聚乙二醇干扰素α-2a的触发NK细胞功能和患者特异性T细胞反

StephenW

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        PLoS One. 2016 Jun 27;11(6):e0158297. doi: 10.1371/journal.pone.0158297. eCollection 2016.
Pegylated Interferon α-2a Triggers NK-Cell Functionality and Specific T-Cell Responses in Patients with Chronic HBV Infection without HBsAg Seroconversion.Bruder Costa J1,2, Dufeu-Duchesne T2,3, Leroy V2,3, Bertucci I4, Bouvier-Alias M5, Pouget N6, Brevot-Lutton O6, Bourliere M7, Zoulim F8,9,10, Plumas J1,11, Aspord C1,11; ANRS HB06 PEGAN study group.
Author information

• 1University Grenoble Alpes, Grenoble, F-38041 France; INSERM, U1209, Immunobiology and Immunotherapy of Chronic Deseases, La Tronche, F-38706 France.
• 2CHU Grenoble, Michallon Hospital, Hepato-gastroenterology unit, Grenoble, F-38043 France.
• 3University Grenoble Alpes, Grenoble, F-38041 France; INSERM, U1209, Analytic Immunology of chronic pathologies, La Tronche, F-38706 France.
• 4ANRS (France REcherche Nord & sud Sida-hiv Hépatites: FRENSH), Paris, France.
• 5Department of Virology, Henri Mondor Hospital, University Paris-Est and Inserm U955, Creteil, France.
• 6Sorbonne Universités, UPMC Univ Paris 06, INSERM, Institut Pierre Louis d'épidémiologie et de Santé Publique (IPLESP UMRS 1136), 75012, Paris, France.
• 7Hepato-gastroenterology department Hospital Saint Joseph, Marseille, 13008 France.
• 8INSERM U1052-CNRS 5286, Cancer Research Center of Lyon (CRCL), Lyon, France.
• 9Hepatology Department, Hospices Civils de Lyon, Lyon, France.
• 10Université de Lyon, Lyon, France.
• 11EFS Rhone-Alpes, R&D Laboratory, La Tronche, F-38701 France.
  

AbstractPegylated interferon α-2a (Peg-IFN-α) represents a therapeutic alternative to the prolonged use of nucleos(t)ide analog (NA) in chronic hepatitis B (CHB) infection. The mechanisms leading to a positive clinical outcome remain unclear. As immune responses are critical for virus control, we investigated the effects of Peg-IFN-α on both innate and adaptive immunity, and related it to the clinical evolution. The phenotypic and functional features of the dendritic cells (DCs), natural killer (NK) cells and HBV-specific CD4/CD8 T cells were analyzed in HBeAg-negative CHB patients treated for 48-weeks with NA alone or together with Peg-IFN-α, before, during and up to 2-years after therapy. Peg-IFN-α induced an early activation of DCs, a potent expansion of the CD56bright NK subset, and enhanced the activation and functionality of the CD56dim NK subset. Peg-IFN-α triggered an increase in the frequencies of Th1- and Th17-oriented HBV-specific CD4/CD8 T cells. Peg-IFN-α reversed the unresponsiveness of patients to a specific stimulation. Most of the parameters returned to baseline after the stop of Peg-IFN-α therapy. Peg-IFN-α impacts both innate and adaptive immunity, overcoming dysfunctional immune responses in CHB patients. These modulations were not associated with seroconversion, which questioned the benefit of the add-on Peg-IFN-α treatment.


PMID:27348813 [PubMed - in process]

StephenW

抽象

发给：
公共科学图书馆之一。 2016年6月27日; 11（6）：e0158297。 DOI：10.1371 / journal.pone.0158297。 eCollection 2016年
聚乙二醇干扰素α-2a的触发NK细胞功能和患者特异性T细胞反应慢性HBV感染的HBsAg没有血清转化。
布鲁德科斯塔J1,2，Dufeu-杜申T2,3，乐华V2,3，贝图西I4，布维尔-M5的别名，POUGET N6，Brevot-Lutton O6，Bourliere M7，Zoulim F8,9,10，普卢默斯J1,11，Aspord C1,11; ANRS HB06佩甘研究小组。
作者信息

