免疫细胞

保护性免疫。因此,虽然要求Tfh细胞在动物模型的结核病感染有明确定义，而对人类的结核感染和对疾病的作用尚不清楚。

To study the distribution of Tfh cells in TB infections, we examined Mtb antigen-specific

induction Tfh cells subsets (defined as CD4+，CXCR5+，PD-1+，ICOS2 or CD4+，CXCR5+，PD-，ICOS+ or CD4+，CXCR5+，PD-1+，ICOS+) in PTB and LTB individuals in an area highly endemic for tuberculosis. We observed that active PTB was characterized by diminished

frequencies of Tfh cells ex vivo and in response to TB antigens and by diminished frequencies Tfh cells producing IL-21, a finding that was reflected in circulating plasma levels of IL-21.

本研究是在结核病感染者中了解Tfh细胞的分布情况,我们检测了Mtb特异性诱导Tfh细胞子集在PTB和LTB中，一个区域高度流行的肺结核。我们观察到体外培养中PTB Tfh细胞的活性特点是频率降低。针对结核抗原和减少Tfh细胞频率产生IL-21,这一发现是反映在循环等离子IL-21水平。 IL-10,CTLA-4 and

PD-L1 each appear to play a role in the Tfh homestasis as well. Our data therefore suggest that central defects in Tfh subset differentiation and/or function is a feature of active pulmonary

tuberculous disease. IL- 10,CTLA-4和PD-L1似乎扮演一个角色在Tfh homestasis。我们的数据表明,中央

缺陷Tfh子集分化和/或功能是活动性肺结核结核性疾病的一个特色。

Study population

We studied a group of 50 individuals; 30 with pulmonary TB(PTB) and 20 individuals with latent TB (LTB). Among the 30 individuals with PTB, 19 of these were also used for antibody blocking studies. Individuals with PTB were diagnosed sputum smear and culture positivity. Individuals with LTB were diagnosed on the basis of being positive in the Quantiferon-TB Gold in Tube (Cellestis) assay but having an absence of pulmonary symptoms concurrent with a normal chest radiograph. All subjects had been bacillus Calmette-Guerin (BCG) vaccinated at birth. All the individuals were HIV negative and blood was collected prior to commencement of anti-TB treatment. 我们研究了一群50个人,30与肺结核(PTB)和20个人和潜在的结核病(LTB)。在PTB的30个人,

其中19也用于抗体阻断研究。PTB的人被诊断为痰涂片和培养积极性。个人LTB被诊断的基础上,积极在Quantiferon-TB黄金管(Cellestis)测定但缺乏肺并发症状有正常胸部x光照片。所有科目都卡介苗(BCG)出生时接种疫苗。所有的个人都是消极和HIV病毒的血液收集毕业典礼前抗结核治疗。

Antigens抗原

Mycobacterial antigens - PPD (Statens Serum Institute,Copenhagen, Denmark), ESAT-6 and CFP–10 (both from NIAIDTB antigen repository at BEI resources) were used as antigenic stimuli, and anti-CD3 antibody was used as positive control. Final concentrations were 10mg/ml for PPD, ESAT-6 and CFP-10 and 5mg/ml for anti-CD3. For antibody ELISA, PPD and MTB whole cell lysate (WCL) were used as the antigens. 分枝杆菌抗原——产后抑郁症(史坦顿血清研究所,哥本哈根,

丹麦),ESAT-6和CFP-10(从NIAIDTB抗原库贝资源)作为抗原刺激,和anti-CD3抗体被用来作为积极的控制。最后10毫克/毫升浓度对产后抑郁症,ESAT-6 anti-CD3 CFP-10和5毫克/毫升。结核分枝杆菌抗体ELISA、产后抑郁症和整个细胞溶解产物(水控制法)作为抗原。

In vitro culture体外培养

Intracellular cytokine staining细胞内因子染色 (ELISA)

Discussion

Infection with Mtb can lead to various outcomes that range from active or chronic pulmonary disease, extra-pulmonary TB and latent TB, that occurs when the initial infection is controlled but not completely eliminated. While a number of host immune mechanisms have been described to play a role in the diverging clinical manifestations of TB infection and disease, the immune

mechanisms that contribute directly to disease pathogenesis are still incompletely understood. Tfh cells are a subset of CD4+ T cells that are indispensable for the generation and maintenance of humoral immunity. 结核分枝杆菌感染可以导致不同的结果,从活跃或慢性肺疾病,肺外结核病和潜伏性结

核病,发生在最初的感染控制,但不能完全消除。虽然描述了一系列的宿主免疫机制发挥作用的不同临床表现的结核感染和疾病的免疫机制,有助于疾病的发病机制仍不完全清楚。Tfh细胞CD4 + T细胞的一个子集,是不可或缺的生成和维护体液免疫。It has recently been demonstrated that CXCR5+ T cell accumulate

within ectopic lymphoid structures associated with TB granulomas in humans,non-human

primates and mice. These lymphoid follicles appear to be important for proper localization of T cells in the granulomas, for the optimal activation of macrophages and for protection against TB disease. Thus, while Tfh cells located within the granulomas are clearly important in the immune response to TB, the role of circulating Tfh cells in human TB infection and disease remains

unexplored. 它最近被证明CXCR5 + T细胞内积累异位淋巴结构在人类与结核肉芽肿,非人类的灵长类动

物和老鼠。这些淋巴滤泡似乎重要的适当的本地化的T细胞肉芽肿,最佳活化的巨噬细胞和预防结核病。因此,虽然Tfh细胞位于肉芽肿在结核病免疫反应,显然是重要的角色循环Tfh细胞在人类结核病感染和疾病仍然是未知的。

