Patients with Autoimmune Anti-Red Blood Cell IgG1 Have Different Total- and Autoantibody -IgG1 Glycosylation Compared to Healthy Controls
Authors: Sonneveld Myrthe E. , De Haas Masja , Koeleman Carolien , Ligthart Peter , Wuhrer Manfred , Van der Schoot C. Ellen , Vidarsson Gestur 
Published: October 2019

Abstract
Introduction
Autoimmune hemolytic anemia (AIHA) is a potentially severe disease in which red blood cells (RBC) are destroyed by autoantibodies. IgG anti-RBC autoantibodies bind at 37°C and can lead to extravascular hemolysis by binding to phagocytes in the spleen.
The composition of the N-linked sugar at position 297 in the IgG-Fc tail influences the binding affinity to IgG Fc receptors (FcγR) on effector cells. By mass spectrometric analysis we recently extensively studied the contribution of this sugar moiety to the clinical effect of RBC and platelet alloantibodies in pregnant women. Compared to the IgG glycoforms detected in plasma of these women, anti-D, anti-K and anti-Human Platelet Antigen 1a were found to be skewed towards IgG1 isoforms with low fucosylation, increasing binding capacity to IgG-Fc receptors type IIIa and IIIb (Kapur et al Blood 2014;123:471-80 & BHJ 2015;166:936-45; Sonneveld et al BJH 2016; 174:310-320 and oral presentation ASH Blood 2015;126:660). This feature has only been described for anti-HIV antibodies, but never for any other immune response. Furthermore, we observed an additional role for the level of bisected GlcNAc (bisection), galactosylation and sialylation of the RBC alloantibody-bound sugar moiety in predicting clinical outcome of HDFN. It is known that in autoimmune diseases the galactosylation and sialylation of plasma IgG1 is lowered, the glycoforms of specific autoantibodies have not yet been studied.
Aim
The aim of this study was to investigate the type of glycoforms of RBC autoantibodies in relation to the plasma IgG1 glycoforms in these patients and occurrence of hemolysis.
Methods
109 patients with a positive direct antiglobulin test (DAT) with anti-IgG were included, of whom blood was sent to our laboratory for diagnostic purposes. From 90 patients clinical data were collected. Based on laboratory markers (hemoglobin (Hb), haptoglobin, lactate dehydrogenase, reticulocyte count, bilirubin level), these patients were divided in three groups: 1. with hemolysis (n=36), 2. no hemolysis (n=33) or 3. possible hemolysis (n=21). RBC-bound autoantibodies were purified by acid elution. This RBC-eluted IgG and total IgG purified from paired plasma samples were subjected to tryptic digestion followed by mass spectrometry for glycoprofiling. Total IgG1 glycosylation patterns from healthy donors were used as controls.
Results
In agreement with the previously described low IgG1-galactosylation and sialylation in patients with autoimmune diseases, we observed that IgG1 purified from plasma of patients with RBC-bound antibodies (a positive DAT) showed decreased galactosylation and sialylation levels compared to healthy controls: average 36% (95% CI, 34-37%) versus 53% (95%CI, 50-56%) (p<0.0001) and 2.4% (95% CI, 1.8-2%) versus 4% (95% CI, 3.4-4.5%), respectively. We now observed that total bisection levels were higher in patients with RBC autoantibodies (19%) versus healthy controls (12%) (p<0.001).
Compared to plasma IgG1 glycosylation, the RBC-bound autoantibodies showed a profile with lower galactosylation, higher sialylation and lower bisecting GlcNAc levels (p=0.0029, p=0.0128 and p<0.0001 respectively). Fc-fucosylation was not different between healthy controls and patients for plasma IgG1 and not for RBC-bound IgG1 fractions (p=0.4986 and p=0.29 resp.).
Analysis of Fc glycoprofiles in relation to Hb levels showed that higher bisection was associated with higher Hb levels for the total group of patients (p<0.001). This correlation was not found analyzing the three patients groups separately, probably due to low sample number.
In conclusion, we found a low total IgG1 Fc-galactosylation in patients with RBC-bound IgG antibodies irrespective of occurrence of hemolysis. This supports the hypothesis that galactosylation is a marker for activation of the immune system in autoimmune diseases. In addition, we found RBC-bound IgG1 to be differently skewed in the composition of the N-linked sugar at position 297 compared to plasma-derived IgG1 in these patients.

Disclosures
No relevant conflicts of interest to declare.