ROTRF Recognition Prize – Recognising Excellence in Organ Transplantation Research

12:55: C1q- and Mannose Binding Lectin (MBL)-dependent Pathways of Complement Activation Are Modulated by IVIg Glycoforms

Dr Barbara Wasowska, Johns Hopkins University, Baltimore, USA

Alloantibodies (AlloAb) contribute to transplant rejection through complement (C) activation. Clinically irreversible graft rejection is correlated with the presence of high levels of AlloAb to donor HLA and deposition of C4d in the graft.

In our previously reported studies we found that mouse mononuclear cells sensitized with specific C-activating Allo-mAb synergized with non-C-activating IgG1 Allo-mAb to deposit C4d. This synergy was significantly decreased in the absence of mannose binding lectin (MBL) and C1q. Reconstitution of C1q/MBL deficient sera restored almost completely the ability to deposit C4d indicating that both C1q and MBL strongly contribute to this synergy in vitro.

IVIg can reverse antibody-mediated graft rejection and prevents its reemergence through the inhibition of C activation. The presence and composition of the sugar moiety attached to the asparagine 297 residue (ASN297) in the Fc fragment of IgG determines its interaction with Fc receptors and MBL. MBL binds to N-acetylglucosamine, mannose, glucose, fucose, but not to galactose, residues in the core structure of ASN297. We propose that IgG-G0 glycoforms of IVIg generated by the removal of sialic acid+galactose from the ASN297 residue in the Fc fragment of IVIg increase its ability to inhibit MBL-dependent pathway of C activation.

In this study, human brain microvascular endothelial cells (BMEC), macrophage U-937 and HLA-typed B cell lines were reacted with human sera containing high levels of C-activating anti-HLA AlloAb. IVIg did not contain antibodies that either bound or activated C on tested target cells. The involvement of C1q- and/or MBL-dependent pathways was measured by the ability of IVIg to inhibit C4d deposition in the presence of normal human sera (NHS), C1q-deficient, MBL-deficient or reconstituted sera (1:1 mixture of C1q-def+MBL-def).

In the presence of NHS, AlloAb from highly sensitized patients caused C4d deposition, which was decreased significantly in the absence of C1q (85-95%) or MBL (70-85%) and restored almost completely (70-90%) in the presence of reconstituted sera. The addition of increasing doses (8, 16, 32 mg/ml) of untreated IVIg (Talecris, Inc) to target cells sensitized with anti-HLA AlloAb resulted in a dose-dependent inhibition of C4d deposition. In the presence of the same doses of IgG-G0 glycoforms of IVIg deposition of C4d on target cells was 3-6-fold lower compared with untreated IVIg.

This study demonstrated that 1) IVIg-dependent inhibition of C activation by anti-HLA antibodies involves both C1q and MBL pathways, and 2) IgG-G0 glycoforms of IVIg are more effective than untreated IVIg in inhibiting C activation by anti-HLA AlloAb. These findings indicate that the carbohydrate content of IVIg glycoforms contributes to significant changes in C-mediated immune responses.

Barbara Wasowska

Dr Barbara Wasowska received her PhD in Immunology at the Polish Academy of Sciences in Warsaw, Poland in 1981. She underwent her initial post-graduate training at the Transplantation Laboratory, University of Helsinki, Finland, the Department of Clinical Immunology, University of Goeteborg, Sweden and the Center for Experimental and Clinical Medicine, Polish Academy of Sciences in Warsaw, Poland. In 1989 she joined the Transplant Research Laboratory at Duke University Medical Center in Durham, and then in 1992, Transplant Research Laboratories at the Beth Israel and Brigham & Women’s Hospitals, Harvard Medical School in Boston, where she completed her post-doctoral fellowships. In 1996 she obtained a junior faculty position at the Johns Hopkins University (JHU) School of Medicine in Baltimore and became an Assistant Professor of Immunopathology in 2000. Besides research, Dr. Wasowska has been involved in the training of basic science and clinical researchers, and teaching immunology and organ transplantation in the JHU Pathobiology Graduate Program.

Dr Wasowska is an internationally recognised expert in transplant immunology. Her research interests focus on studying the mechanisms of antibody-, complement- and cytokine-mediated endothelial cell injury/development of vascular pathology in mouse and human models of cardiac and kidney graft rejection, the role of glycosylation patterns of antibodies in expression of pro- or anti-inflammatory functions and regulation of complement- and FcgR-dependent pathways that link innate and adaptive immune responses in allograft rejection. She is an active member of several professional societies, including The Transplantation Society and the American Society of Transplantation. She serves as a reviewer for the American Journal of Transplantation, Transplantation, Immunobiology and The Open Surgery Journal. In 2010 she served as an invited editor of the section “Mechanisms of Rejection” in the Current Opinion of Organ Transplantation. She has published approximately 100 peer-reviewed articles, reviews and book chapters. Her research has been continuously funded by grants from the National Institute of Health, Roche Organ Transplantation Research Foundation, American Heart Association and Talent Program from Talecris Biotherapeutics, Inc.