Immunological Phenotype of LRBA Deficient Mice

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

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

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

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) under SPF conditions ...... 41 Figure 14: Spleen weight of Lrba-/- mice was higher compared to Lrba +/+ (p=0.9694) and Lrba+/- (p>0.9999) mice ...... 42 Figure 15: Macroscopic aspect of spleens of Lrba-/- and Lrba +/+ mice suggests splenomegaly in Lrba-/- mice after vaccination with a TI antigen (NP-Ficoll) and a TD antigen (NP-CGG). 42 Figure 16: Lrba+/+ and Lrba-/- mice presented normal spleen histology under steady state conditions...... 43 Figure 17: Lrba+/+ and Lrba-/- mice presented normal spleen histology after TD vaccination with NP-CGG ...... 43 Figure 18 Gating strategy for bone marrow B cells...... 45 Figure 19: Normal repartition of B cell subsets on the bone marrow in Lrba-/- mice ...... 45 Figure 20: Gating strategy for B cells in the spleen...... 46 Figure 21: Analysis of the repartition of B cell subsets in the spleen showed no statistically relevant differences in Lrba-/- mice compared to Lrba+/+ and Lrba+/- mice ...... 46 Figure 22: Gating strategy in B cells from the peritoneal cavity ...... 47 Figure 23: B-1a cells in the peritoneal cavity were statistically relevant reduced in Lrba-/- compared to Lrba+/+ mice under steady state conditions (p=0.0238) and after vaccination with the TI antigen NP-Ficoll (p=0.0286)...... 47 Figure 24: Lrba-/- mice produced comparable amounts of IgM and IgG subtypes, but significantly more IgA compared to Lrba+/+ (p=0.0012) and Lrba+/- mice (p=0.0399) under steady state conditions...... 48 Figure 25: IgA titers from intestinal washing showed no statistically relevant differences in Lrba-/- mice compared to Lrba+/+ and Lrba+/- mice under steady state conditions ...... 49 Figure 26: Gating strategy for T cells in the thymus...... 50

Figure 27: Analysis of the repartition of T cell subsets in the thymus under steady state conditions showed no statistically relevant differences in Lrba-/- mice compared to Lrba+/+ mice ...... 51 Figure 28: Gating strategy in spleen cells for T cell subsets ...... 52 Figure 29: Analysis of the repartition of T cell subsets in the spleen under steady state conditions showed no statistically relevant differences in Lrba-/- mice compared to Lrba+/+ mice ...... 52 Figure 30: Analysis of the repartition of T cell subsets in the spleen showed no statistically relevant differences in Lrba-/- mice compared to Lrba+/+ and Lrba+/- mice...... 53 Figure 31: Gating strategy in cells from the peritoneal cavity for T cells ...... 53 Figure 32: T cells in the peritoneal cavity are similar in Lrba-/- compared to Lrba+/+ and Lrba+/- mice. T cells percentages show no differences between the groups and are not affected by vaccination ...... 54 Figure 33: Absence of signs of inflammation in the ileum of young Lrba-/-mice (below four months of age) ...... 55 Figure 34: Absence of signs of inflammation in the ileum of young Lrba-/-mice (above twelve months of age)...... 56 Figure 35: Cell counts of goblet cells, IEL and plasma cells as well as counts of mitotic figures and apoptotic bodies in ileum of Lrba-/- mice were similar to Lrba+/+ and Lrba+/- mice ...... 57 Figure 36: Absence of signs of inflammation in the colon of young Lrba-/- mice (below four months of age)...... 57 Figure 37: Absence of signs of inflammation in the colon of old Lrba-/- mice (above twelve months of age) ...... 58 Figure 38: Young Lrba-/- mice exhibited higher counts of plasma cells in the colon compared to Lrba+/+ mice (p = 0.0454) ...... 59

List of tables Table 1: Transcript variants of LRBA...... 14 Table 2: Devices and materials used to conduct this work ...... 22 Table 3: Reagents used to conduct this work ...... 23 Table 4: Antibodies used to conduct this work ...... 25 Table 5: Buffer recipes used to conduct this work ...... 26 Table 6: Software used to conduct this work ...... 27 Table 7: Primers used for mouse genotyping ...... 29 Table 8: PCR reaction mix for mouse genotyping ...... 29 Table 9: Cycling program for mouse genotyping ...... 30 Table 10: Antibody cocktail 1 used in bone marrow cells ...... 32 Table 11: Antibody cocktail 2 used in bone marrow cells ...... 33 Table 12: Antibody cocktail 1 used in splenocytes ...... 33 Table 13: Antibody cocktail 2 used in splenocytes ...... 33 Table 14: Antibody cocktail 3 used in splenocytes ...... 33 Table 15: Antibody cocktail 1 used in cells from the peritoneal cavity ...... 33 Table 16: Antibody cocktail 2 used in cells from the peritoneal cavity ...... 34 Table 17: Antibody cocktail 3 used in cells from the peritoneal cavity ...... 34 Table 18: Antibody cocktail used in thymocytes ...... 34 Table 19: Upper gel (stacking gel) recipe for SDS-Page ...... 35 Table 20: Lower gel (running gel) recipe for SDS-Page...... 35

Abbreviations

AIHA Autoimmune hemolytic anemia AKAP A-kinase anchor proteins ALPS Autoimmune lymphoproliferative syndrome BAFF B cell activating factor BCR B cell receptor BDCP BEACH domain containing protein BEACH Beige and Chediak Higashi domain BSA Bovine Serum Albumin CGG Chicken gamma globulin CHS Chediak Higashi syndrome CTLA-4 Cytotoxic CVID Common variable immunodeficiency DAPI 4’,6-diamidino-2-phenylindole DN Double negative DP Double positive EBV Epstein barr virus ER Endoplasmic reticulum FCS Fetal calf serum FO Follicular FOXP3 Forkhead box protein 3 FVD Fixable Viabilty Dye GC Germinal center gDNA Genomic Deoxyribonucleic acid GFP Green fluorescent protein HE Hematoxylin eosin HIES Hyper-IgE syndromes HRP Horseraddish peroxidase HSC Hematopoietic stem cell HSCT Hematopoietic stem cell transplantation IBD Inflammatory bowel syndrome IEL Intraepithelial lymphocyte Ig Immunoglobulin IL Interleukin IPEX Immune dysregulation, polyendocrinopathy, enteropathy, X-linked ITP Idiopathic thrombocytopenic purpura LPS Lipopolysaccharide LRBA LPS-responsive beige-like anchor LYST Lysosomal trafficking regulator MHC Major histocompatibility complex MZ Marginal zone NBEA Neurobeachin NBEAL Neurobeachin-like NMDA N-methyl-D-aspartate NP 4-hydroxy nitrophenylacetyl hapten NSMAF Neutral sphingomyelinase activation-associated factor P/S Penicillin/ Streptomycin PAS Periodic acid-Schiff PCR Polymerase chain reaction 7

PH Pleckstrin homology PI3KR4 Phosphoinositide 3-kinase regulatory subunit 4 PID Primary immunodeficiency PVDF Polyvinylidene difluoride RT Room temperature SCID Severe combined immunodeficiency SD Standard deviation SPF Specific pathogen-free TCR T cell receptor TD Thymus dependent TI Thymus independent Treg Regulatory T cells TSAP Thermosensitive alkaline phosphatase WAS Wiskott-Aldrich syndrome WDFY WD and FYVE zinc finger domain containing protein WDR81 WD repeat domain 81

1. Introduction

1.1. Development and activation of B cells B cells are a part of the adaptive immune system, helping to attack a broad range of pathogens, such as bacteria or viruses. As all blood cells, they derive from the hematopoietic stem cell compartment (HSC) in the bone marrow, where the environment for B cell development is provided and its initial stages take place. A common lymphoid progenitor (CLP) cell deriving from the HSC is the origin of the B cell line (Pieper et al., 2013). Further development to the pro-B cell stage occurs by rearrangement first of the heavy and then of the light chain (L-chain) segment of the immunoglobulin gene (LeBien and Tedder, 2008). At the pre-B cell stage, a pre- B cell receptor is expressed on the cell surface, consisting of a µ-heavy chain and a surrogate light chain, fulfilling two major functions: prevention of the expression of other heavy chains, known as allelic exclusion, and initiation of the rearrangement of L-chain genes for further development of a functional B cell receptor (BCR). Rearrangement of light chain gene segments allows the expression of IgM on the cell surface, forming immature B cells (Melchers, 2007). To avoid binding to self-antigens, immature B cells with high autoreactivity are excluded by clonal deletion or repaired by receptor editing through secondary L-chain arrangement in the process of central tolerance (Manjarrez-Orduño et al., 2009). Next, immature B cells are released to the blood stream passing through a transitional 1 and 2 stage, and developing into mature naïve B cells expressing IgD and IgM (Samitas et al., 2010). In the spleen, B cells evolve into either marginal zone (MZ) or follicular (FO) B cells. MZ B cells help to mount a first line of defense upon recognition of bacterial polysaccharides by secreting polyreactive IgM and generating short-lived plasma cells. For there is no support by T cells needed throughout this process, it is called thymus independent (TI). Two subtypes can be discriminated: During TI type I response, non-specific immunoglobulins are produced upon polyclonal B cell activation. In contrast, the TI type II response enables antigen-specific immunoglobulin production, being initiated by crosslinking of Toll-like receptors and BCRs through carbohydrates (Obukhanych and Nussenzweig, 2006).

