T细胞免疫球蛋白和黏蛋白结构域蛋白-3对慢性心力衰竭患者T细胞功能的调控作用

The modulatory activity of T cell immunoglobulin and mucin domain-containing protein 3 on T lymphocytes in patients with chronic heart failure

目的观察慢性心衰患者中T细胞免疫球蛋白和黏蛋白结构域蛋白-3(TIM-3)的表达及其对T细胞的调控作用。方法收集2018年1至10月在郑州大学第一附属医院心血管内科住院治疗的慢性心衰患者86例(心衰组)和在郑州大学第一附属医院接受查体的健康对照32名(健康对照组),分离外周血单个核细胞,流式细胞术检测CD4+T细胞和CD8+T细胞中TIM-3的表达。分选CD4+T细胞和CD8+T细胞,使用抗TIM-3抗体刺激培养。酶联免疫吸附试验检测CD4+T细胞培养上清中干扰素-γ(IFN-γ)、白细胞介素(IL)-4、IL-10、IL-35、IL-17、IL-22水平和CD8^+T细胞培养上清中肿瘤坏死因子(TNF-α)和IFN-γ水平,实时定量PCR法检测CD4+T细胞中转录因子T-bet、GATA-3、FoxP3、RORγt和CD8+T细胞中穿孔素、颗粒酶B mRNA相对表达量。组间比较采用t检验或配对t检验进行。结果心衰组TIM-3^+CD4^+T细胞比例高于健康对照组(3.47%±1.06%比0.92%±0.27%,P<0.001),心衰组TIM-3^+CD8^+T细胞比例亦高于健康对照组(6.12%±1.91%比1.77%±0.63%,P<0.001)。慢性心衰患者存在CD4+T细胞和CD8+T细胞功能不全,表现为心衰组CD4^+T细胞分泌IFN-γ、IL-17和IL-22水平低于健康对照组,分泌IL-10和IL-35水平高于健康对照组(均P<0.05)。心衰组CD4^+T细胞中T-bet和RORγt mRNA相对表达量低于健康对照组(均P<0.01),FoxP3 mRNA相对表达量高于健康对照组(1.93±0.88比0.97±0.28,P=0.031)。心衰组CD8+T细胞分泌TNF-α和IFN-γ水平低于健康对照组(均P<0.05),穿孔素和颗粒酶B mRNA相对表达量亦低于健康对照组(均P<0.05)。抗TIM-3抗体刺激慢性心衰患者纯化的CD4+T细胞,分泌IFN-γ、IL-17和IL-22水平高于无抗TIM-3抗体刺激(均P<0.05),分泌IL-35水平则低于无抗TIM-3抗体刺激[(61±13)ng/ml比(72±17)ng/ml,P=0.029]。抗TIM-3抗体刺激后CD4+T细胞中T-bet和RORγt mRNA相对表达量高于无TIM-3抗体刺激(均P<0.05)。抗TIM-3抗体刺激慢性心衰患者纯化的CD8+T细胞后,分泌TNF-α和IFN-γ水平高于无抗TIM-3抗体刺激(均P<0.01),穿孔素和颗粒酶B mRNA相对表达量亦高于无抗TIM-3抗体刺激(均P<0.05)。结论慢性心衰患者中T细胞表面TIM-3升高表达,TIM-3可能参与了慢性心衰患者T细胞功能不全的调控。

Objective To investigate the expression of T cell immunoglobulin and mucin domain-containing protein 3(TIM-3)in patients with chronic heart failure and its modulatory activity on T lymphocytes.Methods Eighty-six patients with chronic heart failure(CHF group)who were hospitalized in Department of Cardiology,the First Affiliated Hospital of Zhengzhou University between January and October 2018 were enrolled in the study.Meanwhile,thirty-two healthy controls(HC group)who received healthy examination were also selected.Peripheral blood mononuclear cells were isolated,and TIM-3 expression of CD4+and CD8+T cells was investigated by flow cytometry.CD4+and CD8+T cells were purified,and were stimulated by anti-TIM-3 antibody.Interferon-γ(IFN-γ),interleukin(IL)-4,IL-10,IL-35,IL-17,and IL-22 expressions in the supernatants of cultured CD4^+T cells and tumor necrosis factor-α(TNF-α)and IFN-γexpressions in the supernatants of cultured CD8^+T cells were measured by enzyme-linked immunosorbent assay.mRNA expressions of T-bet,GATA-3,FoxP3,and RORγt in CD4^+T cells and perforin and granzyme B in CD8^+T cells were semi-quantified by real-time PCR.Student t test or paired t test was used for comparisons between the two groups.Results TIM-3^+CD4^+T cell percentage significantly increased in CHF group than that of HC group(3.47%±1.06%vs 0.92%±0.27%,P<0.001).TIM-3^+CD8^+T cell percentage also notably elevated in CHF group compared to HC group(6.12%±1.91%vs 1.77%±0.63%,P<0.001).CD4^+T and CD8^+T cells were dysfunctional in chronic heart failure.The levels of IFN-γ,IL-17,and IL-22 secreted by purified CD4^+T cells significantly reduced in CHF group,while IL-10 and IL-35 expressions elevated in CHF group(all P<0.05).The relative mRNA expression levels of T-bet and RORγt in CD4^+T cells remarkably decreased in CHF group than those of HC group(all P<0.01),while relative expression level of FoxP3 mRNA increased in CHF group(1.93±0.88 vs 0.97±0.28,P=0.031).The levels of TNF-αand IFN-γproduced by purified CD8^+T cells notably reduced in CHF group than those of HC group(all P<0.05),and relative mRNA expression levels of perforin and granzyme B also decreased in CHF group(all P<0.05).The levels of anti-TIM-3 antibody stimulation-produced IFN-γ,IL-17,and IL-22 increased(all P<0.05)but IL-35 secretion reduced[(61±13)ng/ml vs(72±17)ng/ml,P=0.029]by CD4^+T cells in CHF group.The relative mRNA expression levels of T-bet and RORγt also elevated in response to anti-TIM-3 antibody stimulation(all P<0.05).Anti-TIM-3 antibody stimulation promoted TNF-αand IFN-γproduction by CD8+T cells in CHF group(all P<0.01).The relative mRNA expression levels of perforin and granzyme B also increased(all P<0.05).Conclusion TIM-3 was increasingly expressed in T cells from patients with chronic heart failure,and might take part in the regulation of T cell dysfunction in chronic heart failure.