The distribution of Tfh cells in TB infection and disease was studied by classifying them into 3 subsets. We first attempted to infer the function of these Tfh subsets expressing different combination of surface markers by examining their response to MTB antigens in those with LTB and those with active PTB. Our data clearly reveals that both PD-1 and ICOS either separately or together mark Tfh cells with similar properties in terms of antigenic responsiveness. Tfh细胞结核感

染和疾病的分布研究了分类成三个子集。我们第一次试图推断出这些Tfh子集的函数表达不同的表面标记组合通过检查他们对结核分枝杆菌抗原的反应在那些PTB LTB和活跃。我们的数据清楚地表明,PD-1和国际单独或一起马克Tfh细胞具有类似属性的抗原反应。Subsequently,the examination of frequencies of

these Tfh subsets revealed asignificant reduction in the frequencies of these subsets in those with PTB. This study demonstrates for the first time, we believe, that there are decreased frequencies of Tfh cells in tuberculosis, data that are in contrast to data from HIV and other viral infections where Tfh frequencies are increased. Thus, PTB appears to be selectively associated with TB - antigen specific deficiency in Tfh cells expressing either ICOS and/or PD-1. One of the major hallmarks of Tfh cells is their ability to produce a variety of cytokines – most notably IL-21. Our examination of the IL-21 expressing Tfh cells in PTB reveals that the diminished frequency of Tfh cells in TB disease translated to diminished function of these cells. 随后,这些Tfh子集的检查频率显

示乳癌的这些子集的频率在PTB。这项研究首次表明,我们相信,有频率的降低Tfh细胞肺结核、数据与其他数据从艾滋病毒和病毒感染Tfh频率增加。因此,PTB似乎是选择性地与结核病——特定抗原缺乏相关国际和/或PD-1 Tfh细胞表达。Tfh细胞的主要特征之一是他们的能力产生多种细胞因子,尤其是IL-21。我们检查IL-21表达Tfh细胞PTB揭示了降低结核病Tfh细胞转换成频率减少这些细胞的功能。 In addition, we

also observed significantly diminished systemic levels of IL-21 in PTB and a significant positive correlation between Tfh cell subset frequencies and IL-21 levels in these individuals suggesting that most of the IL-21 in TB infection and disease is probably derived from circulating Tfh cells. Since there are increasing reports of Tfh cells producing other cytokines including IL-4, IFNc and IL-17, we examined the levels of these cytokines and found lower levels of circulating IFNcin

PTB. Thus,we demonstrate that the dysregulated expression of Tfh cell subsets is actually

associated with functional impairment of these cells in the circulation of PTB individuals. 此外,我

们还观察到显著减少系统性的IL-21 PTB Tfh之间显著正相关细胞频率和IL-21子集水平在这些个人建议的大部分IL-21结核感染和疾病可能是来自循环Tfh细胞。因为越来越多的报道Tfh细胞生产其他细胞因子包括IL-4,IFNc IL-17,我们检查了这些细胞因子的水平,发现低水平的循环IFNcin PTB。因此,我们证明Tfh细胞的表达特异表达基因子集实际上是与这些细胞的功能障碍相关的循环PTB个人。It is possible that

the diminished frequencies in the blood of PTB individuals is a reflection of increased migration to the tissues - lungs or mediastinal lymph nodes. However, since circulating Tfh cells have been shown to consistently exhibit additional functions compared to tissue resident Tfh cells, our data hold importance in terms of potential impact on pathogenesis of TB disease. 血液中有可能减少频率

PTB个人反映增加的移民组织——肺或纵隔淋巴结。然而,由于循环Tfh细胞已被证明始终表现出额外的功能与组织居民Tfh细胞相比,我们的数据保存的重要性对结核病的发病机制的潜在影响。 However,we

have not examined the frequencies of these cells or that of their hallmark cytokines in healthy control individuals (without latent infection) and therefore need to perform these studies in the future to elucidate the role of these cells and cytokines in TB infection.Another hallmark of Tfh cells is their ability to provide B cell help. Although the role of B cells in TB is not clearly understood, it is known that B cell deficient mice appear more susceptible to TB. Studies of

human and non human primate TB granulomas have identified B cell follicle structures that might contribute to the immune response to TB. 然而,我们没有检查的频率,这些细胞或细胞因子在健康的标

志控制个人(没有潜伏性感染),因此需要执行这些研究在未来阐明这些细胞和细胞因子的作用在结核病感染。Tfh细胞的另一个特点是,他们提供B细胞帮助的能力。虽然在结核病B细胞的作用并不清楚,众所周知,B细胞缺陷小鼠更容易出现结核病。研究人类和非人类灵长类动物的结核肉芽肿都发现B细胞滤泡结构,可能导致结核病的免疫反应。While conventional Tfh cells are known to promote all aspects of B cell

function, circulating Tfh cells are known only to specifically promote activated and classical memory B cell and plasma cell formation. However, we did not observe any significant compromise in memory B cell formation in PTB nor were there any perturbations in frequencies of other B cell subsets. Again, we have not estimated the function of these cells directly nor have we estimated the frequency of these cells in healthy control individuals (without latent infection).

