共抑制分子BTLA在HIV慢性感染中的特点及与疾病进展的关系研究
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摘要
T细胞过度活化是HIV-1(以下均简称HIV)慢性感染过程中的一个重要病理特征。近期发现T细胞过度活化与细胞表面共抑制分子如PD-1、CTLA-4和BTLA的表达有密切关系。这些共信号分子途径在T细胞分化的不同阶段、T细胞反应的不同阶段发挥着不同的调节功能,从而维持机体免疫反应处于稳态。近期在对HIV的感染研究中共抑制分子PD-1、CTLA-4和Tim-3受到高度关注。许多研究均发现在HIV、HBV及HCV等慢性病毒感染性疾病中CD4和/或CD8 T细胞功能耗竭与PD-1水平增高有关,体外阻断PD-1信号后可恢复T细胞的功能。近期研究发现CTLA-4也与HIV慢性感染中的CD4 T细胞功能耗竭有关,阻断CTLA-4信号通路后可部分恢复CD4 T细胞的功能。最近发现的共抑制分子BTLA已证实在T细胞免疫耐受中发挥重要作用,并且影响T细胞的活化和T细胞功能,与自身免疫性疾病的发生和疾病进程相关。但BTLA在慢性病毒感染性疾病,尤其是HIV慢性感染中的研究目前还未有报道。那么我们考虑BTLA是否和PD-1、CTLA-4一样参与了HIV慢性感染的发病过程?因此,我们对慢性HIV感染过程BTLA的表达特点、功能意义及与疾病进展的关系进行了研究,以进一步阐明HIV慢性感染的发病机制。
我们纳入了85例HIV慢性感染者,根据CD4细胞计数和HIV病毒载量分为三组:长期无进展者(LTNP)、典型进展者(TP)和AIDS患者,并纳入了26例健康人做为对照。通过BTLA在外周淋巴细胞上频率的流式细胞分析,我们发现BTLA主要表达在B细胞,其次在CD4 T细胞、CD8 T细胞、NKT细胞和单核细胞,而弱表达于NK细胞。通过比较HIV慢性感染者和健康者,发现在HIV慢性感染过程中CD4 T细胞和CD8 T细胞上的BTLA表达明显降低,并随着疾病进展呈进行性下降。为了进一步确证这种降低,我们用实时荧光定量PCR检测了BTLA在mRNA水平的表达,发现在慢性HIV感染者(包括TP和AIDS患者),BTLA mRNA的水平确实比健康对照低。由于PD-1主要调节的细胞是HIV特异性的CD8 T细胞,因此我们也研究了BTLA是否也主要作用于HIV特异性的CD8 T细胞,结果发现不仅HIV特异性的CD8 T细胞上BTLA表达降低,而且CMV-和Flu-特异性的CD8 T细胞上的BTLA也降低,说明BTLA的降低是非HIV特异性的。
我们对CD4 T细胞的BTLA表达水平与HIV疾病进展的相关性进行了研究,发现CD4 T细胞上BTLA的表达水平和平均荧光强度与CD4 T淋巴细胞计数呈正相关,而与HIV病毒载量呈负相关。另外,我们还分析了CD4 T细胞上BTLA表达水平与CD38、PD-1、CD127和Ki67等与疾病进程密切相关的免疫学标志的相关性,结果发现CD4 T细胞上BTLA的表达与CD38、PD-1和Ki67的表达呈负相关,而与CD127的表达呈正相关,这进一步显示HIV感染过程中BTLA的进行性下降与疾病进程密切相关。
根据CD27和CD45RO可将CD4 T细胞分为naive T细胞(Tnaive)、中央记忆性细胞(Tcm)和效应记忆性T细胞(Tem)等三个亚群,我们也检测了BTLA在这些CD4 T细胞亚群上的表达。发现LTNP患者Tnaive亚群和Tem亚群上BTLA仅有微弱下降,而Tcm亚群几乎无下降。在TP和AIDS患者,BTLA在Tnaive、Tcm和Tem三个亚群上的表达均明显降低,特别是在AIDS患者。另外,我们从mRNA水平也验证了BTLA在这些CD4细胞亚群上的降低。这些结果说明,持续的HIV感染导致了BTLA的持续下降,BTLA的持续下降又导致了na?ve CD4 T细胞比例降低和记忆性CD4 T细胞比例升高。
为了调查CD4 T细胞上BTLA的表达是否影响CD4 T细胞的活化状态,我们分析了健康个体中CD4+BTLA+和CD4+BTLA– T细胞的表型特征,发现BTLA+的CD4 T细胞总体表现出幼稚的表型,即Tniave细胞所占比例最高,而Tcm和Tem所占比例较低。BTLA–的CD4 T细胞总体表现出成熟的表型,主要表现在Tnaive细胞所占比例较低,Tcm和Tem占了大多数。在HIV感染者中虽然BTLA+和BTLA–的CD4 T细胞表现出和健康个体较相同的特征,但BTLA+的CD4 T细胞中Tnaive细胞比健康个体比例要低,尤其在AIDS患者,Tnaive细胞比例更低。这些结果提示持续的HIV感染可能改变了BTLA+ CD4 T细胞中初始T细胞和记忆T细胞的组成。进一步分析BTLA+和BTLA– CD4 T细胞上BTLA和细胞活化标志CD38、凋亡标志Fas(CD95)、细胞功能耗竭标志PD-1、功能效应分子穿孔素(perforin),以及细胞增殖标志Ki67的关系发现,HIV慢性感染者BTLA+ CD4 T细胞表达CD38、PD-1、CD95、perforin和Ki67均有明显升高,而表达CD127明显降低,尤其在TP和AIDS患者这种变化更为明显。但是在BTLA– CD4 T细胞上,除了CD38在HIV慢性感染者中表达升高外,其他上述免疫学标志几乎无变化,说明在慢性HIV感染过程中BTLA+ CD4 T细胞分化、成熟等方面比BTLA– CD4 T细胞受到的影响更为明显。
我们在研究中发现在体外加入HIV-1 NL4-3病毒颗粒能够使CD4 T细胞上的BTLA表达降低,而且这种降低与加入的病毒量有量效关系,加入逆转录酶抑制剂齐多夫定后不能逆转BTLA的降低,说明HIV诱导的BTLA表达下降并不是通过HIV基因去影响BTLA的表达,这种调节很可能发生在HIV进入或吸附细胞表面阶段,或HIV分泌的某种蛋白调节了BTLA的下降。
