52 inclusion of non-coding DNA. In contrast, the synthetic DNA cloned in PhIP-Seq is highly uniform resolving these issues. When applied in IBD, preliminary evidence suggests that PhIP-Seq may hold promise for identifying the exact epitopes of antibody-bound proteins (such as Lachnospiraceae flagellins) and for increasing statistical power by processing larger sample numbers.38 Yet PhIP-Seq is limited to protein antigens, with lipids, glycans and post-translational modifications typically not covered. The length of displayed peptides is currently constrained to ca. 90 amino acids (aa), therefore conformational antigens formed by aa stretches further apart would be missed. An alternative approach based on Fluorescence-activated Cell Sorting (FACS) in combination with NGS (termed IgA-SEQ56,57 or BugFACS58) enables the detection of bacterial antigens in a setting closelymatchingnatural conditions. Fecal material, containingboth antibodybound and unbound bacteria, is stained with secondary antibodies, and the bound bacteria are FACS-sorted and subsequently identified via 16S or metagenomic sequencing. This methodology can also be run on panels of pre-cultivated bacteria to test binding of antibodies in other body fluids, e.g. blood.59 While this approach provides the closest resemblance to bacterial antigens in their natural context, it does not inform users about the exact antigens bound, just the bacterial species they originate from. Moreover, when working with panels of pre-cultivated bacteria, not all species of interest may grow under laboratory conditions and the proteins expressed may differ from the proteins expressed upon growth inside of the human gut. Beyond these antigen-focused approaches, a DNA-sequence–based methodology, B cell receptor sequencing (BCRseq), has been applied to analyze antibody repertoires in IBD.60 BCRseq provides information about the clonality and diversity61,62 of antibody-producing B cells by investigating VDJ gene usage and mutations occurring in the variable regions.63 IgA binding to microbial antigens inferred fromVDJ gene usage have revealed surprising similarities between CD and systemic lupus erythematosus.60 Recently, a single-cell-based IgH gene sequencing approach was employed to map the clonotypic landscape of circulating and mucosal B-cells in patients with UC.64 However, BCRseq only provides information about DNA sequences, whereas it is difficult to determine the exact antigens bound and the functional antibody recognition by these BCR repertoires.65 Clinical Potential of Antibody Repertoire Profiling in IBD Over the last few decades, a variety of antibody responses have been profiled in IBD (Table 1) and novel technologies (Figure 2) have deepened our understanding of the exact antigens3,38, bacterial species56 and BCR sequence characteristics60 involved. Despite these advances, several aspects of antibody repertoires in IBD remain incompletely characterized and, from a clinical perspective, recent findings have not been fully translated into direct benefits to patients. While most biomarker discovery efforts in IBD have focused on antibody responses in blood3,38 or systemic BCR repertoires60, fewer studies have compared mucosal56 vs. systemic antibody-binding. Comparing the exact microbial antigens bound in blood and fecal material from IBD patients to those in healthy individuals using high-throughput methods such as peptide arrays or PhIP-Seq could thus shed light on immune crosstalk beyond the gut barrier. In this respect, it might also be useful to analyze antibody classes and subclasses66 separately Chapter 2
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