In comparison, FO B cells elicit a thymus dependent (TD) immune response, for they depend on the activation through T-helper cells during the germinal center reaction in the lymphoid organs. FO B cells recognize protein peptides via their BCR, internalize them and present them on a MHC class II. During the germinal center reaction, this complex can thereupon be recognized by T helper cells through their T cell receptor (TCR) (DeFranco, 1987). As a result, germinal center B cells (GC) undergo immunoglobulin class switching and affinity maturation

9 by somatic hypermutation. Plasma cells are generated which produce high-affinity antibodies (Eibel et al., 2014). Whilst some of the plasma cells are short lived and produce a significant number of antibodies, others develop into long-lived plasma cells, which aid the production of antibodies for several years, residing in adapted niches in the spleen or the bone marrow (Fairfax et al., 2008). Lastly, one part of the FO B cells differentiates into memory B cells, which can quickly generate a population of plasma cells upon reactivation by a known antigen (Tarlinton, 2006). An overview of B cell development is seen in Figure 1.

Figure 1: Stages of B cell development. B cells develop from a hematopoietic stem cell (HSC) in the bone marrow. When passing from the Pro to the Pre B cell stage, a pre-B cell receptor is expressed on the cell surface. The immature stage is marked by the surface expression of IgM. While migrating to the secondary lymphoid organs, B cells pass through a T1 and T2 maturation stage, acquiring additional expression of IgD. Once in the lymphoid organ such as the spleen, the cells develop into marginal zone (MZ) or follicular (FO) B cells. FO B cells engage in the germinal center reaction, where affinity maturation and class switching takes place, resulting in antibody producing plasma cells and memory B cells (after Janeway’s Immunobiology, 2012). This conventional pathway generates MZ and FO B cells, also called B2 cells. In addition to them, a B1 cell subset existing in mice and humans, resides in body cavities such as the peritoneal or pleural cavity. In mice, B1 cells are mainly generated in the fetal liver (Prieto and Felippe, 2017). This subset can further be divided into B-1a and B-1b cells. The B-1a compartment produces natural antibodies, with low affinity and polyreactivity, playing a role in thymus-independent immune response (Carsetti et al., 2004). In contrast, the B-1b compartment can produce antigen-specific antibodies (Baumgarth, 2011).

1.2. Development and activation of T cells Like B cells, T cell precursors also derive from a pluripotent hematopoietic stem cell. Through the peripheral blood they migrate to the thymus cortex where the further development takes place. Signals from the thymus stromal cells allow T cell maturation through rearrangement of their TCR gene segments (Anderson et al., 1996). In order to avoid autoreactivity, a positive and negative selection process takes place in the thymus, eliminating a big percentage of T cells: TCRs, which are compatible with self-MHC-molecules, produce a survival signal by interaction with the thymus stromal cells and are preserved through positive selection (Hogquist et al., 1997). Autoreactive TCRs are excluded through a negative selection process upon emission of cell death signals (Kishimoto and Sprent, 1997).

The maintained, non-autoreactive cells are called double negative cells for they do not express either CD4 nor CD8. Furthermore, they lack CD3, a part of the TCR complex. From this initial state, a small percentage becomes the γ:δ line, remaining double negative even in mature states. The major percentage forms the double positive α:β line, expressing CD4 and CD8 and additionally a TCR complex including CD3. Out of the double positive population single positive T cells, either CD4 or CD8 positive, are generated and exported through the thymus medulla in the periphery (Borowski et al., 2002).

Figure 2: Stages of T cell development. T cell precursors, originating from HSC in the bone marrow, migrate to the thymus for further development. The first stage of double negative cells expresses neither CD4, nor CD8, nor CD3, which is a part of the T cell receptor complex. Two cell lines develop from this stage: a double negative γ:δ line, and – majorly – a CD4+ CD8+, double positive α:β line, additionally expressing a TCR. Next, single positive CD4+ or CD8+ cells are generated and exported into the periphery (after Janeway’s Immunobiology, 2012). Upon contact with antigens, TCRs and the co-receptors CD4 and CD8 recognize MHC- molecules presented by the pathogens. Hereby, CD8+ T cells recognize MHC I and lead to killing of cells carrying pathogens. In contrast, CD4+ T cells recognize MHC II complexes and

11 activate B cells or macrophages to generate a further immune response (Janeway’s Immunobiology, 2012).

1.3. Primary Immunodeficiencies Primary immunodeficiencies (PID) are a heterogeneous group of diseases caused by mutations in a broad range of genes involved in the immune response (Lim and Elenitoba-Johnson, 2004). PIDs are rare disorders, yet their incidence of 1 in 10.000 has increased over time due to the implementation of gene analysis like whole-exome sequencing, revealing numerous mutations in immunological genes (Lim and Elenitoba-Johnson, 2004). More than 300 monogenetic causes have been described to date (Shields and Patel, 2017). Major symptoms include recurrent infections with bacteria, viruses or especially opportunistic germs, as well as autoimmunity, autoinflammatory processes and malignant pathologies, like hematological malignancies (Shields and Patel, 2017). As in many genetic disorders, onset of symptoms mostly occurs at an early age, yet PIDs of the adult patient exist as well, such as common variable immunodeficiency (CVID) (Shields and Patel, 2017). Patients suffering from CVID present with hypogammaglobulinemia – a decrease in IgG serum levels accompanied by low IgM and/or IgA serum titers (Salzer et al., 2012).

The International Union of Immunological Societies (IUIS) Expert Committee of Primary Immunodeficiencies defined nine subgroups of PIDs (Picard et al., 2015):

1. Immunodeficiencies affecting cellular and humoral immunity, including severe combined immunodeficiency (SCID) and combined immunodeficiency (CID) 2. Combined immunodeficiencies with associated or syndromic features, including Wiskott-Aldrich syndrome (WAS) or Hyper-IgE syndromes (HIES) 3. Predominantly antibody deficiencies, like Common variable immunodeficiency disorders (CVID) 4. Diseases of immune dysregulation, such as autoimmune lymphoproliferative syndrome (ALPS) 5. Congenital defects of phagocyte number, function, or both 6. Defects in intrinsic and innate immunity 7. Auto-inflammatory disorders 8. Complement deficiencies 9. Phenocopies of PIDs

In 2012, a CVID-like syndrome due to lack of lipopolysaccharide-responsive beige-like anchor protein (LRBA) was described, known since then as LRBA deficiency (Lopez-Herrera et al., 2012).

1.4. Characteristics of LPS-responsive beige-like anchor (LRBA) protein LPS-responsive beige-like anchor is a widely distributed throughout human tissues multi- domain protein, encoded in the long arm of chromosome 4 (Lopez-Herrera et al., 2012). It was first described by Kerr et al. in 1996, as 7a33 gene, a lipopolysaccharide (LPS)-responsive gene highly expressed in murine B cells, predicted to play a role in receptor-mediated signal transduction due to expression of a domain homologous to SH2 (Kerr et al., 1996). Later, Wang et al. found increased mRNA expression of the 7a33 gene product in murine macrophages and B cells upon LPS activation, as well as features of both BEACH domain containing proteins like CHS1/beige and A-kinase anchor proteins (AKAP), naming it LPS-responsive and beige- like anchor gene (lba, Wang et al., 2001). AKAPs bind to the regulatory subunit of cAMP- dependent Proteinase K (Newlon et al., 1999) and translocate it to distinct intercellular locations, enabling it to phosphorylate substrates such as the N-methyl-D-aspartate (NMDA) receptor (Colledge and Scott, 1999). CHS1/beige (also known as lysosomal trafficking regulator, LYST) plays a role as scaffold protein in fusion/fission events in the context of vesicle trafficking (Tchernev et al., 2005). Mutations in LYST cause the Chediak-Higashi Syndrome, another PID characterized by immunodeficiency, hypopigmentation, progressive neurological defects and bleeding diathesis (Spritz, 1998). Subcellular localization of LRBA was observed in lysosomes, trans-Golgi, endoplasmic reticulum (ER), the plasma membrane, the perinuclear ER and endocytic vacuoles upon LPS stimulation in RAW 264.7 cells, transfected with a vector coding for a GFP-LRBA fusion protein, suggesting a role of LRBA in vesicle trafficking (Wang et al., 2001).

