Naseraldeen Naseba, Michelis Regina, Barhoum Masad, Chezar Judith, Tadmor Tamar, Aviv Ariel, Shvidel Lev, Litmanovich Adi, Shehadeh Mona, Stemer Galia, Shaoul Ety, Braester Andrei
The Institute for Medical Research, Galilee Medical Center, Nahariya, Israel.
Azrieli Faculty of Medicine, Bar Ilan University, Safed, Israel.
Front Immunol. 2021 Feb 11;11:603569. doi: 10.3389/fimmu.2020.603569. eCollection 2020.
Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. One of the treatments offered for CLL is immunotherapy. These treatments activate various cellular and biochemical mechanisms, using the complement system. Recently it was shown that the complement system in CLL patients is persistently activated at a low level through the classical pathway (CP). The mechanism of chronic CP activation involves the formation of IgG-hexamers (IgG-aggregates). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces specific interactions between Fc regions of the IgG monomers, which occur after antigen binding. The present study investigated the formation of IgG-hexamers in CLL patients and normal (non-malignant) controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation. Sera from 30 patients and 12 NC were used for separation of IgG- aggregates. The obtained IgG- aggregates were measured and used for assessment of CP activation. For evaluation of the presence of IgG- aggregates on blood cells, whole blood samples were stained and assessed by flow cytometry. Serum levels of IgG- aggregates were higher in CLL and they activated the complement system to a higher extent than in NC. Alpha 2 macroglobulin (A2M) was identified as the antigen causing the hexamerization/aggregation of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the cell surface. The exact time course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens.
慢性淋巴细胞白血病（CLL）是西方世界成年人中最常见的白血病。针对CLL提供的治疗方法之一是免疫疗法。这些治疗利用补体系统激活各种细胞和生化机制。最近有研究表明，CLL患者的补体系统通过经典途径（CP）持续处于低水平激活状态。慢性CP激活的机制涉及IgG六聚体（IgG聚集体）的形成。根据最近的研究，有序IgG六聚体的形成发生在细胞表面，是IgG单体的Fc区域之间特异性相互作用的结果，这种相互作用在抗原结合后发生。本研究调查了CLL患者和正常（非恶性）对照（NC）中IgG六聚体的形成、它们激活补体的能力、它们以游离形式和细胞结合形式存在的发生率以及导致其形成的抗原的特性。使用30例患者和12例NC的血清分离IgG聚集体。对获得的IgG聚集体进行测量，并用于评估CP激活情况。为了评估血细胞上IgG聚集体的存在情况，对全血样本进行染色并通过流式细胞术进行评估。CLL患者血清中IgG聚集体水平更高，并且它们比NC更能激活补体系统。α2巨球蛋白（A2M）被确定为导致IgG六聚化/聚集的抗原，通过质谱、蛋白质印迹和流式细胞术分析发现它是六聚体结构的一部分。B细胞上存在A2M-IgG六聚体表明它可能在B细胞表面形成，然后脱离成为游离形式。或者，它可能在血浆中形成，然后附着在细胞表面。CLL中A2M-IgG六聚体形成的确切时间进程应进一步研究。本研究结果可能有助于改进当前的免疫治疗方案。