    1University格勒诺布尔阿尔卑斯，格勒诺布尔，F-38041法国; INSERM，U1209，免疫学和慢性Deseases的免疫治疗，拉特龙克，F-38706法国。
    2CHU格勒诺布尔，Michallon医院，肝，胃肠病学单位，格勒诺布尔，F-38043法国。
    3University格勒诺布尔阿尔卑斯，格勒诺布尔，F-38041法国; INSERM，U1209，慢性病症的免疫学分析，拉特龙克，F-38706法国。
    4ANRS（法国RECHERCHE北与SUD思达艾滋病毒Hépatites：FRENSH），法国巴黎。
    病毒学，亨利Mondor医院，大学巴黎东和INSERM U955，克雷泰伊，法国5Department。
    6Sorbonne综合大学，UPMC大学巴黎06，INSERM，研究所皮埃尔路易épidémiologie和de桑特Publique（IPLESP正常市场需求量1136），75012，法国巴黎。
    7Hepato，消化内科医院圣约瑟夫，马赛，法国13008。
    8INSERM U1052-CNRS 5286，里昂的癌症研究中心（CRCL），法国里昂。
    9Hepatology部，收容所Civils里昂，法国里昂。
    10Université里昂，法国里昂。
    11EFS罗纳 - 阿尔卑斯大区，R＆D实验室，拉特龙克，F-38701法国。

抽象

聚乙二醇干扰素α-2a干扰素（聚乙二醇化干扰素α）表示治疗的替代长期使用治疗慢性乙型肝炎（CHB）感染的核苷（酸）类似物IDE（NA）的。导致积极的临床结果的机制尚不清楚。作为免疫应答是病毒控制的关键，我们研究了聚乙二醇干扰素α对先天性和适应性免疫的影响，并将其相关的临床演变。树突状细胞的表型和功能特征（DCS），天然杀伤（NK）细胞和HBV特异性CD4 / CD8 T细胞在48周与NA与聚乙二醇干扰素单独或一起处理HBeAg阴性患者进行分析-α，之前，期间和最多2年治疗后。 PEG-IFN-α诱导的DC中，CD56bright NK子集的强效扩张的早期活化，提高了CD56dim NK子集的激活和功能。 PEG-IFN-α引发Th1-和频率增加的Th17为本HBV特异性CD4 / CD8 T细胞。 PEG-IFN-α逆转患者的无应答于特定的刺激。大部分参数钉-IFN-α治疗停止后回到基线。 PEG-IFN-α的影响先天性和获得性免疫，克服慢性乙肝患者不正常的免疫反应。这些调制没有用血清学转换，而质疑的附加聚乙二醇化干扰素α治疗的好处有关。

StephenW

Discussion

We provide a dynamic description of the innate and adaptive immunological changes during and up to 2 years after the course of an additional Peg-IFN-α therapy in CHB patients compared to patients receiving NA alone, whose clinical outcomes are known to be different. The findings increase the understanding of Peg-IFN-α immunomodulatory effects, and reveal yet unexplored immune effects of the combination therapy. However, as no seroconversion occurred in our cohort of patients, this work questions which function, direct antiviral or indirect immunomodulation, is more relevant in the efficacy of Peg-IFN-α in the treatment of CHB patients in the add-on settings.

Our data bring a larger view of the immunologic changes triggered by Peg-IFN-α therapy, also with a better subsetting definition and the full kinetics of the immune modulations, reinforcing the results of recent clinical trials [17–20, 23]. Firstly, Peg-IFN-α triggered a marked expansion of the CD56bright NK-cell subset accompanied by an activation and an enhanced functionality of the CD56dim NK-cell subset. CD56bright NK cells represent an intermediate stage of NK-cell differentiation, as a precursor of the CD56dim subset. Their unique immunoregulatory role [25] is illustrated by their preferential interactions with DCs, and their important role in early immune responses and in the shaping of subsequent adaptive responses [26]. Expansion of CD56bright NK cells has been described in several diseases, especially in HCV infection [27], in patients with multiple sclerosis treated with IFN-β [28], and interestingly in patients with active systemic lupus erythematosus [29], a disease mediated by elevated type-I IFN levels. Of note, we observed an inverse correlation between the absolute numbers of CD56bright NK cells and the decline in HBsAg titer. The distinct conclusions on NK functionality compared to other studies [19, 20] may be due to the different ways of data analysis, either as absolute numbers of functional NK cells [19] or as percentages of functional cells within the NK population [20], the time point (point of kinetics or the end of treatment) and the groups that were compared (responders versus non-responders, pre- and on-treatment points).

Moreover, Peg-IFN-α triggered as soon as 4 weeks an activation of pDCs and mDCs, which are critical in antiviral responses [30]. Indeed, type-I IFN is essential for DC maturation and activation [31], and to favor the cross-presentation of antigens by DCs [32], which can help promote subsequent cross-priming of antiviral T cells and facilitate viral clearance. Since DCs are essential to activate NKs and antiviral responses [33], modulation of DCs by Peg-IFN-α may affect the subsequent DC-NK cross-talk and potentiate NK-cell and T-cell functions. This hypothesis is supported by the strong correlation observed between the percentages of activated CD69+CD56dim NK cells and CD86+ pDCs. Thus, Peg-IFN-α may restore the pDC/NK cross-talk [6], subsequently enhancing antiviral NK-cell responses that could optimize the elimination of infected hepatocytes.

