aP < 0.05，^bP < 0.001

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CD4⁺CD25⁺foxp3⁺ regulatory T cells promote NK cytotoxicity in acute myeloid leukemia via NKG2D