Objective: To investigate the effects of low molecular peptide of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg-10k) on the production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in peripheral blood T cells and test the feasibility of differential diagnosis between pulmonary tuberculosis (PTB) and latent tuberculosis infection (LTBI) by assessing the number of Mtb-HAg-10k-stimulated IFN-γ-producing T cells.
Methods: Peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood of 10 healthy adults, 6 individuals with LTBI and 13 patients with PTB. The PBMCs were cultured in the presence of Mtb-HAg-10k obtained by ultrafiltration centrifugation, with Mtb-HAg and phytohaemagglutinin (PHA) as the controls. The proportions of TNF-α- and IFN-γ-producing cells in the T cell subsets were detected by flow cytometry (FCM), and the number of IFN-γ-producing cells from patients with PTB and LTBI was detected with ELISPOT.
Results: Flow cytometry showed that Mtb-HAg-10k exposure resulted in a significantly higher proportion of TNF-α-producing γδT cells than that of IFN-γ-producing γδT cells in the PBMCs (P<0.01). Compared with the PBMCs exposed to PHA, the PBMCs exposed to Mtb-HAg-10k exhibited a significantly greater proportion of γδT cells that produced both TNF-α and IFN-γ (P<0.01) but a significantly lower proportion of αβT cells producing both TNF-α and IFN-γ (P<0.01). Mtb-HAg-10k exposure of the PBMCs caused a significant reduction in the number of IFN-γ-producing cells as compared with Mtb-HAg and PHA treatments (P<0.01), and this reduction was more obvious in PBMCs from patients with PTB than in those from individuals with LTBI (P<0.01).
Conclusion: Mtb-HAg-10k can markedly induce γδT cells in the PBMCs to produce TNF-α and IFN-γ, and detection of the number of IFN-γ-producing cells in the PBMCs following Mtb-HAg-10k stimulation helps in the differential diagnosis between pulmonary tuberculosis and latent tuberculosis infection.
目的: 研究结核杆菌耐热抗原小分子多肽(Mtb-HAg-10k)刺激人外周血T细胞产生细胞因子肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)的作用特点及肺结核患者和潜伏性感染者IFN-γ产生细胞的数量差异，初步探讨Mtb-HAg-10k作为诊断抗原鉴别肺结核和潜伏性结核感染的可行性。
方法: 以超滤离心法获得的Mtb-HAg-10k为刺激剂作用于人外周血单个核细胞(PBMCs)，并以Mtb-HAg、植物血凝素PHA作为对照，用多色流式细胞术检测T细胞亚群中TNF-α和IFN-γ产生细胞比例，酶联免疫斑点法检测肺结核患者和潜伏性感染者PBMCs中IFN-γ产生细胞数量。
结果: 流式细胞术检测人外周血Mtb-HAg-10k特异性γδT细胞中TNF-α产生细胞比例显著高于IFN-γ产生细胞比例(P < 0. 0.01)；与PHA刺激组相比，γδT细胞中TNF-α和IFN-γ产生细胞比例增高(P < 0. 0.01)，而αβT细胞中TNF-α和IFN-γ产生细胞比例显著降低；酶联免疫斑点法检测Mtb-HAg-10k刺激的健康者PBMCs中IFN-γ产生细胞数量低于Mtb-HAg刺激组和PHA对照组(P < 0. 0.01)；肺结核患者IFN-γ产生细胞数量低于潜伏性结核感染者(P < 0. 0.01)。
结论: 以Mtb-HAg-10K为刺激剂可使人外周血γδT细胞优势产生TNF-α和IFN-γ，通过酶联免疫斑点法检测PBMCs中IFN-γ产生细胞数量差异有助于鉴别肺结核和潜伏性结核感染。