A functional spleen contributes to afucosylated IgG in humans

Abstract

As a lymphoid organ, the spleen hosts a wide range of immune cell populations, which not only remove blood-borne antigens, but also generate and regulate antigen-specific immune responses. In particular, the splenic microenvironment has been demonstrated to play a prominent role in adaptive immune responses to enveloped viral infections and alloantigens. During both types of immunizations, antigen-specific immunoglobulins G (IgGs) have been characterized by the reduced amount of fucose present on N-linked glycans of the fragment crystallizable (Fc) region. These glycans are essential for mediating the induction of immune effector functions. Therefore, we hypothesized that a spleen may modulate humoral responses and serve as a preferential site for afucosylated IgG responses, which potentially play a role in immune thrombocytopenia (ITP) pathogenesis. To determine the role of the spleen in IgG-Fc glycosylation, we performed IgG subclass-specific liquid chromatography-mass spectrometry (LC-MS) analysis of Fc glycosylation in a large cohort of individuals splenectomized due to trauma, due to ITP, or spherocytosis. IgG-Fc fucosylation was consistently increased after splenectomy, while no effects for IgG-Fc galactosylation and sialylation were observed. An increase in IgG1- and IgG2/3-Fc fucosylation level upon splenectomy has been reported here for the first time, suggesting that immune responses occurring in the spleen may be particularly prone to generate afucosylated IgG responses. Surprisingly, the level of total IgG-Fc fucosylation was decreased in ITP patients compared to healthy controls. Overall, our results suggest a yet unrecognized role of the spleen in either the induction or maintenance of afucosylated IgG responses by B cells.

Figure 1

LC–MS analysis of IgG-Fc glycopeptides. (A) Schematic workflow. The figure was created with BioRender.com (B) Representative LC–MS summed mass spectrum of IgG1 tryptic glycopeptides obtained from a 43 year-old splenectomized ITP male. Annotated are the 11 most abundant IgG1 glycopeptides. Green circle: mannose, yellow circle: galactose, blue square: N-acetylglucosamine, red triangle: fucose, pink diamond: N-acetylneuraminic acid. (C) Scheme of the IgG-Fc glycan structure, common disease-related changes in total IgG and their immunological implications. It shows derived glycosylation traits representing common glycosylation features shared by glycan composition: sialylation—sialylation per antenna of biantennary glycans, fucosylation—fraction of fucosylated glycans, galactosylation—galactosylation per antenna of biantennary glycans, bisection—fraction of glycans with bisecting N-acetylhexosamine. For detailed calculation per IgG subclass see Supplementary Table S2. *Down arrow refers to a commonly observed lower and up arrow to higher proportion of the corresponding IgG glycosylation trait in patients suffering from autoimmunological, infectious and inflammatory diseases compared to healthy controls; ADCC—antibody-depended cellular cytotoxicity.

Figure 2

Effect of splenectomy on IgG1-Fc glycosylation in healthy individuals and ITP patients. Data are shown for healthy individuals with (Healthy Control, n = 38) or without splenectomy (Trauma Splenectomy, n = 76) and ITP patients with (ITP Splenectomy, n = 35) or without splenectomy (ITP, n = 57), matched for age and sex. Compared to the respective controls, IgG1-Fc fucosylation was higher in splenectomized and otherwise healthy individuals (P = 0.026; Wilcoxon-rank sum test), as well as in splenectomized ITP patients (P = 0.017; Wilcoxon-rank sum test). Regarding IgG1-Fc bisection, otherwise healthy individuals showed lower levels (P = 0.008; Wilcoxon-rank sum test), whereas in splenectomized ITP patients this difference was less prominent and did not result in a finding. ITP patients have reduced IgG1-Fc fucosylation compared to healthy controls (P = 0.017; Kruskal–Wallis rank sum test followed by a post-hoc Nemenyi test for pairwise comparison). All other glycosylation features were not significantly different between the groups. *P-values are given for a Wilcoxon-rank sum test, P-values < 0.05; ns, not significant.

Figure 3

Effect of spherocytosis-associated splenectomy on IgG1-Fc glycosylation. Data are shown for healthy individuals without splenectomy (Healthy control, n = 32) and spherocytosis patients with splenectomy (Spherocytosis splenectomy, n = 16). Compared to the respective controls, spherocytosis-associated splenectomy individuals showed lower levels of IgG1-Fc bisection (P = 0.003; Wilcoxon-rank sum test), and trend towards increased IgG1-Fc fucosylation. All other glycosylation features were not significantly different between the groups.