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Investigation of the Binding Profile and Specificity of Monoclonal Iga to Microbiota Communities Under Steady State and Inflammatory Conditions

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Investigation of the Binding Profile and Specificity of Monoclonal Iga to Microbiota Communities Under Steady State and Inflammatory Conditions Investigation of the binding profile and specificity of monoclonal IgA to microbiota communities under steady state and inflammatory conditions Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Johanna Kabbert, M.Sc. aus Berlin, Deutschland Berichter: Univ.-Prof. Dr. rer. nat. Oliver Pabst Univ.-Prof. Dr.-Ing. Lars Blank Tag der mündlichen Prüfung: 01.06.2021 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar. 0 Kabbert J, Benckert J, Rollenske T, Hitch C.A, Clavel T, Cerovic V, Wardemann H and Pabst O, High microbiota reactivity of adult human intestinal IgA requires somatic mutations. J Exp Med (2020) 217 (11): e20200275. https://doi.org/10.1084/jem.20200275 D 82 (Diss. RWTH Aachen University, 2021) 1 Table of Contents Abstract................................................................................................................................ 1 Zusammenfassung .............................................................................................................. 3 1 Introduction .................................................................................................................. 5 1.1 “Guests” in the gut ................................................................................................... 5 1.2 Mucosal surfaces .................................................................................................... 5 1.3 Microbiota composition and distribution in the intestine ........................................... 6 1.4 The microbiota shapes host immunity and physiology ............................................. 7 1.5 Organization of the gut mucosal immune system .................................................... 8 1.6 Origin of B cells and antigen-independent BCR diversification ...............................11 1.7 Structure of antibodies in mice and men .................................................................12 1.8 Luminal transport of intestinal antibodies ................................................................14 1.9 SIgA coats members of the intestinal microbiota ....................................................17 1.10 Effector functions of intestinal SIgA ........................................................................18 1.11 T cell dependent and T cell independent generation of intestinal IgA .....................20 1.12 Generation of affinity-matured IgA responses in Peyer’s patches ...........................22 1.13 How does luminal antigen reach follicles in Peyer’s patches? ................................23 1.14 B cell encounter with antigen in Peyer’s patches ....................................................23 1.15 SHM and CSR in germinal centers require AID activity ..........................................24 1.16 Mechanisms leading to IgA CSR in Peyer’s patches ..............................................26 1.17 Mechanism promoting increased affinity of antibody responses .............................27 1.18 Plasma blast homing to the intestinal lamina propria ..............................................30 2 Aims .............................................................................................................................31 3 Materials and Methods Part I ......................................................................................32 3.1 Animals ..................................................................................................................32 3.2 Human fecal material .............................................................................................32 3.3 Preparation of human tissue samples ....................................................................32 3.3.1 Human tissue samples for monoclonal antibody generation ............................32 3.3.2 Flow cytometry analysis and single B cell sorting ............................................32 3.3.3 PCR amplification and expression vector cloning ............................................33 3.4 Recombinant antibody production and purification .................................................33 3.4.1 Re-transformation of plasmids .........................................................................33 3.4.2 Plasmid purification and Ig gene sequence analysis........................................33 3.4.3 Expression and Protein A based purification of recombinant antibodies ..........34 3.5 Enzyme linked Immunosorbent Assay (ELISA) ......................................................34 3.5.1 Anti-human IgG1 ELISA ..................................................................................34 i 3.5.2 Polyreactivity ELISA of recombinant antibodies ...............................................34 3.6 Deglycosylation of immunoglobulins .......................................................................35 3.6.1 Deglycosylation of recombinant Fc-IgG1 antibodies ........................................35 3.7 Generation of germ-line variants ............................................................................35 3.7.1 Ig sequence analysis of recombinant antibodies .............................................35 3.7.2 Cloning of germ-line variants ...........................................................................35 3.8 Western blot ...........................................................................................................36 3.8.1 Western blot analysis of germ-line variants .....................................................36 3.8.2 Western blot analysis of deglycosylated recombinant antibodies ....................36 3.9 Mass spectrometry (MS) ........................................................................................37 3.9.1 Mass spectrometry of deglycosylated recombinant antibodies ........................37 3.10 Fecal material collection and preparation ...............................................................37 3.10.1 Preparation of fecal material ............................................................................37 3.11 Flow cytometry of antibody-stained bacteria ...........................................................38 3.11.1 Bacterial flow cytometry and bacterial sort purification (FACS) ........................38 3.12 16S rRNA gene amplicon sequencing and analysis ...............................................39 3.12.1 Metagenomic bacterial DNA isolation ..............................................................39 3.12.2 PCR amplification of bacterial DNA .................................................................39 3.12.3 16S rRNA gene amplicon Illumina sequencing ................................................40 3.12.4 16S rRNA gene amplicon analysis ..................................................................40 3.13 Microbiology ...........................................................................................................40 3.13.1 In vitro cultivation of oligoMM12 bacteria .........................................................40 3.14 Data analysis ..........................................................................................................41 3.14.1 Formulas .........................................................................................................41 3.14.2 Statistical analysis ...........................................................................................41 4 Materials and Methods Part II .....................................................................................42 4.1 Animals ..................................................................................................................42 4.2 Intraperitoneal application of Tamoxifen .................................................................42 4.3 FTY720 administration ...........................................................................................42 4.4 Surgical procedures ...............................................................................................43 4.4.1 4-OHT micro-injections of single Peyer’s patches ...........................................43 4.5 Lymphocyte isolation ..............................................................................................44 4.5.1 Tissue preparation and cell isolation ...............................................................44 4.5.2 Cell isolation from blood ..................................................................................44 4.5.3 Cell isolation from lymphoid organs .................................................................45 4.5.4 Lymphocyte isolation from small intestinal lamina propria ...............................45 ii 4.6 Epithelial cell isolation from the small intestine .......................................................45 4.7 Cell stainings for flow cytometry analysis ...............................................................46 4.7.1 Staining of B cells and plasma cells ................................................................46 4.7.2 Staining of small intestinal epithelial cells ........................................................46 4.8 Histology ................................................................................................................47 4.8.1 Tissue preparation for histological analysis .....................................................47 4.8.2 Cryotome tissue sections ................................................................................47
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