体外功能实验显示,用刺激性抗体交联BTLA后,与健康对照相比TP/AIDS患者BTLA对CD4 T细胞活化和分泌细胞因子的抑制功能均受到损伤,这种抑制功能的降低可能是TP/AIDS患者上BTLA表达下降引起的。因此BTLA对CD4 T细胞抑制功能的降低可能是AIDS患者体内免疫系统过度活化的原因之一。以往对人和小鼠的研究表明BTLA对TCR诱导的T细胞增殖有抑制作用,但我们的研究显示,用刺激性抗体交联BTLA后,BTLA对T细胞的增殖表现出较大的异质性,在不同个体可能表现出不同的作用,或者抑制或者促进增殖。在今后的实验中,将进一步明确BTLA对T细胞增殖的影响。
一些研究已经报道了PD-1+ T细胞更容易自发凋亡,我们考虑BTLA介导的信号途径是否对CD4 T细胞的凋亡也有作用。通过对HIV慢性感染者的研究发现,凋亡的CD4 T细胞(即annexin V+ CD4 T细胞)主要是BTLA–细胞,而BTLA+细胞很少有凋亡,且BTLA的表达水平与CD4 T细胞的自发性凋亡呈明显负相关,这些结果显示在HIV感染者中BTLA–CD4 T细胞比BTLA+CD4 T细胞更易发生凋亡。
总之,我们的研究结果显示随着HIV慢性感染疾病进展,CD4 T细胞上BTLA表达逐渐下降,而且BTLA介导的对CD4 T细胞的抑制功能受到损伤,BTLA的功能损伤可能导致了机体的免疫过度活化,免疫活化则有利于HIV的复制,最终促进了疾病的进展。这些结果对于阐明BTLA在HIV慢性感染中的致病机制具有重要意义,也为今后人类治疗HIV感染提供了新的靶点。
Non-specific T cell hyperactivation is associated with HIV-1 disease progression, but its mechanisms are poorly defined. Recent findings have demonstrated that T cell activation is substantially impacted by cell surface-expressing co-inhibitory molecules such as programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuator (BTLA, CD272), as well as other receptors. These co-signaling pathways play distinct and overlapping modulatory roles during sequential (or different) stages of the T-cell response or for different lymphocyte subsets so that immune responses occur in the correct intensity and pattern. Recent studies have highlighted the role of immuno-inhibitory molecules such as PD-1, CTLA-4 and Tim-3, which mediate CD4 and CD8 T cell exhaustion during chronic HIV-1 infection. Although the pioneering findings indicate that co-inhibitory signaling mediates T-cell exhaustion and partially contributes to HIV-1 persistence, it is still unknown whether these inhibitory pathways experience functional deficits that may be responsible for the loss of the delicate balance between immune over-activation and efficient viral containment in chronic HIV-1 infection. BTLA, the most recently recognized member of the CD28 family, which interacts with its ligand, herpesvirus entry mediator (HVEM), predominantly negatively regulates immune responses. Recent investigations further demonstrated that BTLA is down-regulated in allergen-specific immunotherapy-untreated patients with Japanese cedar pollinosis, and BTLA gene polymorphism is associated with a high risk of rheumatoid arthritis development. However, little is known regarding the role of BTLA in viral infection, in particular in chronic HIV-1 infection.
