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

白细胞介素-7（IL-7）是一种在T细胞区室的产生、活化和稳态中起关键作用的细胞因子，目前正作为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感染过程中的作用尚不明确。在HIV感染过程中，IL-7的血浆水平升高，与？在CD 4+和CD 8 + T细胞中，IL-7受体（CD 127）的链。发现血浆IL-7的增加与外周血CD 4 + T细胞计数减少和血浆病毒载量水平升高相关。对这些发现最合理的解释是免疫系统恢复CD 4 + T细胞稳态水平的补偿性努力。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，已经表明，对慢性感染HIV-1的患者早期给予IL-7可能有助于限制CD 4 + T细胞的消耗和/或促进幼稚T细胞从前体库的再生。在SIV慢性感染的猕猴体内实验性注射IL-7显示诱导初始T细胞计数增加，以及CD 4+和CD 8+记忆T细胞的增殖和活化，而不增加病毒载量。由NIAID申办的I期、随机、安慰剂对照、双盲试验（IND #BB 12069）目前正在进行中，用于评估皮下单剂量IL-7在接受抗逆转录病毒治疗的HIV-1感染受试者中的安全性。 由于IL-7在T细胞发育过程中发挥抗凋亡作用，我们研究了其保护T细胞免受HIV-1感染受试者自发凋亡的能力，这些受试者被选择代表疾病的不同阶段。在宽剂量范围（0.5-50 ng/ml）内使用重组人IL-7以试图确定其促存活和增殖活性是否可以解偶联。根据以前的结果，我们发现，在基线水平的自发凋亡是相似的细胞从HIV-1感染的患者和HIV-血清阴性对照，虽然他们成为显着较高的细胞从HIV-1感染的受试者在6天的过程中的离体培养。加入外源性IL-7后，在所有受试HIV-1感染患者（n = 24）的外周血单核细胞（PBMC）中观察到自发性细胞凋亡水平显著降低，这通过两种独立的方法（膜联蛋白V结合和半胱天冬酶3激活）测定。IL-7介导的细胞凋亡的减少与细胞内Bcl-2水平的增加有关。相比之下，IL-7对HIV-1血清阴性受试者PBMC的抗凋亡作用不太一致，不显著。为了进一步了解IL-7介导的细胞凋亡减少的体内意义，我们分析了对IL-7的敏感性与几个人口统计学、临床和免疫学参数之间的相关性，包括年龄、疾病持续时间、CD 4和CD 8计数以及治疗状态。一个显着的负相关性之间的敏感性IL-7的抗凋亡作用和循环CD 4计数，而所有其他参数不相关。通过比较根据每个人口统计学临床和免疫学参数的定义的截止值划分的不同组患者中的IL-7效应，证实了这种相关性。为了验证IL-7是否对CD 4+和CD 8 + T细胞都有保护作用，我们从HIV-1感染患者的外周血中纯化了这两个亚群，并在存在或不存在IL-7的情况下离体培养。这些研究表明，IL-7对来自HIV-1感染受试者的两种T细胞亚群发挥相似量级的保护性抗凋亡作用。 体内使用免疫刺激性细胞因子如IL-2和IL-7的潜在缺点是引发广泛的T细胞活化和增殖的风险，这可能进一步增加在HIV-1感染中发生的已经高水平的免疫刺激，从而为HIV-1复制提供更多的靶细胞。因此，我们研究了使用浓度足以防止自发凋亡的IL-7是否与细胞活化和/或增殖相关。Ki 67抗原（细胞周期进展的特异性标志物）的抗体的细胞内染色表明，IL-7介导的细胞凋亡保护与Ki 67表达在时间上分离。同样地，使用CFSE活体染料或通过流式细胞术进行绝对细胞计数，我们正式证明了IL-7的抗凋亡作用发生在细胞分裂不存在的情况下。此外，我们测量了内源性HIV-1 p24释放水平的PBMC或纯化的CD 4 + T细胞从选定的HIV-1感染的患者的培养物。我们观察到，在没有促有丝分裂刺激的情况下，用IL-7处理在短期离体培养（7-14天）期间没有导致诱导显著水平的内源性HIV-1复制。 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抑制剂治疗HIV-1感染提供了额外的理论依据，特别是在更晚期的疾病患者中，这些患者不太可能用HAART重新获得完全免疫重建，并且其细胞似乎对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.