LRBA is highly conserved throughout species (Cullinane et al., 2013). Particularly in mice, Wang et al. described three isoforms of LRBA, which differ in their 3’-terminus: lba-α (9903bp, 2856 amino acids, currently Lrba-201; ENSMUST00000107635.6) is expressed in spleen and brain. lba-β (9396bp, 2792 amino acids, currently Lrba-204, ENSMUST000000194759.5) is expressed the most widely in brain, spleen, lung and bone marrow and lba-γ (8854bp, 2779 amino acids, currently Lrba-202, ENSMUST00000192145.5) is expressed in spleen, brain and lung (Wang et al., 2001). To date two more protein coding transcript variants in mice are described (Lrba-208, ENSMUST00000212390.1, 2124aa; Lrba-206, ENSMUST00000195524.1, 758aa), as well as three non-protein coding ones. In humans,

13 thirteen protein transcripts are registered in the Ensembl database, of which six translate into proteins (www.ensembl.org):

Table 1: Transcript variants of LRBA.

Name Transcript ID Protein LRBA-201 ENST00000357115.7 2863 aa LRBA-202 ENST00000502839.1 134 aa LRBA-204 ENST00000507224.5 2575aa LRBA-206 ENST00000508606.1 147aa LRBA-207 ENST00000509835.5 1505aa LRBA-209 ENST00000510413.5 2575aa In addition, the murine ortholog shares 90% amino acid identity with human LRBA (www.ensemble.org) whereas Drosophilia and Caenorhabitis elegans share 51% and 35% amino acid identity, respectively (Wang et al., 2001).

1.5. LRBA shares domains with other BEACH family members LRBA is a member of the BEACH domain containing proteins family (BDCPs), characterized by big protein size, a BEACH domain preceded by a pleckstrin homology (PH) domain, and WD40 repeats (Cullinane et al., 2013). Figure 3 shows the structural similarity throughout the family members:

Figure 3: Overview of the BDCP family. The nine members are: Lysosomal-trafficking regulator (LYST), Neurobeachin (NBEA), Neurobeachin-like 1 (NBEAL1), Neurobeachin-like 2 (NBEAL2), Lipopolysaccharide-responsive beige-like anchor protein (LRBA), WD and FYVE zinc finger domain containing protein 3 (WDFY3), WD and FYVE zinc finger domain containing protein 4 (WDFY4), neutral sphingomyelinase activation-associated factor (NSMAF), WD repeat domain 81 (WDR81). All members share the BEACH domain, and almost all the preceding PH domain. NBEA and LRBA show high resemblance (Cullinane et al., 2013).

The BEACH domain (named after Beige and Chediak Higashi) consisting of 280 aa, is highly conserved throughout the BDCP family - with an amino acid sequence identity of 45% or higher (Gebauer et al., 2004) - suggesting to be crucial for the function of BDCP proteins (Jogl et al., 2002). It is located at the C-terminal of the protein (Cullinane et al., 2013) and consists of eleven α helices and seven peptide ε segments, which form a unique three layer folding (Gebauer et al., 2004). The PH domain contains 100 aa and precedes the BEACH domain (Burgess et al., 2009). A variety of different PH domains have been described to date, sharing their 3D structure despite low sequence homology (Rebecchi and Scarlata S., 1998). The PH domain is involved in targeting its host protein to the cytosolic face of the membrane (Cullinane et al., 2013). Interestingly, the BEACH and PH domain are not only in physical proximity, but are connected by a linker region, suggesting that they act as a single unit (Jogl et al., 2002). The WD domain is built of 40 aa repeats, ending in a tryptophan (W) and aspartic acid (D) dipeptide (Neer et al., 1994). Four to sixteen of these repeats create the domain, which is considered to play a role in cell cycle control, transcription regulation, signal transduction, autophagy, apoptosis, and vesicle trafficking (Smith et al., 1999). The Concanavalin-A-like lectin binding domain is present in a multitude of proteins. In BDCPs it is suggested to be involved in oligosaccharide binding, associated with protein trafficking and sorting along in the secretory pathway (Burgess et al., 2009). The DUF1088 domain - domain of unknown function - is only found in Neurobeachin (NBEA) and LRBA, indicating a specific function in these two proteins (Cullinane et al., 2013).

While being widely expressed, particularities of the specific function and acting mechanism of BDCPs remain unknown. A role in vesicular transport, apoptosis, membrane dynamics and receptor signaling has been suggested (Cullinane et al., 2013). In addition connections to autophagy (Simonsen et al., 2004) and apoptosis (Ségui et al., 2001) have been drawn.

Mutations in genes encoding BCDPs have been linked to clinically relevant conditions (Cullinane et al., 2013). NBEA, which is highly expressed in the brain, endocrine cells and platelets (Wang et al., 2000), has been studied in the context of body length, obesity, synaptic spine patterns of neurons in autism, platelet development, and multiple myeloma (Cullinane et al., 2013). It has been hypothesized, that NBEA carries out functions of LRBA in non- immunologic cells, due to their high resemblance, replacing it in patients deficient of LRBA (Lopez-Herrera et al., 2012). Neurobeachin-like 1 (NBEAL1) was shown to be upregulated in glioma (Chen et al., 2004), while mutations in Neurobeachin-like 2 (NBEAL2) cause gray platelet syndrome (Gunay-Aygun et al., 2011). WD and FYVE zinc finger domain containing

15 protein 3 (WDFY3) was associated with neurodegenerative diseases (Cullinane et al., 2013), and WD and FYVE zinc finger domain containing protein 4 (WDFY4) is possibly altered in systemic Lupus erythematosus (Yang et al., 2010). WD repeat domain 81 (WDR81) has been found to be mutated in syndrome of quadrupedal walking, mental retardation and cerebral hypoplasia (Gulsuner et al., 2011). Association to immunodeficiencies have been reported for LYST, the first BDCP to have been described, which is mutated in the Chediak-Higashi syndrome, as described above (chapter 1.4.), with a correspondent phenotype in mice called beige syndrome (Perou et al., 1996), and neutral sphingomyelinase activation-associated factor (NSMAF), with NSMAF deficient mice suffering from discrete immunodeficiency (Montfort et al., 2009). Mutations in LRBA cause a syndrome of immunodeficiency and immune dysregulation (Lopez-Herrera et al., 2012).

1.6. LRBA deficiency

1.6.1. Clinical and immunological phenotype of LRBA deficiency LRBA deficiency is caused by homozygous or compound heterozygous germline mutations in the LRBA gene, resulting in complete absence of the protein (Gámez-Díaz et al., 2016), or residual protein expression, reported in few patients (Revel-Vilk et al., 2015; Levy et al., 2016). Since the first description of LRBA deficiency in 2012 as a primary immunodeficiency presenting with early onset hypogammaglobulinemia, recurrent infections and autoimmune diseases (Lopez-Herrera et al., 2012), several bigger patient cohorts have been described, allowing an extended description of the clinical phenotype (Gámez-Díaz et al., 2016; Alkhairy et al., 2016; Azizi et al., 2017).