Eighty-five HIV-1-infected individuals were enrolled in our study. They were divided into three groups including long-term non-progressors (LTNPs), typical progressors (TPs) and AIDS patients according to CD4 T-cell count and serum HIV-1 RNA load. Twenty-six uninfected subjects were employed as healthy controls (HCs). To define the pattern of BTLA expression in lymphocytes, our experiments showed that BTLA was mainly expressed by peripheral B cells, T cells, NKT cells and monocytes rather than NK cells. In particular, BTLA was found to be constitutively expressed by CD4 T cells and CD8 T cells. We found that BTLA expression levels in both CD4 and CD8 T cells were significantly down-regulated in HIV-1-infected subjects compared with HCs. Importantly, HIV-1-infected individuals displayed large differences in BTLA expression levels in both CD4 and CD8 T cells: LTNPs exhibited higher levels of BTLA expression than did TPs and AIDS patients; while the lowest levels of BTLA expression in both CD4 and CD8 T cells were observed in AIDS patients. The data clearly indicated that chronic HIV-1 infection may lead to overall progressive BTLA down-regulation in CD4 and CD8 T cells. We further analyzed BTLA mRNA expression in HC and HIV-1 infected subjects. It was found that BTLA mRNA expression was also significantly lower in the peripheral blood of TPs and AIDS patients than HCs. Thus, BTLA down-regulation in total T cell subsets is associated with the progression of HIV-1 infection and may serve as a marker of disease progression.
Next, we investigated difference in BTLA expression among HIV-1-, CMV- and Flu-specific pentamer T cells from HIV-1-infected and HC subjects. We observed that BTLA expression in both the CMV- and Flu-specific pentamer cells was significantly reduced in LTNP and TP/AIDS subjects compared with HCs. These data suggest that BTLA expression is also down-regulated in HIV-specific and non-HIV-specific CD8 T cells in progressive HIV infection.
We also analyzed the relationships of BTLA expression in CD4 T cells with two markers of HIV-1 disease progression: plasma viral load and peripheral CD4 T cell count. Our results showed that both BTLA percentage and MFI (mean fluoresce intensity) in total CD4 T cells positively correlated with peripheral CD4 T cell counts, but inversely correlated with plasma viral loads. We further analyzed the associations of BTLA expression with other markers such as CD38, PD-1, CD127 and Ki67, which have been demonstrated to correlate with HIV-1 disease progression. We found that BTLA expression in total CD4 T cells was negatively correlated with CD38, PD-1 and Ki67 expression levels, but positively correlated with CD127 expression in TPs. These data suggest that BTLA down-regulation in CD4 T cells correlates with disease progression in chronic HIV-1 infection.
