Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Identified genera among all strains (N) isolated from duodenal biopsies of four donors as well as a panel of control reference strains. (B) Representative flow cytometry plots showing staining of a bacterial strain with purified serum IgA. (C) Reactivity of purified serum IgA of four donors against all biopsy isolates or control strains (n = 216) given as ΔMFI values determined by flow cytometry. (D) Serum IgA reactivity against strains isolated from each of the donors. Signals are given relative to the maximum ΔMFI value obtained for each IgA sample. Horizontal lines indicate medians, and difference between groups was evaluated by Friedman’s test with Dunn’s multiple comparisons correction. **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Heat map showing serum IgA reactivity of the four donors with all included strains. Each isolate (row) is labeled with its genus. (F) Distribution of IgA-reactive bacteria. For each serum sample (donor), only strains with ΔMFI values >10% of the maximum signal are displayed. Colors indicate genera as in (E). (G) Serum IgA reactivity against isolates of the genus Neisseria . The included isolates belonged to two different species or were unidentified ( Neisseria sp. ). Horizontal lines indicate means, and difference between groups was evaluated by one-way ANOVA with Holm-Sidak multiple comparisons correction. *p < 0.05.

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Isolation, Control, Flow Cytometry, Staining, Purification, Labeling, Bacteria

Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Heat map showing reactivity of serum IgA isolated from a single donor (CD1629) at four discrete time points. Reactivity was assessed against all bacterial strains (rows, n = 216) by flow cytometry. (B) Correlation between mean reactivity and SD across the four time points. For each strain, the coefficient of variation (CV) was calculated as SD divided by mean reactivity. (C) Grouping of strains according to variation in reactivity over time. Only strains with ΔMFI values >10% of the maximum signal in at least one time point are displayed. Colors indicate genera as in (A).

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Isolation, Flow Cytometry

Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Detection of total and bacteria-reactive IgA plasma cells in duodenal biopsy single-cell suspensions by ELISPOT. Images and quantification of spots are shown for two representative isolates. (B and C) Comparison of plasma cell reactivity by ELISPOT (B) and serum IgA reactivity by flow cytometry (C) against individual isolates. Duodenal biopsies and serum samples were obtained from a single donor (CD1629) at two discrete time points. (D) Detection of bacteria-specific IgA plasma cells by staining of duodenal biopsy single-cell suspension with labeled isolates in flow cytometry. (E) Flow cytometry plot showing reactivity of mAb 1629-A01 generated from a single S. parasanguinis- binding plasma cell against the strain that was used for plasma cell isolation. (F) Reactivity of mAb 1629-A01 against a selection of isolates assessed by flow cytometry. The strain that was used for isolation of 1629-A01 is indicated in bold. Error bar indicates SD based on three experiments.

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Bacteria, Clinical Proteomics, Enzyme-linked Immunospot, Comparison, Flow Cytometry, Staining, Suspension, Labeling, Generated, Binding Assay, Cell Isolation, Selection, Isolation

Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Examples of lineage trees showing plasma cell clonotypes spanning gut and BM. Observed sequences are colored according to isotype. Inferred sequences are indicated as smaller white circles, and predicted germline sequences are black. Circle sizes indicate number of cells with identical Ig sequence, and numbers next to edges indicate mutations (nt). (B and C) Overview of Ig isotype usage and number of cells in clonotypes spanning gut and BM in four celiac disease patients. Each clone is represented by one BM bar and one gut bar in (B). Isotype usage among all cells belonging to shared clones is compared to the total populations of BM and gut plasma cells in (C). IgA + cells that could not be assigned to a specific subclass are indicated as IGHA1/2. IgG + cells that could not be assigned to a specific subclass were excluded (in total 34 cells in BM and 1 cell in gut). (D) ELISA binding curves showing reactivity of mAb 558-6BM with LPS of different bacterial sources, purity, and after enzymatic treatment. Error bars represent range of sample duplicates. (E) Western blot showing reactivity of mAb 558-6BM with components of E. coli O55:B5 LPS before and after treatment with mutanolysin. In addition to a band at ∼10 kDa, a high-molecular weight band (indicated with black triangle) is present in the sample without mutanolysin. (F) Comparison of mAb 558-6BM reactivity against E. coli O55:B5 LPS and E. coli Lpp (peptide component) by ELISA. Error bars represent range of sample duplicates. (G) Reactivity of ten mAbs generated from clonotypes spanning gut and BM with a selection of bacterial isolates as determined by flow cytometry.

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Clinical Proteomics, Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Western Blot, High Molecular Weight, Comparison, Generated, Selection, Flow Cytometry

Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Heat map showing IgA reactivity against a selection of bacterial isolates as determined by flow cytometry. Total IgA was purified from serum of healthy donors (HD), untreated celiac disease patients (UCeD) or celiac disease patients treated with a gluten-free diet for at least one year (TCeD) (B) Assessment of IgA integrity by SDS-PAGE after incubation with bacterial culture supernatants. Brain Heart Infusion (BHI) broth alone was used as negative control and supernatant of Streptococcus pneumoniae (S.pn), which is known to express IgA1 protease, was used as positive control. One isolate of Gemella haemolysans (G.hae) was found to have IgA-degrading activity. The effect was observed using purified serum IgA of six individual donors. (C) Detection of IgA heavy chain mobility shift after incubation of purified serum IgA of five donors with supernatant of two isolates of Streptococcus infantis (S.inf) used either alone or in combination.

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Selection, Flow Cytometry, Purification, SDS Page, Incubation, Negative Control, Positive Control, Activity Assay, Mobility Shift