In a cohort of 22 LRBA deficiency patients, Gámez-Díaz et al. found equal distribution of the disease to male and female gender, and a context of consanguineous parents in only 45% of analyzed patients (Gámez-Díaz et al., 2016). Most patients have an early onset of disease, with first symptoms below the age of two (Gámez-Díaz et al., 2016; Azizi et al., 2017) to five years (Alkhairy et al., 2016). Yet the age of onset seems to be variable, possibly because of epigenetic, microbial or environmental modifiers (Gámez-Díaz et al., 2016). As a major symptom, 95% of the patients present with immune dysregulation, mostly enteropathy, autoimmune hemolytic anemia (AIHA) and idiopathic thrombocytopenic purpura (ITP) (Gámez-Díaz et al., 2016). Consistently, Azizi et al. reported enteropathy and autoimmunity in 76% of their 17 patients respectively (Azizi et al., 2017). Comparable frequency of 61% of autoimmune manifestations were found in in the 31-patient cohort reported by Alkhairy et al. Amongst them, AIHA (12 of 31 patients) and ITP (9 of 31 patients) were the most frequent, and autoimmune 16 enteropathy was present in only 2 of 31 patients (Alkhairy et al., 2016). Additional case reports have described an early-onset persistent diarrhea and colitis in the context of LRBA deficiency (Alangari et al., 2012; Serwas et al., 2015), as well as villous atrophy in histopathological samples of the small intestine (Alkhairy et al., 2016). Lévy et al. reported two affected siblings, one suffering from Evans disease (ITP and AIHA), the other one presenting with erosive polyarthritis and type 1 diabetes mellitus (Lévy et al., 2016). Organomegaly was found in 86% (Gámez-Díaz et al., 2016) and 61% (Alkhairy et al., 2016) of reported patients, majorly presenting as splenomegaly (Gámez-Díaz et al., 2016; Azizi et al., 2017). Furthermore, Revel- Vilk et al. reported patients suffering from LRBA deficiency, initially diagnosed as autoimmune lymphoproliferative like syndrome (ALPS), presenting with splenomegaly and lymphadenopathy (Oliveira et al., 2010). Moreover, a majority of LRBA-deficient patients suffer from recurrent infections, mostly of the respiratory tract, like pneumonia, otitis, or sinusitis (71% and 61% (Gámez-Díaz et al., 2016; Alkhairy et al., 2016)). Interestingly the major phenotype described in the first cohort by Lopez-Herrera et. al. – hypo/dysgammaglobulinemia- is only present in about 60% (Gámez-Díaz et al., 2016; Alkhairy et al., 2016) to 80% (Azizi et al., 2017) of the reported patients, suggesting that contrary to initial findings, the most striking phenotype in LRBA-deficient patients is autoimmune manifestation (Gámez-Díaz et al., 2016). Due to the hypogammaglobulinemia and recurrent upper respiratory tract infections many of the patients are diagnosed as CVID-like (Lopez-Herrera et al., 2012). Since autoimmune manifestations are only present in 25% of CVID cases (Park et al., 2008), it creates a suitable parameter for clinical discrimination between CVID and LRBA deficiency. Additionally, neurological disorders, such as myasthenia gravis, cerebral granulomas, demyelination, optic nerve and cerebral/cerebellar atrophy, as well as dermatological impairment like allergic dermatitis or urticaria, have been reported in the context of LRBA deficiency (Alkhairy et al., 2016).

Figure 4: Distribution of major clinical presentations in LRBA-deficient patients. Cohorts by Gámez-Díaz et al. (2016) including 22 patients, Alkhairy et al. (2016) consisting of 31 patients and Azizi et al. (2017) describing 17 patients were compared, showing different percentages in main clinical presentations of LRBA-deficient patients. In conclusion, LRBA deficiency shows a very broad clinical phenotype and thus should be investigated in undiagnosed patients with CVID-like, inflammatory bowel disease (IBD)-like, ALPS-like autoimmune or immune dysregulatory and lymphoproliferative disorders (Gámez- Díaz et al., 2016).

Regarding the immunological phenotype of LRBA-deficient patients, reduction of switched memory B-cells and plasmablasts have been reported (Gámez-Díaz et al., 2016; Alkhairy et al., 2016, Azizi et al., 2017). Consistently, in vitro B cell survival and plasmablast formation upon stimulation with CD40L, IL-4 and IL-21 was reduced (Lopez-Herrera et al., 2012). About 60% of the LRBA patients present with hypogammaglobulinemia (Gámez-Díaz et al., 2016; Alkhairy et al., 2016) and impaired production of specific antibody titers after vaccination was reported (Gámez-Díaz et al., 2016). Suitably, in vitro stimulation of naïve B cells of LRBA patients with anti-IgM, CD40L, BAFF and CpG oligonucleotide resulted in low IgG production (Lopez-Herrera et al., 2012). Besides an impairment in the B cell compartment, reduced numbers of regulatory T cells (Treg) were found in patients lacking LRBA (Gámez-Díaz et al., 2016; Lo et al., 2015 ).

1.6.2. Treatment options Classical immunosuppressive drugs like corticosteroids, methotrexate, cyclosporine A, Rituximab, in addition to immunoglobulin substitution are frequently used to treat LRBA- deficient patients. However, the use of hematopoietic stem cell transplantation (HSCT) and Abatacept treatment have been recently applied in LRBA-deficient patients. Specifically, three HSCT follow-up case reports have been published, showing partial remission in all patients during two (Tesi et al., 2016) and nine years post-HSCT respectively (Seidel et al., 2015). In

18 addition, an excellent clinical improvement after one year post-HSCT was observed in a 10- year-old patient treated with healthy donor sibling HSCT (Sari et al., 2016). Nevertheless, two of four reviewed cases by Gámez-Díaz et al. failed after HSCT, leaving the relevance of HSCT as treatment option unclear. An early intervention might have a positive impact on survival and remission after HSCT (Gámez-Díaz et al., 2016). Another partially successful treatment option, is Abatacept, an Ig-CTLA-4 fusion protein, which improved the overall clinical picture, computed tomography scans, and pulmonary function in 9 patients, reported by Lo et al.(Lo et al., 2015). Cytotoxic T-lymphocyte-associated protein 4 (CTLA4), is a competitor of the costimulatory CD28 on the surface of activated conventional T cells, binding to the T cell ligands CD80 and CD86 expressed on antigen presenting cells (APC) and removing them from the cell surface through a process called transendocytosis (Qurechi et al., 2011). As an Ig- CTLA-4 fusion protein, Abatacept acts as a selective modulator of T cell co-stimulation by interfering with the interaction of CD28 and CD80/86. Consequently, the production of cytokines TNF, IL-1 and IL-6, as well as the B cell activation are modulated, and the T cell response is inhibited (Atzeni et al., 2013).

1.7. The role of LRBA in lymphocytes – a pathogenesis hypothesis for LRBA deficiency Since its discovery, distinct cellular functions, especially in lymphocytes have been assigned to LRBA.

LRBA might act as a negative regulator of apoptosis. An increase of apoptosis was observed in EBV-cell lines of LRBA-deficient patients after serum deprivation for 6 hours (Lopez- Herrera et al., 2012). Furthermore, upregulation of LRBA in certain cancer types, such as kidney, pancreatic, colorectal and lung cancers, was reported, suggesting anti-apoptotic effects of LRBA that allow cell survival under stressful conditions (Wang et al., 2004).

Additionally, autophagy has been studied in the context of LRBA deficiency. Autophagy is the degradation of cellular material in lysosomes, allowing cell survival under stress conditions, like oxidative stress or infection (Levine and Kroemer, 2008). Cells of LRBA-deficient patients were shown to have impaired induction of autophagy upon starvation (Lopez-Herrera et al., 2012). Autophagy is suggested to be important for the homeostasis of the endoplasmic reticulum, energy metabolism and stress response, and thus for the secretion of immunoglobulins and plasmablasts formation (Pengo et al., 2013). Therefore, defective autophagy in LRBA-deficient patients might be the cause of defective plasma cell development and immunoglobulin-secretion (Alkhairy et al., 2016). It might also be linked to autoimmunity, 19 since defective apoptosis is associated to autoimmune diseases, like systemic lupus erythematosus, possibly through insufficient apoptotic cell clearance which can cause inflammation, and can disrupt the tolerance to self-antigens (Levine and Kroemer, 2008).

In 2015, Charbonnier et al. suggested a role of LRBA in Treg functionality since her LRBA- deficient patient, presenting with immune dysregulation, poly-endocrinopathy, enteropathy X- linked (IPEX) syndrome-like phenotype, showed decreased Treg numbers, reduced expression of Treg canonical markers (FOXP3, CD25, CTLA-4) and impaired T cell suppression. In addition, a preferential development towards memory T cells consistent with immune deregulatory manifestation, was observed, as well as increased follicular helper T cell (TFH) + counts and elevated levels of autoantibodies. Moreover, increased apoptosis in Treg and CD4 T cells was found. These findings first linked immune deregulatory symptoms in LRBA- deficient patients to defective Treg functionality and increased Treg apoptosis (Charbonnier et al., 2015). In 2015, Lo et al. reported a significant clinical improvement in 9 LRBA-deficient patients after treatment with Abatacept for 5 to 8 years (CTLA-4-Ig) (Lo et al., 2015). CTLA-

4 was found to be less expressed in LRBA-deficient Treg in a dose dependent manner, yet mRNA levels were normal, suggesting, that LRBA modifies CTLA-4 post-translationally. Furthermore, the amount of CTLA-4 was partly restored by the inhibition of lysosomal degradation, indicating that LRBA plays a role in the recycling of CTLA-4, whereas in LRBA absence, CTLA-4 is degraded in the lysosomes (Lo et al., 2015).

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. Degradation of the internalized complex in the lysosome can be inhibited by binding of LRBA to the cytoplasmatic tail of CTLA-4, thus enabling recirculation of CTLA-4 to the cell surface (Lo et al., 2016).