虽然传统Tfh细胞促进B细胞功能的各个方面,循环Tfh细胞只有专门促进激活和古典知道记忆B细胞和浆细胞的形成。然而,我们没有观察到任何重大妥协在PTB记忆B细胞的形成也不再有任何扰动频率的B细胞的子集。再一次,我们没有直接估计这些细胞的功能我们估计也没有控制这些细胞在健康个体的频率(没有潜伏性感染)。

Although it is known that numerous cytokines can promote Tfh formation, much less is

known about the factors that restrain this process. It has been reported that Tfh cell formation can be suppressed by several cytokines, including IL-10 and TGFb. Indeed, we also detected increased levels of IL-10 in circulation in PTB individuals. Coupled with data on the blockade of IL-10 in PTB individuals, it appears that IL-10 does indeed play an important role in the modulation of Tfh cells in active PTB. On the other hand, TGF (beita)appears to play a negligible role in the regulation of Tfh cells in active TB. Although PD-1 is highly expressed in Tfh cells, little is

known about the role of PD-1 in Tfh cell development and function. 虽然众所周知,许多细胞因子能

促进Tfh形成,不太了解的因素抑制这一过程。据报道,Tfh可以抑制细胞的形成一些细胞因子,包括il - 10和TGFb。实际上,我们还发现增加流通中的il - 10在PTB个人的水平。加上数据的封锁在PTB il - 10个人,看来il - 10确实发挥重要作用的调制Tfh PTB细胞活跃。另一方面,TGF(beita)扮演一个微不足道的角色出现在Tfh细胞活动性结核病的监管。虽然PD-1 Tfh细胞高表达,很少被人所知的角色在Tfh PD-1细胞发育和

功能。 Studies in mice deficient in PD-1 or its ligands report increased numbers of Tfh cells,

suggesting that PD-1/PD-L1 interactions downregulate Tfh generation and/or differentiation [20]. While CTLA-4 is a potent inhibitor of effector T cell differentiation and function [21], its role in regulating Tfh cell expansion is not known. Interestingly, our study reveals an important role for both PD-1 and CTLA-4 signaling in the down modulation of Tfh cell expansion in pulmonary TB since blockade of either of these pathways significantly restored the TB – antigen induced

expansion of Tfh cell subsets in PTB individuals. 研究小鼠缺乏PD-1或其配体报告Tfh细胞数量的增

加,表明PD-1 / PD-L1交互Tfh代表达下调和/或分化[20]。虽然CTLA-4是一个强有力的抑制剂效应T细胞分化和功能[21],它的作用在调节Tfh细胞扩张是未知的。有趣的是,我们的研究揭示了一个重要的角色对于PD-1和CTLA-4下调制信号的Tfh细胞扩张以来,肺结核的封锁这些通路显著恢复结核病——抗原诱导PTB个人Tfh细胞子集的扩张。These data provide novel insights into the role of PD-1 and CTLA-4 on

the regulation of Tfh cells in a human infectio and suggest that imporant new roles for these

molecules apart from their effect on T effector cells. 这些数据提供新颖的见解PD-1的角色和CTLA-4 Tfh细胞调节的人类感染个案和认为重要的新角色这些分子除了影响的效应细胞。

In summary, our data on the Tfh cell distribution and function in pulmonary tuberculosis suggest that compromised Tfh cell numbers and function are a prominent feature of active disease.Although our study was not designed to decipher cause and effect mechanisms of Tfh association with active TB, it nevertheless implicates an important role for this poorly explored subset of CD4+ T cells in active TB. Our study also provides the first comprehensive analysis of the distribution of B cell subsets in pulmonary TB and reveals that compromised B cell subset distribution is not a feature of active TB disease. Our data also suggest that adjunct

immuno-modulation in the form of coinhibitory receptor blockade (or immune checkpoint blockade using CTLA-4 and PD-1 antagonists) could potentially also enhance the protective

immune responses of susceptible individuals. 总之,我们的数据在肺结核Tfh细胞分布和功能表明,妥协

Tfh细胞数量和功能是活动性疾病的显著特征。虽然我们的研究不是为了解释因果机制Tfh与活动性结核病,然而牵连到一个重要的角色对于这糟糕的探索子集CD4 + T细胞在活动性结核病。我们的研究也提供了第一个综合分析B细胞分布的子集在肺结核和显示,妥协B细胞分布不是活动性结核患者的特征子集。我们的数据也表明,兼职的形式immuno-modulation coinhibitory受体封锁(或免疫检查点封锁使用CTLA-4和PD-1拮抗剂)也有可能增强易感个体的保护性免疫反应。