A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy

Abstract

Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

Figure 1

Mass spectrometric analysis of Fc glycopeptides of total IgG1 and anti-HPA-1a IgG1 from 2 pregnant women. One person showed high degrees of fucosylation for total IgG1 (97%; A) and anti-HPA-1a IgG1 (86%; B). The other also showed a high degree of fucosylation for total IgG1 (86%; C), yet a low degree of fucosylation for anti-HPA-1a IgG1 (9%; D). Major fucosylated glycoforms are labeled in red, and nonfucosylated glycoforms are labeled in blue or are unlabeled. Blue square indicates N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose; purple diamond, N-acetylneuraminic acid; pep, peptide moiety; and asterisk, contaminant. Triple-protonated glycopeptide signals were observed throughout. For the assignment of the glycopeptides signals, see Table 1. The level of galactosylation, sialylation, bisection (bisecting N-acetylglucosamine), and fucosylation were calculated according to the following formulas: Galactosylation = (G1F + G1FN + G1FS + G1FNS + G1) × 0.5 + G2F + G2FN + G2FS + G2FNS + G2 + G2S. Agalactosylated structures = G0F + G0FN + G0. Digalactosylated structures = G2F + G2FN + G2FS + G2FNS + G2 + G2S. Sialylation = G1FS + G2FS + G1FNS + G2FNS + G2S. Bisection = G0FN + G1FN + G2FN + G1FNS + G2FNS. Fucosylation = G0F + G1F + G2F + G0FN + G1FN + G2FN + G1FS + G2FS.

Figure 2

Anti-HPA-1a antibodies in FNAIT display a pronounced lowering of Fc fucosylation. Relative expression levels of major IgG-Fc Asn297 glycoforms for both total IgG1 (x-axis) and antigen-specific IgG1 (y-axis) for 48 FNAIT anti-HPA-1a serum samples (A-D). Serum populations were analyzed for Fc galactosylation (A), sialylation (B), bisection (C), and fucosylation (D). The statistical outcome between 2-tailed paired Student t test analysis of total IgG1 vs specific antibodies is listed in each panel. The diagonal, dotted line represents the equal ratio between total IgG1 and the specific antibody.

Figure 3

FNAIT anti-HPA-1a antibodies with decreased fucosylation are present years after delivery. The degree of anti-HPA-1a Fc fucosylation (specific IgG1 fucosylation minus total IgG1 fucosylation) is plotted against the time after delivery, for 7 anti-HPA-1a FNAIT samples (period of 6 weeks to 7 years postdelivery).

Figure 4

Anti-HLA antibodies in RT do not display a pronounced lowering of Fc fucosylation. Relative expression levels of major IgG-Fc Asn297 glycoforms for both total IgG1 (x-axis) and antigen-specific IgG1 (y-axis) for 13 RT anti-HLA class I serum samples (A-D). Serum populations were analyzed for Fc galactosylation (A), sialylation (B), bisection (C), and fucosylation (D). The statistical outcome between 2-tailed paired Student t test analysis of total IgG1 vs specific antibodies is listed in each panel. The diagonal, dotted line represents the equal ratio between total IgG1 and the specific antibody.

Figure 5

Absence of IgG-Fc core fucose enhances platelet phagocytosis through FcγRIII on PMNs and monocytes. Platelets were haptenized with TNP; labeled for phagocytosis with pHrodo; opsonized with either isotype antibody, anti-TNP low- or high-fucose antibodies; subjected to phagocytosis by either neutrophils (PMNs; A), FcγRIIIa⁺ monocytes (B), or FcγRIIIa^– monocytes (C) as effector cells; and expressed as anti-TNP–specific phagocytosis (Mean Fluorescence Intensity (MFI) anti-TNP − MFI isotype IgG). Anti-TNP IgG1 antibodies with low fucose displayed increased phagocytosis compared with highly fucosylated anti-TNP antibodies using PMNs or FcγRIIIa⁺ monocytes (A-B, respectively) as effector cells, but not with FcγRIIIa^– monocytes (C). Platelets were labeled with pHrodo, opsonized with 35 AU of anti-HPA-1a antibodies from maternal FNAIT sera, and subjected to phagocytosis by neutrophils (D). Phagocytosis was expressed as anti-HPA-1a–specific phagocytosis (MFI anti-HPA-1a from maternal FNAIT sera − MFI isotype IgG from NHS). Statistical analyses: 1-tailed unpaired Student t test (A-C) and Pearson correlation (D). *P ≤ .05; **P ≤ .01. NS, nonsignificant.

Figure 6

Degree of fucosylation correlates with neonatal platelet counts in FNAIT and clinical severity. Degree of fucosylation was compared with neonatal platelet counts in FNAIT directly after delivery (A), as well as with the clinical disease severity score (B). The scores are as follows: 0 indicates no symptoms; 1, mild symptoms (petechiae); 2, moderate symptoms (other bleeding, including organ bleeding); and 3, severe symptoms (intracranial hemorrhages) (B). Statistical analyses were performed using Spearman rank correlation, with a positive correlation in panel A (ρ = 0.320) and a negative correlation in panel B (ρ = −0.377). *P ≤ .05.