Through examination of the distribution of BTLA expression in Tna?ve, Tcm and Tem subsets on the basis of CD45RO and CD27 expression, we found that CD4 T cells from HIV-1-infected TPs and AIDS patients were constituted with higher percentages of Tcm and Tem subsets and lower percentage of Tnaive subsets than those from the HC and LTNP subjects. These data probably indicate that chronic HIV-1 infection leads to a progressive decline in naive CD4 T cell subsets but a continuous increase in memory CD4 T cell subsets, and thus altered the distribution of memory CD4 T cell population. More importantly, we found that BTLA expression was more dominant in Tna?ve cells, but gradually decreased in the Tna?ve, Tcm and Tem populations regardless of disease status. BTLA expression was significantly down-regulated in all of three CD4 T cell subsets in HIV-1-infected subjects in comparison to the HCs. These data indicate that persistent HIV-1 infection leads to a progressive loss of BTLA expression as an early change, which precede a reduction of the na?ve CD4 T cell subset and an expansion of the memory CD4 T cell subset.
To determine whether BTLA expression in CD4 T cells influences their activation status, we analyzed BTLA+ and BTLA– CD4 T cell phenotypic profiles in HC and HIV-1-infected subjects. It was found that HC subjects displayed a juvenile phenotype in BTLA+CD4 T cells which were constituted with large percentage of the Tnaive and small percentage of the Tcm and Tem, while BTLA– CD4 T cells that displayed a relatively mature phenotype indicated by large percentage of the Tcm and Tem and small percentage of the Tniave subsets. Interestingly, this distribution was disturbed within BTLA+ but not BTLA– CD4 T cells in HIV-1-infected subjects. Interestingly, this distribution was disturbed within BTLA+ but not BTLA– CD4 T cells in HIV-1-infected subjects. We found that BTLA+ CD4 T cells were constituted with a smaller fraction of Tna?ve subset and more of the Tem subset, in particular in HIV-infected TP and AIDS patients. These data suggest that persistent HIV-1 infection might substantially alter the profiles of memory subsets within BTLA+ CD4 T cells.
We further analyzed the associations of BTLA expression with CD38, CD95, PD-1, Ki67, and perforin. Data showed that chronic HIV-1 infection markedly increased CD38, CD95, PD-1, Ki67 and perforin expression and decreased CD127 expression in BTLA+ CD4 T cell subsets, in particular TPs and AIDS patients, in contrast with HCs. For the BTLA– CD4 T cell population, HIV-1 infection seldom influences the expression of these markers, except for CD38. This comprehensive analysis suggests that HIV-1 infection significantly skews BTLA+ rather than BTLA– CD4 T cells towards differentiation and maturation. Thus, HIV-1 infection not only reduces BTLA expression, but also significantly impacts the immune status of BTLA+ CD4 T cells. We found that HIV-1 NL4-3 viral could down-regulate BTLA expression in CD4 T cells in vitro. In addition, HIV-1 NL4-3-induced BTLA down-regulation in CD4 T cells was viral load-dependent. BTLA down-regulation in CD4 T cells was more pronounced when the cells were cultured with high doses of the virus supernatant. Notably, we found that addition of the anti-viral agent AZT failed to reverse HIV-1-induced BTLA loss at the indicated time point. These data indicate that HIV-1 virus can directly stimulate CD4 T cells to down-regulate BTLA expression, which may reduce HVEM-mediated inhibition and cause more non-specific activation of T cells via a positive loop.
The BTLA-mediated inhibitory pathway plays an important role in maintaining basic immune tolerance by inhibiting T cell activation, cytokine production and proliferation. In order to investigate the functional relevance of diminished BTLA expression in CD4 T cells, we used the agonistic anti-BTLA mAb to compare the BTLA-mediated functional inhibition of CD4 T cells in HIV-1-infected subjects and uninfected persons. We found that BTLA-mediated suppression of CD4 T cell activation (marked by CD25, CD38 and CD69) and cytokine production (IL-2 and INF-γ) was significantly impaired during chronic HIV-1 infeciton.
Some studies showed that BTLA cross-linking can inhibit the proliferation of T cells. But in our study we found that inhibition of T cell proliferation exhibited a high heterogeneity. BTLA cross-linking inhibited T cells proliferation in some subjects but promoted proliferation in other subjects. So further studies should be performed to determine the exact function of BTLA in influencing the T cell proliferation.