The low CTLA-4 levels in LRBA-deficient patients explain the high similarity of the clinical phenotype that LRBA deficiency has with CTLA-4 insufficiency: Patients who carry heterozygous mutations in CTLA-4, mainly present with enteropathy, hypogammaglobulinemia, granulomatous lung disease and respiratory infections, and CD4+ T- cell infiltrations in intestine, lung, bone marrow, central nervous system and kidneys. Just like

LRBA-deficient patients, CTLA-4 deficient subjects lack CTLA-4 on their Treg and Treg suppression is impaired (Schubert et al., 2014). Thus, the resulting lack of CTLA-4 in LRBA deficiency might be crucial for pathogenesis.

1.8. Objectives of this thesis Since LRBA deficiency is a very rare disease with approximately 70 cases reported to date, access to patients’ samples for answering our research questions is very challenging. In addition, human samples normally present high variability which might interfere with the development of the phenotype such as: additional mutations, exposure to a diversity of pathogens, diet, among others. To avoid these confounders, a murine Lrba-/- model was used aiming to:

• Investigate the basal antibody titers in Lrba-/- mice, since about 50% of the LRBA- deficient patients present with hypogammaglobulinemia. • Evaluate the different B and T cell subsets in Lrba-/- mice upon immunization with a thymus independent and thymus dependent antigen, since LRBA-deficient patients present with abnormalities in both compartments with a reduced response to vaccines. • Perform a histopathological screening of spleen, small intestine and colon in Lrba-/- mice, under basal condition and upon immunization, since LRBA-deficient patients present with lymphocytic infiltrations and immunoproliferation in the spleen and inflammation in the intestine.

2. Materials and methods

2.1. Materials

2.1.1. Devices and materials Table 2: Devices and materials used to conduct this work

Device / material Manufacturer Product number 96 Well Microplate Half Area Corning 3690 Axio Cam camera for histology Zeiss MRc Cryomold Standard Sakura 4557 Cryostat Leica CM1850 Electrophoresis power supply EV231 Consort Electrophoresis chamber Mini-PROTEAN Tetra System BIO-RAD FACS Canto II BD Bioscience Falcon 40µm cell strainer Fisher Scientific 08-771-1 Freezer -20°C MF290 SG Dometic Greiner 96 Well Plates, not sterile Sigma-Aldrich M2186 HERAcell 150i CO2 incubator ThermoFisher Scientific Heraeus Fresco 21 centrifuge ThermoFisher Scientific Hereaus Megafuge 16R centrifuge ThermoFisher Scientific Hereaus Megafuge 40R centrifuge ThermoFisher Scientific Injekt Solo single use syringe, 20 ml Braun 4606205V LSE Vortex Mixer Corning Magnetic mixer MZ Hei-Mix L Heidolph Microscope for histology Leica DM2500 Microscope Primo Vert Zeiss Microtainer SST tubes BD 365951 Microwave Panasonic MiniSpin Eppendorf Multiskan FC ThermoFisher Scientific Nanodrop 2000c Spectrophotometer Thermofisher Scientific Neubauer Chamber Marienfeld Superior pH-Meter 765 Calimatic Knick Omnifix F Solo 1 ml syringes Braun 9161406V Pipettes 2,10,20,200,1000 µl Gilson Safe 2020 cell culture hood ThermoFisher Scientific Scale EWB Kern Shaking device KS260 basic IKA Special accuracy weighing machine ABT 220-5DM Kern Sterican single use hypodermic needles, 20-gauge Braun 4657519 Sterican single use hypodermic needles, 26-gauge Braun 4657683 Thermomixer compact Eppendorf UV Transilluminator Professional System Phase GmbH Waterbath ED Julabo

2.1.2. Chemicals and reagents Table 3: Reagents used to conduct this work

Reagent Manufacturer Product Application Number Acetic acid Sigma-Aldrich 33209 TAE buffer for gel electrophoresis Acrylamide/Bis-Acrylamide Sigma-Aldrich A3699 WB gels Agarose SERVA 11404.07 Gel electrophoresis Ammoniumpersulfat (APS) Sigma-Aldrich A3678 WB gels Aqua ad iniectabilia Braun 144518091 PCR (Destillated water) BD Cytofix/Cytoperm BD Biosciences 554714 Intracellular staining for FACS Fixation/Permeabilization Kit BD FACSClean BD Biosciences 340345 FACS BD FACSFlow Sheath Fluid BD Biosciences 342003 FACS BD FACSRinse BD Biosciences 340346 FACS BD FACSShutdown Solution BD Biosciences 334224 FACS -Mercaptoethanol Sigma-Aldrich 63689 Sample buffer for WB Bovine Serum Albumine Sigma-Aldrich A2153 Blocking for ELISA Bromphenolblue sodium salt Sigma-Aldrich 114405 Sample buffer for WB, loading buffer for gel electrophoresis Cell Lysis Solution Qiagen 158906 gDNA extractio cOmplete ULTRA Mini EDTA- Roche 05892791001 Protein lysis for WB free Easy pack DAPI ThermoFisher D1306 FACS staining Scientific Diethanolamine Sigma-Aldrich D8885 Diethanolamine buffer for ELISA Dipotassiumphosphate Carl Roth P749.1 Coating buffer for ELISA DNA Hydration Solution Qiagen 158914 gDNA extraction DNA ladder 1kb Peqlab PEQL25- Gel electrophoresis 2030 DNA ladder 500kb Peqlab PEQL25- Gel electrophoresis 2330 EDTA 0.02 solution Sigma-Aldrich E8008 TAE buffer EDTA disodium salt SERVA 11280.02 TAE buffer Ethanol Sigma-Aldrich 32205 gDNA extraction Ethanol 99% denaturated with SAV Liquid ETO-5000- Cleaning MEK 5l Produktion 99-1 Ethidiumbromide Carl Roth 2218.2 Gel electrophoresis Exonuclease I New England Biolabs M0293L Digestion for sequencing Fetal Calf Serum Life Technologies 10270106 Complete medium Freund‘s adjuvant complete Sigma-Aldrich F588a-10mL Immunization Freund‘s adjuvant incomplete Sigma-Aldrich F5506-10mL Immunization Gelatin from bovine Sigma-Aldrich G9391 ELISA Gel Red Biotium 41003 Gel electrophoresis Glycerol Sigma-Aldrich G5516 Sample Buffer for WB Glycine Sigma-Aldrich G8898 Running and transfer buffer for WB HoTStarTaq Qiagen 203203 PCR Hydrochloric acid 1 mol/L VWR International A14341000 pH adjustment in buffers Hydrochloric acid 5 mol/L AppliChem A2668 pH adjustment in buffers Incidin Ecolab 3011510 Bench cleaning Isopropanol Sigma-Aldrich 59300 gDNA extraction LPS Sigma-Aldrich L2880 Immunization LumiGLO and Peroxide Cell Signaling 7003S Membrane development WB Lympholyte M Cedarlane CL5031 Isolation of mononuclear cells

Reagent Manufacturer Product Application Number Magnesium chloride Sigma-Aldrich 208337 Diethanolamine buffer for ELISA Methanol Sigma-Aldrich 34860 Membrane activation for WB Milkpowder Carl Roth T145.1 Blocking for WB Monopotassiumphosphate Carl Roth 3904.1 Coating buffer for ELISA Multicore Buffer 10x Promega R999A Digestion for sequencing NP140 AppliChem A194,0250 RIPA buffer NP-BSA Biosearch N-5050L Immunization, coating buffer Technologies for ELISA NP-CGG Biosearch N5055A-1 Immunization Technologies NP-Ficoll Biosearch F-1420-10 Immunization Technologies NuPage 3-8% Tris-Acetate ThermoFisher EA0375BOX Western Blot gel Protein Gels Scienific NuPage Sample buffer LDS 4X Life technologies 1386564 Sample buffer for Western Blot NuPage Transfer buffer 20X ThermoFisher NP0006 Transfer Buffer for Western Scientific Blot NuPage Tris-Acetate SDS ThermoFisher LA0041 Running Buffer for Western Running Buffer Scientific Blot Penicillin/Streptomycin Sigma-Aldrich P0781 Complete medium Phosphate Buffered Saline Sigma-Aldrich D8537, Washing steps (PBS) L1835 Phosphatase Inhibitor Sigma-Aldrich Chemie P5726#1ML Protein extraction Cocktail 2 1 ml Phosphatase Inhibitor Sigma-Aldrich Chemie P0044#1ML Protein extraction Cocktail 3 1 ml Phosphate Substrate Sigma-Aldrich S0942 ELISA Pierce BCA Kit Thermo Scientific 23227 Protein quantification Ponceau S Sigma-Aldrich P3504 Western Blot Protein Precipitation Solution Qiagen 158910 gDNA extraction Proteinase K Carl Roth 7528.1 Liard’s lysis buffer for gDNA extraction RBC lysis buffer eBioscience 00-4333-57 Red cell lysis for cell isolation for FACS RNase AWAY Surface Fisher Scientific 7000 RNA extraction Decontamination RNeasy Mini Kit Qiagen 74106 RNA extraction Roti-Histofix 4% Carl Roth P08.74 Formalin fixation RPMI 1640 + L-Glutamine GIBCO 21875091 Complete medium SignalFire Plus ECL Reagent A Cell Signaling 12630 S Membrane development for Western Blot Sodium Dodecyl Sulfate Carl Roth 2326.3 Buffers ultrapure Sodium azide Sigma-Aldrich 71289 Diethanolamine buffer for ELISA Sodium bicarbonate Sigma-Aldrich S5761 Sodium chloride Sigma-Aldrich S9625-1KG PBS, liard’s lysis buffer, TBS Sodium hydroxide Applichem 1099130001 pH adjustment Sodium phosphate monobasic Sigma-Aldrich 13472-35-0 PBS dihydrare Spectra Multicolor Broad Thermo Scientific 26634 WB Range Protein Ladder Spectra Multicolor High Range Thermo Scientific 26625 WB Protein Ladder Super Signal West Femto Thermo Scientific 34095 Membrane development for WB Substrate

