Protective Effect of IL-7 against Spontaneous T-Cell Apo

白介素7(IL-7)是一种细胞因子，在T细胞亚群的产生、激活和稳态中起关键作用，目前正在作为HIV-1感染患者免疫重建的潜在治疗剂进行临床研究。IL-7在HIV-1感染过程中的作用仍不确定。在HIV感染过程中，血浆中IL-7水平的升高已被记录在案，同时伴随着在CD4+和CD8+T细胞中IL-7受体(CD127)？链的表达减少。研究发现，血浆IL-7的升高与外周血中CD4+T细胞数量的减少和血浆病毒载量的升高有关。对这些发现最合理的解释是免疫系统为恢复CD4+T细胞的体内平衡水平所做的补偿性努力。然而，很明显，在晚期HIV-1疾病患者中，这种补偿努力是不够的。因此，对慢性感染HIV-1的患者早期使用IL-7可能有助于限制CD4+T细胞的耗竭和/或促进前体细胞池中的初始T细胞的再生。体内注射IL-7对慢性感染SIV的猕猴的实验表明，在不增加病毒载量的情况下，诱导幼稚T细胞数量增加，以及CD4+和CD8+记忆T细胞的增殖和激活。由NIAID赞助的第一阶段随机、安慰剂对照、双盲试验(IND#BB12069)目前正在进行，以评估皮下单剂IL-7对正在接受抗逆转录病毒治疗的HIV-1感染者的安全性。 由于已知IL-7在T细胞发育过程中发挥抗凋亡作用，我们在选择代表疾病不同阶段的HIV-1感染受试者的队列中，研究了它保护T细胞免受自发性凋亡的能力。重组人IL-7的剂量范围很广(0.5-50 ng/ml)，试图确定它的生存和增殖活性是否可以解偶联。根据先前的结果，我们发现HIV-1感染患者和HIV血清阴性对照的细胞在基线时的自发凋亡水平相似，尽管在6天的体外培养过程中，HIV-1感染患者的细胞自发凋亡水平显著高于HIV-1感染患者。加入外源性IL-7后，通过两种独立的方法(Annexin V结合和caspase 3激活)，观察到所有被测试的HIV-1感染者(n=24)外周血单个核细胞(PBMC)的自发凋亡水平显著降低。IL-7介导的细胞凋亡减少与细胞内Bcl2水平升高有关。相比之下，IL-7对HIV-1血清阴性受试者的PBMC的抗凋亡作用不太一致，也不显著。为了进一步了解IL-7介导的细胞凋亡减少在体内的意义，我们分析了对IL-7的敏感性与几个人口统计学、临床和免疫学参数的相关性，包括年龄、病程、CD4和CD8计数和治疗状态。对IL-7抗凋亡作用的敏感性与循环中的CD4计数呈显著负相关，而所有其他参数均不相关。通过比较IL-7在不同组患者中的影响，证实了这种相关性，这些患者根据每个人口统计学、临床和免疫学参数的定义截止值进行分组。为了验证IL-7是否对CD4+和CD8+T细胞都有保护作用，我们从HIV-1感染者的外周血中纯化了这两个亚群，并在有或没有IL-7的情况下进行了体外培养。这些研究表明，IL-7对HIV-1感染者的两个T细胞亚群具有相似程度的保护性抗凋亡作用。 在体内使用免疫刺激细胞因子如IL-2和IL-7的一个潜在缺点是触发广泛的T细胞激活和增殖的风险，这可能进一步增加HIV-1感染中已经很高的免疫刺激水平，从而为HIV-1复制提供更多的靶细胞。因此，我们调查了IL-7的使用浓度是否与细胞激活和/或增殖有关。用Ki67抗原的抗体进行细胞内染色，显示IL-7介导的抗凋亡作用与Ki67的表达暂时分离。Ki67抗原是细胞周期进展的特异性标志。同样，使用CFSE活性染料或流式细胞术的绝对细胞计数，我们正式证明了IL-7的抗凋亡作用发生在没有细胞分裂的情况下。此外，我们还检测了选定的HIV-1感染患者PBMC或纯化的CD4+T细胞培养中内源性HIV-1p24的释放水平。 我们记录的IL-7的抗凋亡活性为考虑将这种细胞因子作为免疫治疗剂用于治疗HIV-1感染提供了额外的理由，特别是在患有更晚期疾病的患者中，他们不太可能通过HAART恢复完全的免疫重建，其细胞似乎对IL-7的抗凋亡作用更敏感。

Interleukin-7 (IL-7), a cytokine that plays a key role in the generation, activation and homeostasis of the T-cell compartment, is currently under clinical investigation as a potential therapeutic agent for the immune reconstitution of HIV-1-infected patients. The role of IL-7 in the course of HIV-1 infection is still uncertain. Increased plasma levels of IL-7 have been documented in the course of HIV infection, in association with a reduced expression of the ?-chain of the IL-7 receptor (CD127) in both CD4+ and CD8+ T cells. The increase in plasma IL-7 was found to correlate with reduced peripheral CD4+ T-cell counts and higher levels of plasma viral load. The most plausible interpretation of these findings is a compensatory effort by the immune system to restore homeostatic levels of CD4+ T cells. However, it is evident that in patients with advanced HIV-1 disease this compensatory effort is insufficient. Thus, it has been suggested that the early administration of IL-7 to patients chronically infected with HIV-1 may help to limit the depletion of CD4+ T cells and/or to promote the regeneration of naive T cells from the precursor pool. Experimental injection of IL-7 in vivo in macaques chronically infected with SIV was shown to induce an increase in naive T-cell counts, as well as proliferation and activation of both CD4+ and CD8+ memory T cells, without augmenting the viral load. A phase I, randomized, placebo-controlled, double-blind trial (IND # BB12069) sponsored by NIAID is currently under way for evaluating the safety of subcutaneous single dose IL-7 in HIV-1-infected subjects who are receiving antiretroviral treatment. Since IL-7 is known to exert antiapoptotic effects during the course of T-cell development, we have investigated its ability to protect T cells from spontaneous apoptosis in a cohort of HIV-1-infected subjects selected to represent different stages of disease. Recombinant human IL-7 was used over a wide dose range (0.5-50 ng/ml) in an