Some investigations have demonstrated that PD-1+ T cells are more prone to apoptosis. So we considered that whether BTLA signaling were associated with CD4 T cell apoptosis. We found that annexin V+ CD4 T cells were mainly the BTLA– cells, and BTLA+ CD4 T cells were seldom to apoptosis. Also BTLA expression on CD4 T cells was negatively with annexin V+ CD4 T cells. These data indicate that BTLA–CD4 T cells are more prone to apoptosis that BTLA+CD4 T cells.
Taken together, these data indicate that chronic HIV-1 infection not only progressively down-regulate in BTLA expression in CD4 T cells but also leads to an impairment of BTLA-mediated inhibitory functions on CD4 T cells. This functional loss of BTLA might facilitate immune hyper-activation, which is potentially favorable for HIV-1 proliferation and persistence. Our findings likely indicate that BTLA may present a novel predictor for early progression, which provide further insight into the mechanisms by which non-specific immune activation disrupts T-cell homeostasis and contributes to AIDS disease progression.
我们纳入了85例HIV慢性感染者,根据CD4细胞计数和HIV病毒载量分为三组:长期无进展者(LTNP)、典型进展者(TP)和AIDS患者,并纳入了26例健康人做为对照。通过BTLA在外周淋巴细胞上频率的流式细胞分析,我们发现BTLA主要表达在B细胞,其次在CD4 T细胞、CD8 T细胞、NKT细胞和单核细胞,而弱表达于NK细胞。通过比较HIV慢性感染者和健康者,发现在HIV慢性感染过程中CD4 T细胞和CD8 T细胞上的BTLA表达明显降低,并随着疾病进展呈进行性下降。为了进一步确证这种降低,我们用实时荧光定量PCR检测了BTLA在mRNA水平的表达,发现在慢性HIV感染者(包括TP和AIDS患者),BTLA mRNA的水平确实比健康对照低。由于PD-1主要调节的细胞是HIV特异性的CD8 T细胞,因此我们也研究了BTLA是否也主要作用于HIV特异性的CD8 T细胞,结果发现不仅HIV特异性的CD8 T细胞上BTLA表达降低,而且CMV-和Flu-特异性的CD8 T细胞上的BTLA也降低,说明BTLA的降低是非HIV特异性的。
我们对CD4 T细胞的BTLA表达水平与HIV疾病进展的相关性进行了研究,发现CD4 T细胞上BTLA的表达水平和平均荧光强度与CD4 T淋巴细胞计数呈正相关,而与HIV病毒载量呈负相关。另外,我们还分析了CD4 T细胞上BTLA表达水平与CD38、PD-1、CD127和Ki67等与疾病进程密切相关的免疫学标志的相关性,结果发现CD4 T细胞上BTLA的表达与CD38、PD-1和Ki67的表达呈负相关,而与CD127的表达呈正相关,这进一步显示HIV感染过程中BTLA的进行性下降与疾病进程密切相关。
根据CD27和CD45RO可将CD4 T细胞分为naive T细胞(Tnaive)、中央记忆性细胞(Tcm)和效应记忆性T细胞(Tem)等三个亚群,我们也检测了BTLA在这些CD4 T细胞亚群上的表达。发现LTNP患者Tnaive亚群和Tem亚群上BTLA仅有微弱下降,而Tcm亚群几乎无下降。在TP和AIDS患者,BTLA在Tnaive、Tcm和Tem三个亚群上的表达均明显降低,特别是在AIDS患者。另外,我们从mRNA水平也验证了BTLA在这些CD4细胞亚群上的降低。这些结果说明,持续的HIV感染导致了BTLA的持续下降,BTLA的持续下降又导致了na?ve CD4 T细胞比例降低和记忆性CD4 T细胞比例升高。
为了调查CD4 T细胞上BTLA的表达是否影响CD4 T细胞的活化状态,我们分析了健康个体中CD4+BTLA+和CD4+BTLA– T细胞的表型特征,发现BTLA+的CD4 T细胞总体表现出幼稚的表型,即Tniave细胞所占比例最高,而Tcm和Tem所占比例较低。BTLA–的CD4 T细胞总体表现出成熟的表型,主要表现在Tnaive细胞所占比例较低,Tcm和Tem占了大多数。在HIV感染者中虽然BTLA+和BTLA–的CD4 T细胞表现出和健康个体较相同的特征,但BTLA+的CD4 T细胞中Tnaive细胞比健康个体比例要低,尤其在AIDS患者,Tnaive细胞比例更低。这些结果提示持续的HIV感染可能改变了BTLA+ CD4 T细胞中初始T细胞和记忆T细胞的组成。进一步分析BTLA+和BTLA– CD4 T细胞上BTLA和细胞活化标志CD38、凋亡标志Fas(CD95)、细胞功能耗竭标志PD-1、功能效应分子穿孔素(perforin),以及细胞增殖标志Ki67的关系发现,HIV慢性感染者BTLA+ CD4 T细胞表达CD38、PD-1、CD95、perforin和Ki67均有明显升高,而表达CD127明显降低,尤其在TP和AIDS患者这种变化更为明显。但是在BTLA– CD4 T细胞上,除了CD38在HIV慢性感染者中表达升高外,其他上述免疫学标志几乎无变化,说明在慢性HIV感染过程中BTLA+ CD4 T细胞分化、成熟等方面比BTLA– CD4 T细胞受到的影响更为明显。