Reagent Manufacturer Product Application Number Thermo sensitive Alkaline Promega M9910 Digestion for sequencing Phosphatase Tissue Tek OCT Compound Sakura 4583 Cryo fixation Tris for buffer solutions Applichem A13791000 Buffers Trypan blue solution Sigma-Aldrich T8154 Cell counting Tween 20 Sigma-Aldrich P1379 Washing steps for WB Water, nuclease free Thermo Scientific R0582 PCR

2.1.3. Antibodies Table 4: Antibodies used to conduct this work

Antibody Manufacturer Product number Anti-murine LRBA antiserum By Prof. Kilimann Anti-mouse HRP Santa Cruz Sc-2096 Anti-rabbit HRP Santa Cruz Sc-2077 Anti-Tubulin HRP Abcam Ab108629 Goat anti-mouse IgA, Human ads-UNLB Southern Biotech 1040-01 Goat anti-mouse IgG-PE/Cy7 Biolegend 405315 Goat anti-mouse IgG-PE Southern Biotech 1030-9 Goat anti-mouse IgG1, Human ads-UNLB Southern Biotech 1070-01 Goat anti-mouse IgG2b, Human ads-UNLB Southern Biotech 1090-01 Goat anti-mouse IgG2c, Human ads-UNLB Southern Biotech 1079-01 Goat anti-mouse IgG3, Human ads-UNLB Southern Biotech 1100-01 Goat anti-mouse IgM, Human ads-UNLB Southern Biotech 1020-01 Hamster anti-mouse CD3-PE Biolegend 100307 Mouse Immunoglobulin Panel (ELISA standards) Southern Biotech 5300-01 Rat anti-mouse B220-APC/Cy7 Biolegend 103224 Rat anti-mouse B220-FITC BD Biosciences 553087 Rat anti-mouse B220-PE BD Biosciences 553089 Rat anti-mouse CD1d-PE BD Biosciences 553846 Rat anti-mouse CD138-APC BD Biosciences 558626 Rat anti-mouse CD138-APC Biolegend 142505 Rat anti-mouse CD138-BV421 BD Biosciences 562610 Rat anti-mouse CD21/CD35-APC Biolegend 123412 Rat anti-mouse CD23-PE BD Biosciences 01235B Rat anti-mouse CD25-APC Biolegend 102011 Rat anti-mouse CD335-APC Biolegend 137607 Rat anti-mouse CD335-FITC Biolegend 137605 Rat anti-mouse CD4-PE/Cy7 Biolegend 100422 Rat anti-mouse CD4-V500 BD Biosciences 560782 Rat anti-mouse CD43-APC BD Biosciences 560663 Rat anti-mouse CD5-APC BD Biosciences 550035 Rat anti-mouse CD5-FITC BD Biosciences 553020 Rat anti-mouse CD8a-APC H7 BD Biosciences 560182 Rat anti-mouse FoxP3-AlexaFluor 488 BD Biosciences 560403 Rat anti-mouse IgD-FITC eBioscience 11-5993-82 Rat anti-mouse IgM-PerCP eBioscience 46-5790-52 Rat anti-mouse IgM-PerCP eFluor710 eBioscience 45-5790-82 Rat anti-mouse TER-119-FITC Biolegend 116205

2.1.4. Buffers Table 5: Buffer recipes used to conduct this work

Experiment Buffers Ingedients PBS 10X • 11.2 g NaH2PO4 • distilled water up to 2 L • pH adjustment to 7.2 to 7.4 • 350.6 g NaCl • final volume 4 L DNA extraction Liard’s Lysis buffer • 200 mM NaCL • 100 mM Tris HCl pH 8.3 • 5 mM EDTA • 0.2% SDS ELISA Coating Buffer 10X • 500 ml K2HPO4 0.5 M • pH adjustment to 8.0 (adjust with KH2PO4 0.5 M) Diethanolamine buffer • 97 ml Diethanolamine • 100 mg MgCl2 • distilled water up to 800 ml • pH adjustment to 9.8 • final volume 1 L Western Blot 5X Running Buffer • 75.5 g Tris • 360.33 g Glycine • 25 g SDS • final volume 5 L Transfer Buffer • 302.5 g Tris • 375 g Glycine • 5 g SDS • pH adjustment to 8.3 (adjust with HCl) • final volume 5 L 10X TBS • 80 g Tris • 80 g NaCl • 2 g KCl • pH adjustment to 7.4 (adjust with HCl) • final volume 1L 6X Sample Buffer (Lämmli) • 3.75 ml 1 M Tris HCl pH 6.83 • 6 ml Glycerol • 0.6 ml β-Mercaptoethanol • 2 ml H2O • 2 mg Bromphenolblue • 1 g SDS • final volume 12 ml RIPA Buffer • 2 ml 100% NP140 • 10 ml 10X PBS • 2 ml 10X SDS • 156 ml Milipore H2O • 20 ml 5X Sodiumdeoxycholate • final volume 200 ml 4X Upper Gel Buffer • 18.17 g Tris • 16 ml 10% SDS • pH adjustment to 6.8 (adjust with HCl) • final volume 300 ml 4X Lower Gel Buffer • 109.03 g Tris • 24 ml 10% SDS • pH adjustment to 8.8 (adjust with HCl) • final volume 600 ml

Experiment Buffers Ingedients Gel elecrophoresis Loading buffer • 42% sucrose • 0.15% Bromphenolblue • 0.15% Xylencyanol • 0.3% Orange G • In TAE 1X Tris-Acetate-EDTA (TAE) • 48.4 g Tris 10X • 11.4 ml Acetic acid • 3.7 g EDTA disodium salt • final volume 1 L

2.1.5. Software Table 6: Software used to conduct this work

Software Application

BD FACSDIVA Acquisition of Flow cytometry data FlowJo vX.0.7 Analysis of flow cytometry data

GraphPad Prism 6 Statistical analysis and graph compilation MS Excel 2010 Analysis ELISA data

Nanodrop 2000 Measurement of nucleic acid sample concetrations

Phase Gel Documentation Acquisition of gel electrophoresis results Sequencher Analysis of sequencing data

SkanIt ThermoFisher Scientific Acquisition of ELISA data

2.2. Methods

2.2.1. Genotyping LRBA deficiency is a very rare disease with about 70 diagnosed patients worldwide, thus complicating sample acquisition. In addition, patients present heterogenous conditions due to other possibly modifying mutations and environmental factors. Investigation of a Lrba-/-mouse model allows elimination of these possible confounders and provides sufficient sample numbers.

For this work we used a murine Lrba knockout model, designed by Prof. Manfred Kilimann (Max Planck Institute for Experimental Medecine, Göttingen, Germany) and Taconis Artemis GmbH (Cologne, Germany). A targeting vector containing exons three and four, as well as Zs green and a Neomycin resistance gene as selection items was used. The entire construct was flanked by LoxP sites. Successfully recombined vectors through homologous recombination with the wild type genomic locus, were screened for with Zs-green negativity and resistance to Neomycin treatment. After effective integration of the targeting vector, excision of exon four was achieved by ubiquitous expression of the Cre recombinase. Hereby a frame shift after 149 amino acids was caused, resulting in a null mutation with less than 1% protein expression in Western Blot (personal communication from Prof. Manfred, Kilimann, Figure 6).

