Immunoglobulin G Fragment Crystallizable Glycosylation After Hematopoietic Stem Cell Transplantation Is Dissimilar to Donor Profiles

Abstract

Immunoglobulin G (IgG) fragment crystallizable (Fc) N-glycosylation has a large influence on the affinity of the antibody for binding to Fcγ-receptors (FcγRs) and C1q protein, thereby influencing immune effector functions. IgG Fc glycosylation is known to be partly regulated by genetics and partly by stimuli in the microenvironment of the B cell. Following allogeneic hematopoietic stem cell transplantation (HSCT), and in the presence of (almost) complete donor chimerism, IgG is expected to be produced by, and glycosylated in, B cells of donor origin. We investigated to what extent IgG glycosylation in patients after transplantation is determined by factors of the donor (genetics) or the recipient (environment). Using an IgG subclass-specific liquid chromatography-mass spectrometry method, we analyzed the plasma/serum IgG Fc glycosylation profiles of 34 pediatric patients pre-HSCT and at 6 and 12 months post-HSCT and compared these to the profiles of their donors and age-matched healthy controls. Patients treated for hematological malignancies as well as for non-malignant hematological diseases showed after transplantation a lower Fc galactosylation than their donors. Especially for the patients treated for leukemia, the post-HSCT Fc glycosylation profiles were more similar to the pre-HSCT recipient profiles than to profiles of the donors. Pre-HSCT, the leukemia patient group showed as distinctive feature a decrease in sialylation and in hybrid-type glycans as compared to healthy controls, which both normalized after transplantation. Our data suggest that IgG Fc glycosylation in children after HSCT does not directly mimic the donor profile, but is rather determined by persisting environmental factors of the host.

Keywords: N-glycan; fragment crystallizable glycosylation; hematopoietic stem cell transplantation; immune reconstitution; immunoglobulin G.

Figure 1

Glycan structures found on immunoglobulin G (IgG)-fragment crystallizable (Fc). Both hybrid-type and diantennary complex-type glycan structures were found on the IgG Fc glycopeptides in our study. Hybrid-type glycans varied by the presence of a sialic acid and/or a bisecting N-acetylglucosamine (GlcNAc). Diantennary complex-type glycans were found with zero to two galactoses, zero to two sialic acids, and the presence or absence of a bisecting GlcNAc and/or a core fucose. Monosaccharide linkages were based on literature (6, 13, 20, 21). Green circle: mannose, yellow circle: galactose, blue square: GlcNAc, red triangle: fucose, pink diamond: N-acetylneuraminic acid (Neu5Ac/sialic acid).

Figure 2

Representative mass spectra of IgG1 glycopeptides. Annotated are the 15 most abundant IgG1 glycoforms detected for a 7.5 years old, male, acute myeloid leukemia patient (A) before hematopoietic stem cell transplantation (HSCT), (B) 12 months after HSCT and (C) his 24 years old, healthy, male donor. Triply charged glycopeptides are shown, the proposed glycan structures are based on literature (6, 13, 20, 21). Green circle: mannose, yellow circle: galactose, blue square: N-acetylglucosamine (GlcNAc), red triangle: fucose, pink diamond: N-acetylneuraminic acid (Neu5Ac). Differences in relative abundances of glycoforms as compared to the patient pre-HSCT are indicated by red (lower than in pre-HSCT patient) and blue (higher than in pre-HSCT patient) arrows.

Figure 3

Immunoglobulin G (IgG) fragment crystallizable (Fc) galactosylation in all patients, compared to their donors and age-matched healthy controls. IgG Fc galactosylation on (A) IgG1, (B) IgG2/3, and (C) IgG4 is different between patients before hematopoietic stem cell transplantation (HSCT) (Pre) and their donors (D). No changes were observed between patients pre- and 12 months post-HSCT (12 m). In general, patients before and after HSCT have lower galactosylation than age-matched healthy controls (H), although this was not statistically significant for IgG4 galactosylation. 6 m: patients 6 months after HSCT. Patients and donors were compared by Wilcoxon signed-rank tests, healthy controls and patients with Mann–Whitney U tests. p-Values <0.015 were considered statistically significant after 5% FDR correction (as indicated by the asterisks above the plots). For IgG1 galactosylation, n (H, Pre, 6 m, 12 m, D) = 81, 32, 34, 34, 29, for IgG2/3 galactosylation, n (H, Pre, 6 m, 12 m, D) = 69, 32, 32, 33, 29, for IgG4 galactosylation, and n (H, Pre, 6 m, 12 m, D) = 59, 27, 15, 17, 28, respectively.

Figure 4

IgG1 fragment crystallizable (Fc) galactosylation in all patients stratified on diagnose, compared to their donors and age-matched healthy controls. IgG1 Fc galactosylation in children (A) with non-malignant hematological diseases and (B) with malignant hematological diseases. H: age-matched healthy controls, Pre: patients prior to hematopoietic stem cell transplantation (HSCT), 6 m: patients 6 months after HSCT, 12 m: patients 12 months after HSCT, D: donors prior to HSCT. Patients and donors were compared by Wilcoxon signed-rank tests, healthy controls and patients with Mann–Whitney U tests. p-Values <0.015 were considered statistically significant after 5% FDR correction (as indicated by the asterisks above the plots). For non-malignant hematological diseases, n (H, Pre, 6 m, 12 m, D) = 18, 16, 18, 18, 14, for malignant hematological diseases, n (H, Pre, 6 m, 12 m, D) = 16, 16, 16, 16, 15, respectively.

Figure 5

IgG1 fragment crystallizable (Fc) glycosylation features in patients with malignant hematological diseases, compared to their donors and age-matched healthy controls. IgG1 (A) hybrid-type glycans and (B) sialylation differ between healthy controls (H) and patients before hematopoietic stem cell transplantation (HSCT) (Pre). In addition, IgG1 (A) hybrid-type glycans and (B) sialylation are different between patients and donors (D), and IgG1 (A) hybrid-type glycans and (C) sialylation per galactose change within the patients after HSCT. 6 m: patients 6 months after HSCT, 12 m: patients 12 months after HSCT. Patients and donors were compared by Wilcoxon signed-rank tests, healthy controls and patients with Mann–Whitney U tests. p-Values <0.015 were considered statistically significant after 5% FDR correction (as indicated by the asterisks above the plots). For all IgG1 glycosylation features in patients treated for malignant hematological diseases, n (H, Pre, 6 m, 12 m, D) = 16, 16, 16, 16, 15, respectively.