我们在研究中发现在体外加入HIV-1 NL4-3病毒颗粒能够使CD4 T细胞上的BTLA表达降低,而且这种降低与加入的病毒量有量效关系,加入逆转录酶抑制剂齐多夫定后不能逆转BTLA的降低,说明HIV诱导的BTLA表达下降并不是通过HIV基因去影响BTLA的表达,这种调节很可能发生在HIV进入或吸附细胞表面阶段,或HIV分泌的某种蛋白调节了BTLA的下降。
体外功能实验显示,用刺激性抗体交联BTLA后,与健康对照相比TP/AIDS患者BTLA对CD4 T细胞活化和分泌细胞因子的抑制功能均受到损伤,这种抑制功能的降低可能是TP/AIDS患者上BTLA表达下降引起的。因此BTLA对CD4 T细胞抑制功能的降低可能是AIDS患者体内免疫系统过度活化的原因之一。以往对人和小鼠的研究表明BTLA对TCR诱导的T细胞增殖有抑制作用,但我们的研究显示,用刺激性抗体交联BTLA后,BTLA对T细胞的增殖表现出较大的异质性,在不同个体可能表现出不同的作用,或者抑制或者促进增殖。在今后的实验中,将进一步明确BTLA对T细胞增殖的影响。
一些研究已经报道了PD-1+ T细胞更容易自发凋亡,我们考虑BTLA介导的信号途径是否对CD4 T细胞的凋亡也有作用。通过对HIV慢性感染者的研究发现,凋亡的CD4 T细胞(即annexin V+ CD4 T细胞)主要是BTLA–细胞,而BTLA+细胞很少有凋亡,且BTLA的表达水平与CD4 T细胞的自发性凋亡呈明显负相关,这些结果显示在HIV感染者中BTLA–CD4 T细胞比BTLA+CD4 T细胞更易发生凋亡。
总之,我们的研究结果显示随着HIV慢性感染疾病进展,CD4 T细胞上BTLA表达逐渐下降,而且BTLA介导的对CD4 T细胞的抑制功能受到损伤,BTLA的功能损伤可能导致了机体的免疫过度活化,免疫活化则有利于HIV的复制,最终促进了疾病的进展。这些结果对于阐明BTLA在HIV慢性感染中的致病机制具有重要意义,也为今后人类治疗HIV感染提供了新的靶点。
Non-specific T cell hyperactivation is associated with HIV-1 disease progression, but its mechanisms are poorly defined. Recent findings have demonstrated that T cell activation is substantially impacted by cell surface-expressing co-inhibitory molecules such as programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuator (BTLA, CD272), as well as other receptors. These co-signaling pathways play distinct and overlapping modulatory roles during sequential (or different) stages of the T-cell response or for different lymphocyte subsets so that immune responses occur in the correct intensity and pattern. Recent studies have highlighted the role of immuno-inhibitory molecules such as PD-1, CTLA-4 and Tim-3, which mediate CD4 and CD8 T cell exhaustion during chronic HIV-1 infection. Although the pioneering findings indicate that co-inhibitory signaling mediates T-cell exhaustion and partially contributes to HIV-1 persistence, it is still unknown whether these inhibitory pathways experience functional deficits that may be responsible for the loss of the delicate balance between immune over-activation and efficient viral containment in chronic HIV-1 infection. BTLA, the most recently recognized member of the CD28 family, which interacts with its ligand, herpesvirus entry mediator (HVEM), predominantly negatively regulates immune responses. Recent investigations further demonstrated that BTLA is down-regulated in allergen-specific immunotherapy-untreated patients with Japanese cedar pollinosis, and BTLA gene polymorphism is associated with a high risk of rheumatoid arthritis development. However, little is known regarding the role of BTLA in viral infection, in particular in chronic HIV-1 infection.
