Immune characterization of the HBHA-specific response in Mycobacterium tuberculosis-infected patients with or without HIV infection

IntroductionRD1-based Interferon-γ Release Assays (IGRAs) cannot distinguish latent from active tuberculosis (TB) disease. Conversely, a positive response to heparin-binding haemagglutinin (HBHA)-based IGRAs, among TB-infected subjects, correlates with Mycobacterium tuberculosis (Mtb) containment and low risk of TB progression. The aim of this study was to characterize HBHA-immune responses in HIV-infected and uninfected subjects with active TB or latent TB infection (LTBI).Methods49 subjects were prospectively enrolled: 22 HIV-uninfected (13 TB, 9 LTBI) and 27 HIV-infected (12 HIV-TB, 15 HIV-LTBI). Whole blood and peripheral blood mononuclear cells were stimulated with HBHA and RD1 antigens. Interferon (IFN)γ release was evaluated by ELISA whereas cytokine profile [IFNγ, tumor necrosis (TNF)α, interleukin (IL)2] and phenotype (CD45RA, CCR7) by flow cytometry.ResultsAmong LTBI individuals, HBHA stimulation induced IFNγ release in all the HIV-uninfected, while, only 4/15 HIV-infected responded. Within the active TB, only 5/13 HIV-uninfected and 1/12 HIV-TB patients responded. Interestingly, by cytometry we showed that CD4+ T-cells response to HBHA was significantly impaired in the HIV-infected subjects with TB or LTBI compared to the HIV-uninfected subjects. The phenotype of HBHA-specific CD4 T-cells showed a predominantly central memory (CM) and effector memory (EM) phenotype without differences among the groups. Differently, HBHA-specific CD8+ T-cells, showed mainly a CM and naïve phenotype in LTBI group while TB, HIV-LTBI and HIV-TB groups were characterized by EM or terminally differentiated phenotypes. Interestingly, differently than what observed for RD1, the cytokine profile of HBHA-specific T-cells evaluated by cytometry showed that the CD4+ T-cells were mostly monofunctional. Conversely, CD8-specific T-cells were mostly monofunctional for both HBHA and RD1 stimulations.ConclusionsThese results characterize the impact of HIV infection in CD4- and CD8-specific response to HBHA in both LTBI and TB patients. HIV infection impairs the CD4 response to HBHA and likely this may lead to an impairment of TB control.

引言
基于RD1的γ干扰素释放试验（Interferon-γ Release Assays, IGRAs）无法区分潜伏性结核病与活动性结核病。与之相反，在结核分枝杆菌（Mycobacterium tuberculosis, Mtb）感染者中，针对肝素结合血凝素（heparin-binding haemagglutinin, HBHA）的γ干扰素释放试验阳性结果与结核分枝杆菌定植控制及结核病进展低风险相关。本研究旨在明确合并与未合并HIV感染的活动性结核病及潜伏性结核感染（latent TB infection, LTBI）受试者体内针对肝素结合血凝素的免疫应答特征。

方法
本研究前瞻性入组49名受试者：22名HIV阴性者（13名活动性结核病患者、9名潜伏性结核感染者），27名HIV感染者（12名HIV相关结核病患者、15名HIV相关潜伏性结核感染者）。采用肝素结合血凝素与RD1抗原对全血及外周血单个核细胞进行刺激。通过酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay, ELISA）检测γ干扰素（Interferon, IFNγ）释放水平，同时借助流式细胞术分析细胞因子谱[γ干扰素、肿瘤坏死因子（Tumor Necrosis Factor, TNF）α、白细胞介素（Interleukin, IL）2]及细胞表型（CD45RA、CCR7）。

结果
在潜伏性结核感染者中，所有HIV阴性受试者经肝素结合血凝素刺激后均可检测到γ干扰素释放，而HIV感染者中仅4/15出现应答。在活动性结核病患者中，HIV阴性者仅5/13、HIV相关结核病患者仅1/12出现应答。值得注意的是，流式细胞术结果显示，与HIV阴性受试者相比，合并结核病或潜伏性结核感染的HIV感染者体内针对肝素结合血凝素的CD4+ T细胞应答显著受损。肝素结合血凝素特异性CD4+ T细胞的表型以中央记忆型（Central Memory, CM）和效应记忆型（Effector Memory, EM）为主，各组间无显著差异。与之不同的是，潜伏性结核感染组的肝素结合血凝素特异性CD8+ T细胞主要表现为中央记忆型和初始表型，而结核病组、HIV相关潜伏性结核感染组及HIV相关结核病组则以效应记忆型或终末分化表型为特征。有趣的是，与RD1的检测结果不同，流式细胞术分析显示肝素结合血凝素特异性CD4+ T细胞大多为单功能细胞；反之，针对肝素结合血凝素与RD1抗原刺激的CD8+特异性T细胞均大多为单功能细胞。

结论
本研究结果明确了HIV感染对潜伏性结核感染及活动性结核病患者体内针对肝素结合血凝素的CD4+及CD8+特异性T细胞应答的影响。HIV感染会削弱针对肝素结合血凝素的CD4+ T细胞应答，这可能会损害结核病的防控效果。