Chelucci C, Hassan H J, Locardi C, Bulgarini D, Pelosi E, Mariani G, Testa U, Federico M, Valtieri M, Peschle C
Department of Hematology-Oncology and Virology, Istituto Superiore di Sanità, Rome, Italy.
Blood. 1995 Mar 1;85(5):1181-7.
Uni- or multi-lineage suppression of hematopoiesis is observed in the majority of acquired immunodeficiency syndrome (AIDS) patients. The mechanism(s) underlying these abnormalities is not understood: particularly, the human immunodeficiency virus (HIV) infection of hematopoietic progenitor and stem cells (HPCs/HSCs) is highly controversial. We report that CD34+ HPCs from adult peripheral blood (PB) are in part CD4+ and susceptible to in vitro HIV infection. Primitive CD34+ HPCs were approximately 80% purified from PB. Double labeling for CD34 and CD4 membrane antigens was shown for 5% to 20% of the purified cells, thus suggesting their potential susceptibility to HIV-1 infection. The enriched HPC population, challenged with purified or unpurified HIV-1 strains, was cloned in unicellular methylcellulose culture. The single colonies generated by erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), and granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-GEMM) were analyzed for the presence of HIV, ie, for gag DNA, tat mRNA, and p24 protein by PCR, reverse transcription PCR (RT-PCR), and enzyme-linked immunosorbent assay, respectively. In the first series of experiments incubation of HPCs with HIV-1 at multiplicities of infection (MOI) ranging from 0.01 to 10 TCID50/cell consistently yielded an 11% to 17% infection efficiency of BFU-E-generated colonies, thus indicating the sensitivity of HPCs to in vitro HIV infection. An extensive series of experiments was then performed on HPCs challenged with HIV at 0.1 MOI level. In the initial studies proviral gag sequences were detected in 9.2% of 121 analyzed CFU-GM colonies. In further experiments tat mRNA was monitored in 17% and 23% of BFU-E and CFU-GM colonies, respectively, but never in CFU-GEMM clones. Finally, 12% of CFU-GM clones and rare erythroid bursts were shown to be positive for the p24 viral protein. In control studies, purified HPCs grown in liquid suspension culture were induced to terminal unilineage erythroid, monocytic, or granulocytic differentiation: monocytes were consistently HIV-infected, whereas mature-terminal erythroblasts and granulocytes were not. Our observations indicate that a minority of primitive HPCs, but not of the multipotent type, is susceptible to in vitro HIV infection. These observations may reflect on the in vivo hematopoietic impairment in AIDS patients; more important, they provide an experimental model for studies on HIV hematopoietic infection and in vitro tests for anti-HIV HSC gene therapy.
在大多数获得性免疫缺陷综合征（艾滋病）患者中观察到造血功能的单系或多系抑制。这些异常现象背后的机制尚不清楚：特别是，造血祖细胞和干细胞（HPCs/HSCs）的人类免疫缺陷病毒（HIV）感染极具争议性。我们报告称，来自成人外周血（PB）的CD34⁺ HPCs部分为CD4⁺，且易受体外HIV感染。从PB中大约纯化出了80%的原始CD34⁺ HPCs。5%至20%的纯化细胞显示出CD34和CD4膜抗原的双重标记，这表明它们可能易受HIV-1感染。用纯化或未纯化的HIV-1毒株攻击富集的HPC群体后，在单细胞甲基纤维素培养中进行克隆。对由红系爆式集落形成单位（BFU-E）、粒细胞-巨噬细胞集落形成单位（CFU-GM）和粒细胞-红系-巨噬细胞-巨核细胞集落形成单位（CFU-GEMM）产生的单个集落进行分析，以检测HIV的存在，即分别通过聚合酶链反应（PCR）、逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定法检测gag DNA、tat mRNA和p24蛋白。在第一系列实验中，将HPCs与HIV-1以感染复数（MOI）为0.01至10 TCID50/细胞进行孵育，BFU-E产生的集落的感染效率始终为11%至17%，这表明HPCs对体外HIV感染敏感。然后对以0.1 MOI水平用HIV攻击的HPCs进行了一系列广泛的实验。在最初的研究中，在121个分析的CFU-GM集落中有9.2%检测到了前病毒gag序列。在进一步的实验中，分别在17%和23%的BFU-E和CFU-GM集落中监测到了tat mRNA，但在CFU-GEMM克隆中从未检测到。最后，12%的CFU-GM克隆和罕见的红系爆式集落显示p24病毒蛋白呈阳性。在对照研究中，在液体悬浮培养中生长的纯化HPCs被诱导向终末单系红系、单核系或粒系分化：单核细胞始终被HIV感染，而成熟终末红细胞和粒细胞则未被感染。我们的观察结果表明，少数原始HPCs，而非多能型HPCs，易受体外HIV感染。这些观察结果可能反映了艾滋病患者体内的造血功能损害；更重要的是，它们为研究HIV造血感染和抗HIV HSC基因治疗的体外试验提供了一个实验模型。