Eighty-five HIV-1-infected individuals were enrolled in our study. They were divided into three groups including long-term non-progressors (LTNPs), typical progressors (TPs) and AIDS patients according to CD4 T-cell count and serum HIV-1 RNA load. Twenty-six uninfected subjects were employed as healthy controls (HCs). To define the pattern of BTLA expression in lymphocytes, our experiments showed that BTLA was mainly expressed by peripheral B cells, T cells, NKT cells and monocytes rather than NK cells. In particular, BTLA was found to be constitutively expressed by CD4 T cells and CD8 T cells. We found that BTLA expression levels in both CD4 and CD8 T cells were significantly down-regulated in HIV-1-infected subjects compared with HCs. Importantly, HIV-1-infected individuals displayed large differences in BTLA expression levels in both CD4 and CD8 T cells: LTNPs exhibited higher levels of BTLA expression than did TPs and AIDS patients; while the lowest levels of BTLA expression in both CD4 and CD8 T cells were observed in AIDS patients. The data clearly indicated that chronic HIV-1 infection may lead to overall progressive BTLA down-regulation in CD4 and CD8 T cells. We further analyzed BTLA mRNA expression in HC and HIV-1 infected subjects. It was found that BTLA mRNA expression was also significantly lower in the peripheral blood of TPs and AIDS patients than HCs. Thus, BTLA down-regulation in total T cell subsets is associated with the progression of HIV-1 infection and may serve as a marker of disease progression.
Next, we investigated difference in BTLA expression among HIV-1-, CMV- and Flu-specific pentamer T cells from HIV-1-infected and HC subjects. We observed that BTLA expression in both the CMV- and Flu-specific pentamer cells was significantly reduced in LTNP and TP/AIDS subjects compared with HCs. These data suggest that BTLA expression is also down-regulated in HIV-specific and non-HIV-specific CD8 T cells in progressive HIV infection.
We also analyzed the relationships of BTLA expression in CD4 T cells with two markers of HIV-1 disease progression: plasma viral load and peripheral CD4 T cell count. Our results showed that both BTLA percentage and MFI (mean fluoresce intensity) in total CD4 T cells positively correlated with peripheral CD4 T cell counts, but inversely correlated with plasma viral loads. We further analyzed the associations of BTLA expression with other markers such as CD38, PD-1, CD127 and Ki67, which have been demonstrated to correlate with HIV-1 disease progression. We found that BTLA expression in total CD4 T cells was negatively correlated with CD38, PD-1 and Ki67 expression levels, but positively correlated with CD127 expression in TPs. These data suggest that BTLA down-regulation in CD4 T cells correlates with disease progression in chronic HIV-1 infection.
Through examination of the distribution of BTLA expression in Tna?ve, Tcm and Tem subsets on the basis of CD45RO and CD27 expression, we found that CD4 T cells from HIV-1-infected TPs and AIDS patients were constituted with higher percentages of Tcm and Tem subsets and lower percentage of Tnaive subsets than those from the HC and LTNP subjects. These data probably indicate that chronic HIV-1 infection leads to a progressive decline in naive CD4 T cell subsets but a continuous increase in memory CD4 T cell subsets, and thus altered the distribution of memory CD4 T cell population. More importantly, we found that BTLA expression was more dominant in Tna?ve cells, but gradually decreased in the Tna?ve, Tcm and Tem populations regardless of disease status. BTLA expression was significantly down-regulated in all of three CD4 T cell subsets in HIV-1-infected subjects in comparison to the HCs. These data indicate that persistent HIV-1 infection leads to a progressive loss of BTLA expression as an early change, which precede a reduction of the na?ve CD4 T cell subset and an expansion of the memory CD4 T cell subset.