attempt to determine whether its prosurvival and proliferative activities can be uncoupled. In accordance with previous results, we found levels of spontaneous apoptosis at baseline were similar in cells from HIV-1-infected patients and HIV-seronegative controls, although they became significantly higher in cells from HIV-1-infected subjects during the course of 6 days of ex vivo culture. Upon addition of exogenous IL-7, a dramatic reduction in the level of spontaneous apoptosis was observed in peripheral blood mononuclear cells (PBMC) from all the HIV-1-infected patients tested (n = 24), as determined by two independent methods (annexin V binding and caspase 3 activation). The reduction of apoptosis mediated by IL-7 was associated with an increase in the intracellular level of Bcl-2. By contrast, IL-7 had a less consistent, not significant, antiapoptotic effect on PBMC from HIV-1-seronegative subjects. To gain further insights into the in vivo significance of IL-7-mediated apoptosis reduction, we analysed the correlation between the sensitivity to IL-7 and several demographic, clinical and immunological parameters, including age, disease duration, CD4 and CD8 counts and treatment status. A significant inverse correlation was documented between the sensitivity to the antiapoptotic effect of IL-7 and the circulating CD4 counts, while all the other parameters were not correlated. This correlation was confirmed by comparison of the IL-7 effects in different groups of patients divided according to a defined cut-off value for each demographic clinical and immunological parameter. To verify whether the protective effects of IL-7 occurred on both CD4+ and CD8+ T cells, we purified the two subpopulations from the peripheral blood of HIV-1-infected patients and cultured them ex vivo in the presence or absence of IL-7. These studies demonstrated that IL-7 exerts a protective anti-apoptotic effect of similar magnitude on both T-cell subpopulations from HIV-1-infected subjects. A potential drawback with the in vivo use of immunostimulatory cytokines such as IL-2 and IL-7 is the risk of triggering widespread T-cell activation and proliferation, which could further increase the already high level of immunologic stimulation that occurs in HIV-1 infection and thereby provide more target cells for HIV-1 replication. Thus, we investigated whether the use of IL-7 at concentrations that are sufficient to protect from spontaneous apoptosis was associated with cellular activation and/or proliferation. Intracellular staining with an antibody to the Ki67 antigen, a specific marker of cell cycle progression, showed that IL-7-mediated protection from apoptosis was temporally dissociated from Ki67 expression. Likewise, using the CFSE vital dye or absolute cell counting by flow cytometry, we formally demonstrated that the anti-apoptotic effect of IL-7 occurred in the absence of cell division. Moreover, we measured the levels of endogenous HIV-1 p24 release in cultures of PBMC or purified CD4+ T cells from selected HIV-1-infected patients. We observed that treatment with IL-7, in the absence of mitogenic stimulation, did not result in the induction of significant levels of endogenous HIV-1 replication during short-term ex vivo culture (7-14 days). The antiapoptotic activity that we documented for IL-7 provides an additional rationale for consideration of this cytokine as an immunotherapeutic agent in the treatment of HIV-1 infection, particularly in patients with more advanced disease who are less likely to regain full immune reconstitution with HAART and whose cells appear to be more sensitive to the anti-apoptotic effects of IL-7.