We also revealed key effects of Peg-IFN-α on adaptive immunity. We observed the elicitation of HBc- and HBs-specific CD4 and CD8 immune responses during Peg-IFN-α therapy. This is in contrast with prior observations that Peg-IFN-α did not improve peripheral HBV-specific T-cell responses [17, 22]. The discrepancy may be due to the time of analysis (very early versus late), and/or the use of frozen cells stimulated once with the peptide pools, whereas we performed two rounds of stimulation on freshly isolated PBMCs. Our data are consistent with the previously described restoration of HBV-specific T-cell responses following Peg-IFN-α therapy [23, 34, 35]. In addition, we have shown for the first time the elicitation of Th17 cells upon Peg-IFN-α treatment, preceding the induction of Th1-oriented immunity. Although IL17-producing T cells are described as pro-inflammatory cells and have been associated with liver damage [36], plasma IL17A levels and Th17 cell frequency are negatively correlated with viral load in patients with CHB infection [37]. Such a Th17 profile is revealed only upon specific stimulation with HBV-derived peptide pools and only in the Peg-IFN-α group, suggesting that Th17 cells are either HBV-specific T cells or that HBV-specific T cells subsequently activate other T cells to produce IL17. Interestingly, Th17 cells have been involved in the establishment of long-term immune memory and for promoting B-cell class switch [38]. Thus, they may regulate cellular and humoral antiviral immune responses, therefore favoring the elicitation of HBV-specific immune responses crucial for the immune control of HBV infection.

By potently modulating both the virus fitness and immunity, Peg-IFN-α appears to trigger a systemic immune activation and overcome the functional impairments to subsequently drive antiviral immunity. Despite evident immunologic changes, patients did not achieve HBsAg seroconversion in this cohort. In the overall ANRS HB06 PEGAN trial, HBsAg clearance at W48 has been achieved in only 8% of the patients treated by Peg-IFN-α (7/90) (Bourliere et al. J Hepatol 62, S2, S249, 2015). HBsAg clearance was associated with a low baseline HBs Ag titers and a history of HBeAg seroconversion prior to the inclusion into the trial, which suggests a potential spontaneous anti-viral immune activation. Immune modulations triggered by the therapy might even though improve clinical parameters. The two patients who underwent the highest decline in HBsAg during Peg-IFN-α therapy had the highest level of activated pDCs and NK cells at the end of treatment. The inverse correlation between the absolute numbers of CD56bright NKs and the down-regulation of HBsAg suggested a relationship between the immunologic and virological changes. Strikingly, we identified immunologic changes that persisted for up to 2 years after the cessation of the treatment in the absence of HBsAg seroconversion, as illustrated by the reversion of the unresponsiveness to the peptide-loaded pDCs.

Peg-IFN-α therapy profoundly affects both innate and adaptive immunity, but only transiently and without leading to HBsAg seroconversion. By identifying yet unexplored immune effects of the combination therapy, our study illustrates the pleiotropic action of IFN-α. However, as shown by the other groups, most of the immune changes returned to baseline after the cessation of Peg-IFN-α treatment. The immune alterations induced by Peg-IFN-α treatment failing in the majority of cases to induce seroconversion, they are probably less determinant than the direct antiviral effect of this cytokine. Our work questioned the benefit of the add-on Peg-IFN-α treatment over the NA or Peg-IFN-α monotherapies.

StephenW

讨论

我们期间和长达2年慢性乙型肝炎患者的额外钉-IFN-α治疗的过程中相比于接收到的NA单独，其临床结果是已知的不同的患者后提供的先天和适应性免疫的变化的动态描述。调查结果增加了聚乙二醇化干扰素α免疫调节作用的认识，揭示联合治疗的未开发但免疫效果。然而，如发生在我们的患者队列无血清转化，这种工作问题，这些问题的功能，直接抗病毒或间接免疫调节，是在聚乙二醇干扰素-α在治疗慢性乙型肝炎患者的疗效附加设置更加相关。

我们的数据带来的由聚乙二醇干扰素-α治疗引发的免疫学变化较大视图，还具有更好的子集定义和免疫调制的全动力学，增强最近的临床试验[17-20，23]的结果。首先，PEG-IFN-α触发伴随着激活和CD56dim NK细胞子集的增强功能性的CD56bright NK细胞亚群的显着扩展。 CD56bright NK细胞代表NK细胞分化的中间阶段，作为CD56dim子集的前体。其独特的免疫调节作用[25]是通过它们与树突优惠的相互作用，以及它们在早期免疫反应的重要作用，并在随后的适应性反应[26]成型示出。 CD56bright NK细胞的扩张已在几种疾病进行了描述，特别是在HCV感染[27]，在患者与IFN-β的[28]，以及有趣的患者活性系统性红斑狼疮[29]，一种疾病介导的治疗多发性硬化升高的I型干扰素的水平。值得注意的是，我们观察到CD56bright NK细胞的绝对数量和在HBsAg的滴度的下降之间的逆相关。对NK功能不同的结论相对于其他的研究[19,20]，可能是由于在不同的方式的数据分析，无论是官能NK细胞[19]作为绝对数值或作为NK细胞群体中功能​​性细胞的百分比[20]中，时间点（动力学或治疗结束的点），并进行了比较，该组（应答与非应答者，前和在治疗点）。