To determine whether BTLA expression in CD4 T cells influences their activation status, we analyzed BTLA+ and BTLA– CD4 T cell phenotypic profiles in HC and HIV-1-infected subjects. It was found that HC subjects displayed a juvenile phenotype in BTLA+CD4 T cells which were constituted with large percentage of the Tnaive and small percentage of the Tcm and Tem, while BTLA– CD4 T cells that displayed a relatively mature phenotype indicated by large percentage of the Tcm and Tem and small percentage of the Tniave subsets. Interestingly, this distribution was disturbed within BTLA+ but not BTLA– CD4 T cells in HIV-1-infected subjects. Interestingly, this distribution was disturbed within BTLA+ but not BTLA– CD4 T cells in HIV-1-infected subjects. We found that BTLA+ CD4 T cells were constituted with a smaller fraction of Tna?ve subset and more of the Tem subset, in particular in HIV-infected TP and AIDS patients. These data suggest that persistent HIV-1 infection might substantially alter the profiles of memory subsets within BTLA+ CD4 T cells.
We further analyzed the associations of BTLA expression with CD38, CD95, PD-1, Ki67, and perforin. Data showed that chronic HIV-1 infection markedly increased CD38, CD95, PD-1, Ki67 and perforin expression and decreased CD127 expression in BTLA+ CD4 T cell subsets, in particular TPs and AIDS patients, in contrast with HCs. For the BTLA– CD4 T cell population, HIV-1 infection seldom influences the expression of these markers, except for CD38. This comprehensive analysis suggests that HIV-1 infection significantly skews BTLA+ rather than BTLA– CD4 T cells towards differentiation and maturation. Thus, HIV-1 infection not only reduces BTLA expression, but also significantly impacts the immune status of BTLA+ CD4 T cells. We found that HIV-1 NL4-3 viral could down-regulate BTLA expression in CD4 T cells in vitro. In addition, HIV-1 NL4-3-induced BTLA down-regulation in CD4 T cells was viral load-dependent. BTLA down-regulation in CD4 T cells was more pronounced when the cells were cultured with high doses of the virus supernatant. Notably, we found that addition of the anti-viral agent AZT failed to reverse HIV-1-induced BTLA loss at the indicated time point. These data indicate that HIV-1 virus can directly stimulate CD4 T cells to down-regulate BTLA expression, which may reduce HVEM-mediated inhibition and cause more non-specific activation of T cells via a positive loop.
The BTLA-mediated inhibitory pathway plays an important role in maintaining basic immune tolerance by inhibiting T cell activation, cytokine production and proliferation. In order to investigate the functional relevance of diminished BTLA expression in CD4 T cells, we used the agonistic anti-BTLA mAb to compare the BTLA-mediated functional inhibition of CD4 T cells in HIV-1-infected subjects and uninfected persons. We found that BTLA-mediated suppression of CD4 T cell activation (marked by CD25, CD38 and CD69) and cytokine production (IL-2 and INF-γ) was significantly impaired during chronic HIV-1 infeciton.
Some studies showed that BTLA cross-linking can inhibit the proliferation of T cells. But in our study we found that inhibition of T cell proliferation exhibited a high heterogeneity. BTLA cross-linking inhibited T cells proliferation in some subjects but promoted proliferation in other subjects. So further studies should be performed to determine the exact function of BTLA in influencing the T cell proliferation.
Some investigations have demonstrated that PD-1+ T cells are more prone to apoptosis. So we considered that whether BTLA signaling were associated with CD4 T cell apoptosis. We found that annexin V+ CD4 T cells were mainly the BTLA– cells, and BTLA+ CD4 T cells were seldom to apoptosis. Also BTLA expression on CD4 T cells was negatively with annexin V+ CD4 T cells. These data indicate that BTLA–CD4 T cells are more prone to apoptosis that BTLA+CD4 T cells.
Taken together, these data indicate that chronic HIV-1 infection not only progressively down-regulate in BTLA expression in CD4 T cells but also leads to an impairment of BTLA-mediated inhibitory functions on CD4 T cells. This functional loss of BTLA might facilitate immune hyper-activation, which is potentially favorable for HIV-1 proliferation and persistence. Our findings likely indicate that BTLA may present a novel predictor for early progression, which provide further insight into the mechanisms by which non-specific immune activation disrupts T-cell homeostasis and contributes to AIDS disease progression.
引文
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