DOI: 10.5061/dryad.tmpg4f4xg
title: The antigen causing IgG hexamerization in patients with Chronic lymphocytic leukemia (CLL) is Alpha 2 macroglobulin (A2M)

description: 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. According to recent studies,
 formation of ordered IgG-hexamers occurs on cell surfaces via 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-hexamers.
 The obtained IgG-hexamers were measured and used for assessment of CP
 activation. For evaluation of the presence of IgG-hexamers on blood cells,
 whole blood samples were stained and assessed by flow cytometry. Serum
 levels of IgG-hexamers 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 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 timecourse 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.
description: Cell-free IgG were separated
 from sera/plasma using a commercial kit for total IgG extraction, based on
 affinity chromatography, according to the manufacturer's instructions
 (Protein G HP SpinTrapTM, GE Healthcare). The IgG separated by this kit
 includes all IgG molecules, i.e. monomers and hexamers. According to the
 manufacturer, other immunoglobulins (IgM etc.) are not separated by the
 kit. The total IgG fraction separated using the protein G kit was
 transferred to a filtration column with a cutoff of 1000KDa (Vivaspin,
 Sartorius), and centrifuged for 3 minutes at 4K g at 4ºC. Due to the high
 molecular weight of IgG-hexamers, &gt;1000KDa, they are retained on
 top of the column while monomeric IgG move to the bottom. The IgG-hexamers
 prepared from patients' sera were separated by SDS-PAGE and silver
 stained (with ProteoSilver TM plus, Sigma). The heavy (γ) and light chains
 were identified by their molecular mass and high abundance. All other
 protein bands were excised from the gels and subjected to mass
 spectrometry at the Smoler Protein Research Center (Technion – Israel
 Institute of Technology, Haifa, Israel).