Fig 2: CD IgA1 N and O-glycosylations do not recapitulate conventional IgA glycosylation patterns AFull MS spectrum of glycopeptides released from CD IgA1 trypsin digest. Main N340-glycoforms of the IgA1 glycopeptide [332–353] (LAGKPTHVNVSVVMAEVDGTCY) are annotated using CFG nomenclature. Appendix Table S4 summarizes a list of N-glycopeptides identified. # is the peptide [43–76] of immunoglobulin kappa constant chain (UniProtKB P01834). *Isobaric structures not differentiated by MS/MS experiments (not exhaustive N-glycans illustrations). (HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR).BO-glycoforms identified for CD.CO-glycoforms identified for UC. Data information: *corresponds to IgA1 peptide [264–273] (WLQGSQELPR). #shows contamination by other multiply charge species covering the glycoforms signals. Details related to O-glycoforms are given in Appendix Table S7. CD: n = 3; UC: n = 1 (biological replicates). All patient samples have been tested in technical duplicates.

Fig 3: Putative model for altered subclass-dependent selection of the microbiota by IgA in IBDWhile in the healthy gut, IgA1 have mostly neutralizing and/or bacteriostatic properties, IgA2 undergoes reverse-transcytosis via Dectin-1 and Siglec-5 to deliver antigens to the immune cells of the PP. This mechanism is dependent on glycosylations, notably sialylations for Siglec-5 recognition. This allows for efficient elimination of pathogens and opportunists. IgA-mediated entrapment of commensal bacteria into the mucus is also glycan-dependent, wherein bacteria, IgA and mucin glycosylation are required. In CD (middle panel), luminal binding of commensal by- and RT of IgA1, would promote higher antigen load to the lamina propria and establishment of responses against both commensals and opportunists, which would ultimately favor opportunist's growth. Mechanisms of IgA1 retro-transport remain to be determined. In addition, de-sialylation of IgA1 limits its effector function, and would affect adequate commensal selection. Tissular IgA2, in turn, is less efficient at neutralizing opportunists, with increased binding to commensals during active disease, which would favor opportunists again. In UC (right panel), absence of RT for IgA2 and overall loss of anti-glycan reactivity would lead to impaired tolerogenic responses to commensals but also impaired responses against pathogens and opportunists, resulting in opportunist's resurgence within the microbiota.

Fig 4: Levels of Ig isotypes against the SARS-CoV-2 spike and RBD vary in plasma or serum samples from COVID-19 convalescent individuals.Total Ig, IgM, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 against (a) spike and (b) RBD in specimens from 29 COVID-19 convalescent individuals, 13 COVID-19 uninfected contemporaneous samples, and pre-pandemic controls were detected using the following secondary Abs: rabbit biotinylated-anti-human total Ig (Abcam, catalog #ab97158) at 2 µg/mL, mouse biotinylated-anti-human IgG1 Fc (Invitrogen #MH1515) at 4 µg/mL, mouse biotinylated-anti-human IgG2 Fc (Southern Biotech #9060–08) at 1 µg/mL, mouse biotinylated-anti-human IgG3 Hinge (Southern Biotech #9210–08) at 3 µg/mL, mouse biotinylated-anti-human IgG4 Fc (Southern Biotech #9200–08) at 4 µg/mL, mouse biotinylated-anti-human IgA1 Fc (Southern Biotech #9130–08) at 4 µg/mL, mouse biotinylated-anti-human IgA2 Fc (Southern Biotech #9140–08) at 4 µg/mL or goat biotinylated-anti-human IgM (Southern Biotech #2020–08) at 3 µg/mL. The samples were tested at a dilution of 1:200 and data are shown as mean MFI + standard deviation (SD) of duplicate measurements from at least two independent experiments. The pre-pandemic controls are shown as mean MFI + SD of 12 samples (Pre, black bar). The horizontal red dotted line represents the cut-off value determined as the mean + 3 SD of 12 pre-pandemic samples for each of the isotypes. (c) Percentages of responders above the cut-off for each spike- or RBD-specific Ig isotype.

Fig 5: CD and UC IgA have distinct anti-glycan reactivity profiles A, BHeatmaps displaying standardized log2-transformed signal intensities per glycan for IgA1 (A) and IgA2 (B) in non-IBD, CD and UC groups, plotted by hierarchical clustering of Euclidean distance.C–FVolcano plots of log2-transformed fold changes for IgA1 anti-glycan reactivity in CD (C) and UC (D) groups, and IgA2 anti-glycan reactivity for CD (E) and UC (F) compared to non-IBD IgA1 and IgA2. For CD, P-values of differentially targeted glycan motifs are listed in Appendix Table S8 and those of UC in Appendix Tables S9 and S10. Significance threshold was placed at P-value < 0.05 (indicated by the dotted line in C–F); For IgA1, Non-IBD: n = 4; CD: n = 4; UC: n = 4. For IgA2, n = 6; CD: n = 6; UC: n = 6. IgA1 and IgA2 were taken matched for the same patient. All patient samples (biological replicates) have been tested in technical duplicates. A list of the glycans from each cluster is provided as Appendix Tables S11 and S12.