Bonanno Giuseppina, Mariotti Andrea, Procoli Annabella, Corallo Maria, Scambia Giovanni, Pierelli Luca, Rutella Sergio
Department of Gynaecology, Catholic University Medical School, Rome, Italy.
BMC Immunol. 2009 Aug 27;10:46. doi: 10.1186/1471-2172-10-46.
Umbilical cord blood (UCB) is enriched with transplantable CD34+ cells. In addition to CD34-expressing haematopoietic stem cells (HSC), human UCB contains a rare population of CD34-lineage- cells endowed with the ability to differentiate along the T/NK pathway in response to interleukin (IL)-15 and a stromal cell support. IL-21 is a crucial regulator of NK cell function, whose influence on IL-15-induced differentiation of CD34-lineage- cells has not been investigated previously. The present study was designed and conducted to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34-lineage- cells.
CD34-lineage- cells were maintained in liquid culture with Flt3-L and SCF, with the addition of IL-15 and IL-21, either alone or in combination. Cultures were established in the absence of feeder cells or serum supplementation. Cytokine-treated cells were used to evaluate cell surface phenotype, expression of molecular determinants of lymphoid/NK cell differentiation, secretion of IFN-gamma, GM-CSF, TNF-alpha and CCL3/MIP-1alpha, and cytolytic activity against NK-sensitive tumour cell targets. CD34-lineage- cells proliferated vigorously in response to IL-15 and IL-21 but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins. CD34-lineage- cells expanded by IL-21 in combination with IL-15 acquired lymphoid morphology and killer-cell immunoglobulin-like receptor (KIR)-CD56+CD16-/+ phenotype, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells expressed high levels of mRNA for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRgamma genes. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-gamma, GM-CSF and CCL3/MIP-1alpha, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules.
This study underpins a novel role for IL-21 in the differentiation of pseudo-mature lytic NK cells in a synergistic context with IL-15, and identifies a potential strategy to expand functional NK cells for immunotherapy.
脐带血（UCB）富含可移植的CD34+细胞。除了表达CD34的造血干细胞（HSC）外，人脐带血还含有一种罕见的CD34谱系阴性细胞群体，该群体在白细胞介素（IL）-15和基质细胞支持下具有沿T/NK途径分化的能力。IL-21是NK细胞功能的关键调节因子，其对IL-15诱导的CD34谱系阴性细胞分化的影响此前尚未研究。本研究旨在探讨IL-21是否可以替代基质细胞需求，并促进IL-15诱导的人脐带血CD34谱系阴性细胞向NK细胞分化。
CD34谱系阴性细胞在添加了Flt3-L和SCF的液体培养基中培养，并单独或联合添加IL-15和IL-21。培养体系在无饲养细胞或血清补充的情况下建立。用细胞因子处理的细胞来评估细胞表面表型、淋巴样/NK细胞分化分子决定因素的表达、IFN-γ、GM-CSF、TNF-α和CCL3/MIP-1α的分泌，以及对NK敏感肿瘤细胞靶标的细胞溶解活性。CD34谱系阴性细胞对IL-15和IL-21有强烈增殖反应，但对单独的IL-21无反应，并上调磷酸化的Stat1和Stat3蛋白。IL-21与IL-15联合培养扩增的CD34谱系阴性细胞获得了淋巴样形态和杀伤细胞免疫球蛋白样受体（KIR）-CD56+CD16-/+表型，与准成熟NK细胞一致。IL-21/IL-15分化的细胞表达高水平的Bcl-2、GATA-3和Id2（NK细胞发育所需的主开关）的mRNA，并含有未重排的TCRγ基因。从功能角度看，IL-21/IL-15处理的细胞分泌大量的IFN-γ、GM-CSF和CCL3/MIP-1α，并在与NK敏感肿瘤靶标接触时表达细胞表面CD107a，这是NK分泌颗粒胞吐作用的一种测量方法。
本研究证实了IL-21在与IL-15协同作用下，在准成熟裂解性NK细胞分化中的新作用，并确定了一种扩增功能性NK细胞用于免疫治疗的潜在策略。
