Afucosylation of HLA-specific IgG1 as a potential predictor of antibody pathogenicity in kidney transplantation

Abstract

Antibody-mediated rejection (AMR) is the leading cause of graft failure. While donor-specific antibodies (DSAs) are associated with a higher risk of AMR, not all patients with DSAs develop rejection, suggesting that the characteristics of alloantibodies determining their pathogenicity remain undefined. Using human leukocyte antigen (HLA)-A2-specific antibodies as a model, we apply systems serology tools to investigate qualitative features of immunoglobulin G (IgG) alloantibodies including Fc-glycosylation patterns and FcγR-binding properties. Levels of afucosylated anti-A2 antibodies are elevated in seropositive patients, especially those with AMR, suggesting potential cytotoxicity via FcγRIII-mediated mechanisms. Afucosylation of both glycoengineered monoclonal and naturally glycovariant polyclonal serum IgG specific to HLA-A2 drives potentiated binding to, slower dissociation from, and enhanced signaling through FcγRIII, a receptor widely expressed on innate effector cells, and greater cytotoxicity against HLA-A2⁺ cells mediated by natural killer (NK) cells. Collectively, these results suggest that afucosylated DSA may be a biomarker of AMR and contribute to pathogenesis.

Keywords: ADCC; IgG; afucosylation; antibody-mediated rejection; donor-specific antibody; effector function; glycosylation; transplantation.

Graphical abstract

Figure 1

Subclass distribution of HLA-specific antibodies Characterization of HLA-specific antibodies binding to HLA-A2 (A) and HLA-A1 (B) antigens across IgG subclasses in HLA-A2-positive (hollow black circles; n = 30–32) and control (hollow gray circles; n = 18) individuals. HLA-A2-specific BB7.2 (square) and HIV-specific VRC01 (triangle) mAb subclass controls for HLA-A2 reactivity are shown for each subclass: IgG1 (light blue), IgG2 (orange), IgG3 (dark blue), and IgG4 (red). Buffer only blank (cross) and pooled IVIG (diamond) are shown in brown and purple, respectively. Serum samples were tested at a 1:100 dilution. Data shown are representative of two technical replicates. Solid red lines indicate group median. Differences between groups were evaluated using ordinary two-way ANOVA adjusted for multiple comparisons using Bonferroni’s test (∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, respectively).

Figure 2

Fc glycosylation of HLA-A2-specific antibodies (A and B) Representative extracted ion chromatograms and mass spectra illustrating the observed variability between HLA-A2-specific (A) and total (B) IgG1 Fc glycosylation patterns of the same patient. (C) Volcano plot displaying the log10-fold change (x axis) and −log10 p value (y axis) of individual IgG1 (blue circle) and IgG2/3 (maroon square) glycoforms between total and HLA-A2-specific IgG1 and IgG2/3, respectively. (D) Violin plots showing the relative prevalence of glycans on bulk and HLA-A2-specific IgG1 (top) and IgG2/3 (bottom), respectively. Statistical analysis was performed using a paired two-tailed Student’s t test (∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, respectively).

Figure 3

Impact of HLA-A2-specific mAb fucosylation on FcγRIIIa binding, signaling, and cytotoxic activity per subclass (A) FcγRIIIa signaling in a reporter cell line assay with unmodified and afucosylated HLA-A2 mAbs of varying subclasses. (B) Death (percentage of cytotoxicity) of HLA-A2+ target cells following coculture with NK cells in the presence or absence of antibodies of varying specificity, subclass, and fucosylation status. Connecting lines indicate curve fit models. Error bars indicate mean and SD of duplicates. Dotted horizontal line represents ADCC activity in the absence of antibody. (C) Schematic figure of the BLI experiment to define FcγRIIIa off rates from HLA-A2-specific antibodies. Illustration created with http://BioRender.com. (D) FcγRIIIa V158 association with and dissociation from unmodified and afucosylated HLA-A2 mAbs. Dissociation rate (K_D) values are shown in inset.

Figure 4

Associations of serum-derived HLA-A2-specific antibody fucosylation with FcγRIIIa binding and signaling (A) Spearman’s correlation (R_S) between IgG1 fucosylation and FcγRIIIa dissociation rate (n = 13). (B) FcγRIIIa binding characterization in high (n = 8), medium (n = 8), and low (n = 8) fucose samples and negative controls (n = 18). Serum samples were tested at a 1:500 serum dilution. Statistical analysis was performed using ordinary one-way ANOVA adjusted for multiple comparisons using Tukey’s test. Solid lines indicate group median. Data shown are representative of two technical replicates. (C and D) Spearman’s correlations between IgG1 fucosylation (n = 24) (C) and FcγRIIIa signaling (n = 31) (D) with FcγRIIIa binding (left) and HLA-A2-specific IgG levels (mean fluorescence intensity [MFI]) (right). Patients with AMR (red), patients without AMR (green), and patients with no AMR information (black) are indicated in color.

Figure 5

HLA-A2-specific IgG1 afucosylation is associated with AMR (A) Violin plot showing relative prevalence of fucose on HLA-A2-specific IgG1 in individuals with AMR (n = 10) and without AMR (n = 9). A Mann-Whitney U test was used to compare the two groups. (B) Receiver operating characteristic (ROC) curve and area under the curve (AUC) depicting performance of AMR status classification across increasing IgG1 fucose prevalence thresholds. (C) Number of subjects in which HLA-A2-specific IgG1 comprise a DSA plotted by fucose content as tertiles (low, medium, high) and AMR status. Statistical significance defined by Fisher’s exact test.