Figure 6: Protein deficiency caused by Cre recombinase mediated excision of a LoxP flanked exon in Lrba. A recombinant allele containing an exon four flanked by LoxP sites was achieved by homologous recombination of Lrba wild type allele with a targeting vector. The floxed region was cut out by ubiquitously expressed Cre recombinase, resulting in a frame shift after 149 amino acids. This led to absence of Lrba protein expression. Genotyping was performed on genomic DNA (gDNA) previously extracted from tail biopsies, by incubating the specimen in 300 µl of Liard’s lysis buffer with Proteinase K at a concentration of 100 µg/ml (Cat.No. 7528.1, Carl Roth) at 56 °C with agitation overnight. The samples were then centrifuged for 5 min at 200 g, and the supernatant containing the gDNA was transferred 28 into 300 µl of 100% Isopropanol for DNA precipitation by swirling until a visible precipitate was obtained, followed by a washing step with 300 µl of 70% ethanol with centrifugation at 200 g for 10 min. The DNA pellets were dried at RT before resuspension in 30 to 50 µl of DNA Hydration solution. DNA concentration and purity were measured with NanoDrop 2000c Spectrophotometer. Next, PCR amplification of 50 ng of DNA was performed using the primers and conditions shown below (Figure 7, Table 7 to 9). The Ex3F forward primer anneals with exon 3 of Lrba+/+, Lrba+/- and Lrba-/-mice. Ex4R reverse primer anneals with exon 4, present only in Lrba+/+and Lrba+/- mice, since Lrba-/- mice harbor a deletion of exon 4. A second reverse primer Int4R, anneals with intron 4 present in all three genotypes of mice, yet amplification is observed only in Lrba+/- and Lrba-/-mice. PCR products were separated on a 1.5% agarose gel electrophoresis containing ethidium bromide during 30 min at 100 V, followed by 40 min at 130 V. Lrba+/+ mice present a band of 528 bp, Lrba-/- mice present a band of 377 bp, and Lrba+/- mice show both (Figure 7).

Figure 7: Multiplex PCR strategy for mice genotyping. Whilst all three primers - Ex3F, Ex4R, and Int4R – anneal with Lrba+/+ mice, resulting in the amplification of the shorter fragment of 528 bp between Ex3F and Ex4R, only primer Ex3F and Int4R can anneal in Lrba-/- mice due to deletion of exon 4, producing a truncated fragment of 377 bp.

Table 7: Primers used for mouse genotyping

Name Sequence Mouse Lrba Ex3F 5’-GAAAGTTGACAGTATGATTGCAGG-3’ Mouse Lrba Int4R 5’-CTAAGGAGGATGGCTCTAACC-3’ Mouse Lrba Ex4R 5’-CATTGTCCTTTATCTCCTTGAA-3’

Table 8: PCR reaction mix for mouse genotyping

PCR Reaction Mix PCR buffer 10X 2.5 µl MgCl2 1.5 µl dNTPs 0.5 µl Primer Ex3F 0.25 µl Primer Ex4R 0.25 µl Primer Int4R 0.25 µl Taq Polymerase 0.125 µl H2O 18.5 µl gDNA 1 µl 29

Table 9: Cycling program for mouse genotyping

Temperature Duration Repetitions 95 °C 5 min 95 °C 30 sec 58 °C 30 sec 35x 72 °C 45 sec 72 °C 10 min 4 °C Eternal

2.2.2. Sequencing To confirm the genotype, the samples were sequenced additionally. For this purpose, 1.5 µl of PCR product was digested with 0.1 µl of thermosensitive alkaline phosphatase (TSAP) and 0.5 µl of exonuclease in a cycler at 37 °C for 15 min and at 80 °C for 15 min. The product (2 µl) and the forward primer (2 µl) were added to an Eppendorf tube. Sequencing was performed by GATC. The results were analyzed with Sequencher software.

2.2.3. Immunization Lrba-/- mice, Lrba+/- mice and Lrba+/+ littermates of two to four months of age were vaccinated with NP-Ficoll (50 µg in 100 µl PBS) to elicit a TI type 2 immune response, as well as NP- CGG (50 µg in 10 µl of complete Freund’s adjuvant and 180 µl PBS) to elicit a TD immune response. A control group was injected with 100 µl PBS. On day 30, a booster injection of their corresponding antigen at the same concentration was applied to all mice, as depicted in Figure 8. After 52 days of the first immunization, the mice were sacrificed for terminal blood withdrawal and organ extraction.

Our mouse protocol was approved by the Regierungspräsidium Freiburg according to § 8 Abs. 1 Tierschutzgesetz (G-16/19). None of the interventions performed on living mice were executed by myself.

Figure 8: Timeline of mouse immunization protocol. Lrba+/+, Lrba+/- and Lrba-/- mice were immunized with intraperitoneal injection of PBS, NP-Ficoll or NP-CGG on day 0. A booster injection was performed on day 30. After 52 days the mice were sacrificed, and the organs were collected.

2.2.4. Preparation of murine organs Mice were anesthetized in a chamber with isoflurane followed by terminal blood withdrawal from the retro-orbital plexus with heparinized glass capillaries. Approximately 500 µl blood were obtained. Next, mice were sacrificed by cervical dislocation and the following organs were extracted.

Cells from the peritoneal cavity After cleaning the mouse’s skin with 70% ethanol, a small incision in the center of the skin overlying the peritoneal wall was performed, in order to remove the skin from the abdomen, keeping the peritoneum intact. By injecting 5 ml of cold PBS in the peritoneal cavity followed by massaging the abdomen for about 10 seconds, peritoneal cells were collected with a Pasteur pipette after slightly opening the peritoneum. The samples were centrifuged at 800 g for 10 min, and pellets were resuspended in RPMI+FCS+P/S. Cell survival and quantification was determined by diluting the cells in a suspension of 1:10 in Trypan blue and transferring 10 µl into a Neubauer chamber. According to the cell count, the medium volume could be adjusted for further usage.

Thymus The thoracic cavity was carefully opened with scissors and the thymus located behind the sternum was extracted, smashed and filtered through a 70 µm cell strainer (Cat.No. 08-771-1, Fisher Scientific) by using the plunger of a syringe. The flow-through cell suspension was centrifuged at 800 g for 10 min, and resuspended in 1 ml of RPMI+FCS+P/S. To obtain isolated mononuclear cells, the cell suspension was gently added in a 1:1 ratio to Lympholyte M (Cat.No. CL5031, Cedarlane), creating two different layers, and centrifuged at 1200 g for 20 min without break. The mononuclear cells formed in the interphase of medium and Lympholyte M and could be aspirated with a pasteur pipette. After another centrifugation step at 800 g for 10 min, the cells were resuspended in 1 ml of RPMI+FCS+P/S and counted as described above for further usage.

Spleen The spleen was dissected from the abdominal cavity and single cell suspensions were achieved by using a cell strainer of 70 µm as described above, followed by a centrifugation step at 800 g for 10 min. Cell pellets were resuspended in RPMI+FCS+P/S. Isolation of mononuclear cells and cell counting was performed as described above.

Bone marrow Bone marrow cells were collected from two femurs after flushing the bone lumen with 1 ml of 31

RPMI+FCS+P/S through a syringe. Centrifugation, isolation of mononuclear cells and counting was performed as above.

2.2.5. Cell staining for flow cytometry Flow cytometry was used to characterize the cell phenotyping of the isolated cells from spleen, thymus, lymph nodes, peritoneal cavity and bone marrow. Specific extra- and intracellular antigens were detected with fluorescent antibodies in order to discriminate the different lymphocyte cell subsets. According to their fluorescence, antibody mixes were established (Table 10 to 18) and 0.5*106 cells were incubated with 100 µl of the antibody mixture for external antigen detection for 20 min at 4 °C in the dark, then washed twice with PBS + 3% FCS (FACS buffer) and resuspended in 250 µl of FACS buffer for either flow cytometry analysis or intracellular staining. In case of the latter, 100 µl of BD Cytofix/Cytoperm Fixation/Permeabilization (Cat.No. 554714, BD Biosciences) was added to the cells and incubated for 20 min at 4 °C in the dark, in order to allow intracellular diffusion of further antibodies. Next, the intracellular antibodies were added and incubated for 30 min at 4 °C in the dark, followed by two washing steps with Perm/Wash buffer and resuspension in 250 µl of FACS buffer for analysis in BD FACS Canto II. Data analysis was performed with FlowJo vX.0.7 software. Gating strategy was performed as follows: cells were first gated on lymphocytes (SSC-A vs. FSC-A) and singlets (FSC-H vs. FSC-A). Then, living lymphocytes were gated based on DAPI (DAPI vs. SSC-A).

In order to collect enough data to perform statistical analysis, data from two series of experiments using the immunization protocol were pooled, one performed by myself, the other kindly provided by Dr. Laura Gámez-Díaz and Dr. Sophie Jung.