此外，PEG-IFN-α尽快4周触发的pDC和的mDC，这是在抗病毒反应[30]临界的活化。事实上，I型干扰素是DC成熟和活化[31]必要的，并且有利于通过树突抗原的交叉呈递[32]，它可以帮助促进抗病毒的T细胞的后续交叉引发和促进病毒的清除。由于DC是必不可少的激活NK可以和抗病毒反应[33]，由钉-IFN-αDC的调节可以影响随后的DC-NK串扰和加强NK细胞和T细胞的功能。这种假设被激活的CD69 + CD56dim NK细胞和CD86 +的pDCs的百分比之间观察到的强相关性的支持。因此，PEG-IFN-α可以恢复的pDC / NK串音[6]，随后提高，可以优化感染的肝细胞的消除病毒NK细胞应答。

我们还透露在适应性免疫聚乙二醇化干扰素α的关键作用。我们钉-IFN-α治疗期间观察HBc-和特定乙型肝炎表面CD4和CD8免疫应答的诱导。这是在与现有的意见，即钉-IFN-α并未改善末梢HBV特异性T细胞反应[17，22]对比。这种差异可能是由于分析的时间（非常早期与晚期），和/或使用与所述肽库刺激一次，而我们进行上新鲜分离的PBMC两轮刺激冷冻细胞。我们的数据与的HBV特异性T细胞应答以下钉-IFN-α治疗[23，34，35]前面描述的恢复一致。此外，我们已经表明，第一次Th17细胞在聚乙二醇干扰素-α处理的诱导，Th1细胞为本免疫的诱导前。虽然IL17的T细胞被描述为促炎性细胞和已与肝损伤[36]，血浆IL17A水平和Th17细胞的频率相关联的负CHB患者感染[37]病毒载量相关。这样的Th17的信息仅在与HBV衍生肽池特定刺激和仅在钉-IFN-α组显示，表明Th17细胞要么HBV特异性T细胞或HBV特异性T细胞随后激活其他T细胞产生IL17。有趣的是，Th17细胞已经参与建立长期免疫记忆，并促进B细胞级交换机[38]。因此，它们可以调节细胞和体液的抗病毒的免疫反应，因此有利于对HBV感染的免疫控制关键HBV特异性免疫应答的诱导。

通过有力地调节两个病毒健身和免疫力，PEG-IFN-α似乎触发全身免疫激活和克服功能障碍随后开车抗病毒免疫。尽管有明显的免疫变化，患者没有达到在这个队列乙肝表面抗原血清学转换。在整体ANRS HB06佩甘试验中，在W48 HBsAg清除已经在只有8％的由聚乙二醇干扰素α（7/90）（Bourliere等肝脏病学杂志62，S2，S249，2015）治疗的患者来实现的。 HBsAg清除用低基线HBs抗体滴度的抗原和前纳入庭审中，这表明潜在的自发的抗病毒免疫激活HBeAg血清学转换的历史有关。由治疗引发的免疫调制甚至可能虽然提高临床参数。谁接受聚乙二醇化干扰素α治疗过程中HBsAg的跌幅最高的2例患者激活pDC细胞和NK细胞在治疗结束时的最高水平。 CD56bright NK可以的绝对数量和HBsAg的下调之间的逆相关提出的免疫学和病毒学变化之间的关系。引人注目的是，我们确定了持续了长达2年在没有的HBsAg血清转换的治疗停止后，由无应答的肽负载的pDCs的逆转所示免疫学变化。

PEG-IFN-α治疗深刻地影响着先天性和获得性免疫，但只有短暂和不导致乙肝表面抗原血清学转换。通过识别联合疗法还未知免疫效果，我们的研究说明了IFN-α的多效性作用。然而，如由其它基团，大多数的免疫变化钉 - IFN-α治疗停止后回到基线。诱导钉-IFN-α治疗在大多数情况下，不能诱导血清转化免疫变化，它们比该细胞因子的直接抗病毒效果行列式可能更少。我们的工作提出质疑附加聚乙二醇化干扰素α治疗在NA或PEG-IFN-α单一疗法的好处。

StephenW

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