Aus dem Centrum für Chronische Immundefizienz des Universitätsklinikums Freiburg im Breisgau Immunological phenotype of LRBA-deficient mice INAUGURAL – DISSERTATION zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert–Ludwigs–Universität Freiburg im Breisgau Vorgelegt 2018 von Fiona Isabel Jäger geboren in Karlsruhe Dekan: Prof. Dr. Norbert Südkamp 1. Gutachter: Prof. Dr. Bodo Grimbacher 2. Gutachter: Prof. Dr. Hanspeter Pircher Jahr der Promotion: 2019 1 Table of content List of figures ............................................................................................................................. 4 List of tables ............................................................................................................................... 6 Abbreviations ............................................................................................................................. 7 1. Introduction ............................................................................................................................ 9 1.1. Development and activation of B cells ........................................................................ 9 1.2. Development and activation of T cells .......................................................................... 11 1.3. Primary Immunodeficiencies ......................................................................................... 12 1.4. Characteristics of LPS-responsive beige-like anchor (LRBA) protein ......................... 13 1.5. LRBA shares domains with other BEACH family members ........................................ 14 1.6. LRBA deficiency ........................................................................................................... 16 1.6.1. Clinical and immunological phenotype of LRBA deficiency ................................. 16 1.6.2. Treatment options ................................................................................................... 18 1.7. The role of LRBA in lymphocytes – a pathogenesis hypothesis for LRBA deficiency 19 1.8. Objectives of this thesis ................................................................................................. 21 2. Materials and methods ......................................................................................................... 22 2.1. Materials ........................................................................................................................ 22 2.1.1. Devices and materials ............................................................................................. 22 2.1.2. Chemicals and reagents ........................................................................................... 23 2.1.3. Antibodies ............................................................................................................... 25 2.1.4. Buffers ..................................................................................................................... 26 2.1.5. Software .................................................................................................................. 27 2.2. Methods ......................................................................................................................... 28 2.2.1. Genotyping .............................................................................................................. 28 2.2.2. Sequencing .............................................................................................................. 30 2.2.3. Immunization .......................................................................................................... 30 2.2.4. Preparation of murine organs .................................................................................. 31 2.2.5. Cell staining for flow cytometry ............................................................................. 32 2.2.6. Western Blot ........................................................................................................... 34 5.2.7. ELISA ..................................................................................................................... 36 2.2.8. Histopathology analysis .......................................................................................... 36 2.2.9. Statistical analysis ................................................................................................... 37 3. Results .................................................................................................................................. 38 3.1. Genotyping of Lrba-/- mice ............................................................................................ 38 2 3.2. Sequencing of Lrba-/- mice ............................................................................................ 39 3.3. Lrba-/- mice do not express LRBA in the spleen ........................................................... 40 3.4. Lrba-/- mice present low body weight and signs of splenomegaly ................................ 41 3.5. Lrba-/- mice have a normal B cell compartment, except for low percentages of B-1a B cells ....................................................................................................................................... 44 3.6. Lrba-/- exhibit normal IgM and IgG titers in steady state conditions and increased IgA levels ..................................................................................................................................... 48 3.7. Lrba-/- mice have a normal T cell compartment ............................................................ 50 3.8. Intestinal histopathology of Lrba-/- mice showed no sign of inflammation, but young Lrba-/- mice had elevated plasma cell counts in the colon .................................................... 55 4. Discussion ............................................................................................................................ 60 4.1. Overall development and organomegaly in Lrba-/- mice ............................................... 61 4.2. B cell development in Lrba-/- mice ................................................................................ 62 4.3. Antibody production in Lrba-/- mice .............................................................................. 64 4.4. Investigation of the T cell compartment in Lrba-/- mice ................................................ 65 4.5. Investigation of intestinal histopathology in Lrba-/- mice ............................................. 66 4.6. Summary, limitations and outlook ................................................................................. 68 Abstract .................................................................................................................................... 70 Zusammenfassung .................................................................................................................... 71 References ................................................................................................................................ 72 List of publications ................................................................................................................... 79 Eidesstattliche Versicherung .................................................................................................... 80 Erklärung zum Eigenanteil/ Declaration of own contribution ................................................. 81 Acknowledgements .................................................................................................................. 83 3 List of figures Figure 1: Stages of B cell development.. ................................................................................. 10 Figure 2: Stages of T cell development .................................................................................... 11 Figure 3: Overview of the BDCP family. ................................................................................ 14 Figure 4: Distribution of major clinical presentations in LRBA-deficient patients ................. 18 Figure 5: CTLA-4 recycling is controlled by LRBA. CTLA-4, a competitor of CD28, binds CD80 and CD86, removing them from the cell surface through transendocytosis. ................. 20 Figure 6: Protein deficiency caused by Cre recombinase mediated excision of a LoxP flanked exon in Lrba. ............................................................................................................................ 28 Figure 7: Multiplex PCR strategy for mice genotyping. .......................................................... 29 Figure 8: Timeline of mouse immunization protocol. .............................................................. 30 Figure 9: Scheme of a horizontal electro blotting apparatus. ................................................... 35 Figure 10: Gel electrophoresis after genotyping PCR to distinguish Lrba+/+, Lrba+/-, and Lrba- /- mice........................................................................................................................................ 38 Figure 11: Lrba-/- mice lack exon 4 in sequencing analysis. .................................................... 39 Figure 12: Splenocytes (stimulated with 1ng/ml LPS overnight) of Lrba-/- mice do not express LRBA compared to Lrba+/+ and Lrba+/- mice. ........................................................................ 40 Figure 13: Lrba-/- mice presented with lower body weight compared to Lrba+/+ (p=0.1261) and Lrba+/- mice (p=0.8626)