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Abstract：
BACKGROUND: Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown.
METHODS: Mtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni-NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs.
RESULTS: In this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αβ T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb.
CONCLUSIONS: Activated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.
METHODS: Mtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni-NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs.
RESULTS: In this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αβ T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb.
CONCLUSIONS: Activated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.
摘要：
背景：结核分枝杆菌耐热抗原（Mtb-HAg）是一种肽抗原，在121°C高压灭菌20分钟后，从分枝杆菌细胞质释放到结核分枝杆菌（Mtb）减毒H37Ra菌株的上清液中。Mtb-HAg可以在体外特异性诱导γδT细胞增殖。然而,目前尚不清楚Mtb-HAg的确切组成和负责其功能的蛋白质抗原。
方法：对从MtbH37Ra菌株中提取的Mtb-HAg进行LC-MS质谱分析。通过基因工程技术，以pET-28a为质粒，在大肠杆菌中重组表达了12种鉴定的蛋白质组分，并通过Ni-NTA琼脂糖树脂纯化，以刺激来自不同健康个体的外周血单核细胞（PBMC）。通过流式细胞术确定γδT细胞和主要γδT细胞亚群类型的增殖以及TNF-α和IFN-γ的产生。使用MACS分离柱分离纯化其增殖的γδT细胞，和MtbH37Ra感染的THP-1与分离和纯化的γδT细胞共培养，以通过计数CFU来定量分枝杆菌活力。
结果：在这项研究中，减毒MtbH37Ra菌株的Mtb-HAg通过LC-MS质谱分析，共鉴定出564种蛋白质。对鉴定的蛋白质级分的分析表明，主要的蛋白质成分包括热休克蛋白和Mtb特异性抗原蛋白。其中10种蛋白的重组表达通过大肠杆菌基因工程技术成功地刺激来自不同健康个体的PBMCs,但是两种蛋白质，EsxJ和EsxA,没有表达。流式细胞仪检测结果显示,与IL-2对照相比，HspX,GroEL1和GroES特异性诱导γδT细胞扩增，以Vγ2δ2T细胞为主要亚群，以及抗微生物细胞因子TNF-α和IFN-γ的分泌。相比之下,HtpG,DnaK,GroEL2,HbhA,Mpt63,EsxB,和EsxN不能促进γδT细胞增殖和TNF-α和IFN-γ的分泌。上述重组蛋白均不能诱导αβT细胞分泌TNF-α和IFN-γ。此外,TNF-α,产生IFN-γ的γδT细胞抑制细胞内Mtb的生长。
结论：Mtb-HAg成分HspX诱导激活的γδT细胞，GroES,GroEL1产生TNF-α，IFN-γ调节巨噬细胞以抑制细胞内Mtb生长。这些数据为后续研究Mtb-HAg体外诱导γδT细胞增殖的机制奠定了基础。以及预防和治疗疫苗和快速诊断试剂的开发。
方法：对从MtbH37Ra菌株中提取的Mtb-HAg进行LC-MS质谱分析。通过基因工程技术，以pET-28a为质粒，在大肠杆菌中重组表达了12种鉴定的蛋白质组分，并通过Ni-NTA琼脂糖树脂纯化，以刺激来自不同健康个体的外周血单核细胞（PBMC）。通过流式细胞术确定γδT细胞和主要γδT细胞亚群类型的增殖以及TNF-α和IFN-γ的产生。使用MACS分离柱分离纯化其增殖的γδT细胞，和MtbH37Ra感染的THP-1与分离和纯化的γδT细胞共培养，以通过计数CFU来定量分枝杆菌活力。
结果：在这项研究中，减毒MtbH37Ra菌株的Mtb-HAg通过LC-MS质谱分析，共鉴定出564种蛋白质。对鉴定的蛋白质级分的分析表明，主要的蛋白质成分包括热休克蛋白和Mtb特异性抗原蛋白。其中10种蛋白的重组表达通过大肠杆菌基因工程技术成功地刺激来自不同健康个体的PBMCs,但是两种蛋白质，EsxJ和EsxA,没有表达。流式细胞仪检测结果显示,与IL-2对照相比，HspX,GroEL1和GroES特异性诱导γδT细胞扩增，以Vγ2δ2T细胞为主要亚群，以及抗微生物细胞因子TNF-α和IFN-γ的分泌。相比之下,HtpG,DnaK,GroEL2,HbhA,Mpt63,EsxB,和EsxN不能促进γδT细胞增殖和TNF-α和IFN-γ的分泌。上述重组蛋白均不能诱导αβT细胞分泌TNF-α和IFN-γ。此外,TNF-α,产生IFN-γ的γδT细胞抑制细胞内Mtb的生长。
结论：Mtb-HAg成分HspX诱导激活的γδT细胞，GroES,GroEL1产生TNF-α，IFN-γ调节巨噬细胞以抑制细胞内Mtb生长。这些数据为后续研究Mtb-HAg体外诱导γδT细胞增殖的机制奠定了基础。以及预防和治疗疫苗和快速诊断试剂的开发。