Table 10: Antibody cocktail 1 used in bone marrow cells

Antibody Fluorochrome Laser FACSCanto Channel IgD FITC Blue 530/30 IgM PerCP-eFluor710 Blue 670 LP IgG Pe-Cy7 Blue 780/60 CD34 APC Red 660/20 B220 APC-Cy7 Red 780/60 CD138 BV421 Violet 450/50 FVD eFluor506 Violet 510/50

Table 11: Antibody cocktail 2 used in bone marrow cells

Antibody Fluorochrome Laser FACSCanto Channel IgM PerCP-eFluor710 Blue 670 LP IgG Pe-Cy7 Blue 780/60 CD43 APC Red 660/20 B220 APC-Cy7 Red 780/60

Table 12: Antibody cocktail 1 used in splenocytes

Antibody Fluorochrome Laser FACSCanto Channel PNA FITC Blue 530/30 NP PE Blue 585/42 IgM PerCP-eFluor710 Blue 670 LP IgG PE-Cy7 Blue 780/60 CD138 APC Red 660/20 B220 APC-Cy7 Red 780/60 DAPI DAPI Violet 450/50

Table 13: Antibody cocktail 2 used in splenocytes

Antibody Fluorochrome Laser FACSCanto Channel IgD FITC Blue 530/30 CD23 PE Blue 585/42 IgM PerCP-eFlour710 Blue 670 LP IgG PE-Cy7 Blue 780/60 CD21 APC Red 660/20 B220 APC-Cy7 Red 780/60 DAPI DAPI Violet 450/50

Table 14: Antibody cocktail 3 used in splenocytes

Antibody Fluorochrome Laser FACSCanto Channel CD3 PE Blue 585/42 CD335 FITC Blue 530/30 CD25 APC Red 660/20 CD8 APC H7 Red 780/60 CD4 V500 Violet 525/20 DAPI DAPI Violet 450/50

Table 15: Antibody cocktail 1 used in cells from the peritoneal cavity

Antibody Fluorochrome Laser FACSCanto Channel B220 FITC Blue 530/30 CD1d PE Blue 585/42 IgM PerCP-eFluor710 Blue 670 LP CD5 APC Red 660/20 DAPI DAPI Violet 450/50

Table 16: Antibody cocktail 2 used in cells from the peritoneal cavity

Antibody Fluorochrome Laser FACSCanto Channel CD5 FITC Blue 530/30 IgG PE Blue 585/42 IgM PerCP Red 695/40 B220 APC-Cy 7 Red 780/60 CD183 APC Red 660/20 DAPI DAPI Violet 450/50

Table 17: Antibody cocktail 3 used in cells from the peritoneal cavity

Antibody Fluorochrome Laser FACSCanto Channel CD3 PE Blue 585/42 CD335 FITC Blue 530/30 CD25 APC Red 660/20 CD8 APC H7 Red 780/60 CD4 V500 Violet 525/20 DAPI DAPI Violet 450/50

Table 18: Antibody cocktail used in thymocytes

Antibody Fluorochrome Laser FACSCanto Channel CD3 PE Blue 585/42 CD335 FITC Blue 530/30 CD25 APC Red 660/20 CD4 V500 Violet 525/20 CD8 APC H7 Red 780/60 DAPI DAPI Violet 450/50

2.2.6. Western Blot Spleen cells were collected as described above and washed twice with PBS at 800 g for 10 min. Cell pellets were resuspended in 15 µl of RIPA buffer per 300.000 cells, containing protease inhibitor cocktail (1:200, Cat.No. 05892791001, Roche) and phosphatase inhibitor cocktail 1 and 2 (1:100, Cat.No. P5726, P0044, Sigma Aldrich). After incubation on ice for 5 min, samples were centrifuged for 10 min at 6000 g at 4 °C). Protein quantification was determined with Pierce BCA Kit (Cat. No. 23227, Thermo Scientific) according to manufacturer’s instructions. According to a standard curve the amount of protein per sample was determined. Next, 20 µg of proteins were incubated with Lämmli Buffer and heated at 70 °C for 10 min for further analysis or storage at -20 °C.

Proteins were size fractioned by SDS-Polyacrylamide-gel electrophoresis (SDS-PAGE) by loading 10 to 20 µl of the protein samples onto a 8%-10% gradient gel (see gel recipes in Table 19 and 20) and running at 75 V for 20 min followed by 100 V for another 90-120 min.

Table 19: Upper gel (stacking gel) recipe for SDS-Page

Ingredient Amount H2O 3.1 ml 4X upper gel buffer 1.3 ml 30% Acrylamide 650 µl 10% APS 50 µl TEMED 5 µl Final volume 5 ml (2 gels)

Table 20: Lower gel (running gel) recipe for SDS-Page

Ingredient 8% gel 10% gel

H2O 4.8 ml 4.2 ml 4X lower gel buffer 2.5 ml 2.5 ml 30% Acrylamide 2.7 µl 4.7 ml 10% APS 100 µl 100 µl TEMED 5 µl 5 µl Final volume 10 ml (2 gels) 10 ml (2 gels)

To enable the detection of separated proteins with antibodies, the proteins were blotted from the gel on to a polyvinylidene difluoride (PVDF) membrane, by electrophoretic transfer in a wet system for 90 min at 45 V. The gel, membrane, filter and cellulose filter paper were arranged in a transfer chamber as seen in Figure 9.

Figure 9: Scheme of a horizontal electro blotting apparatus. (www.hos.ufl.edu/meteng/HansonWebpagecontents/Proteinprotocols.html (03.01.2018)). Background caused by unspecific antibody binding was eliminated by blocking the PVDF membrane with 5% milk diluted in TBS +1% Tween 20 (TBS-T) for an hour at room temperature. Next, the membrane was incubated with the primary anti-LRBA, affinity purified serum from rabbit (1:650 in 2.5% milk in TBS-T, kindly provided by Prof. Kilimann) at 4 °C overnight. The next day, after three washing steps with TBS-T for 5 min each, the membrane was incubated for two hours at room temperature with a secondary HRP anti-rabbit antibody diluted 1:3000 in 2.5% milk in TBS-T. Next, three additional washing steps with TBS-T were 35 performed. Finally, the chemiluminescence kits Lumi Glow and Super Signal Femto were used according to manufacturer’s instructions. They include luminol, which is oxidized by the horseradish peroxidase, resulting in detectable light emission. The band size of LRBA is expected at 317 kDa. As loading control, the housekeeping protein Tubulin was used, with a predicted band size of 50 kDa.

5.2.7. ELISA Serum was obtained by blood centrifugation at 700 g for 7 min, and stored at -80 °C until usage. To collect samples from the small intestine, it was washed with intestinal wash buffer (PBS, 0.5 M EDTA, 100 mM phenylmethylsulfonyl fluoride). These samples were centrifuged and filtrated through a 22 µm filter. Levels of IgG subsets (IgG1, IgG2b, IgG2c, IgG3), IgM and IgA were measured as follows. First, polystyrene plates (Cat.No. 3690, Corning) were coated with 10 µg/ml of NP-BSA (Cat.No. N-505L-100, Biosearch technologies) in order to detect NP antibodies. Coated plates were incubated at 37 °C over night, washed four times with PBS, and blocked with PBS 1% BSA for 30 min at 37 °C. After two washing steps, the plates were incubated with serial dilutions of the sera in 0.1% PBST with 0.5% gelatin and incubated for two hours at 37 °C. Then, plates were washed three times with PBS, and incubated for 90 min with alkaline phosphatase-conjugated antibody in a dilution of 1:1000 in PBS with 1% gelatin. Additional three washing steps were performed, followed by a 30 min incubation in the dark with 1 mg/ml of para-nitrophenylphosphate substrate dissolved in Diethanolamine buffer. During this time, the conversion from nitrophenylphosphate to p-nitrophenylphosphate produced a yellow color. Its intensity is proportional to the amount of protein. Finally, absorbance was measured in the MultiSkan FC at 405 nm.

The experiment was performed by Dr. Laura Gámez-Díaz, who kindly provided the data for statistical analysis, performed by myself.

2.2.8. Histopathology analysis Upon organ extraction, spleens were snap frozen in cryomolds, using Tissue Tek OCT compound and dry ice. Samples were stored at -80 °C until analysis. The frozen specimens were cut at the Leica cryostat in slices of 10 µm. Ilea and colons were fixed in phosphate- buffered 4% formaldehyde solution (Roti-Histofix 4%, Carl Roth), paraffin embedded and sectioned in 3.5 µm slices. Haematoxylin and eosin staining (HE) and periodic acid Schiff (PAS) staining was performed by the Department of pathology, University clinic Freiburg. Overall slice analysis and cell counting of plasma cells, intraepithelial lymphocytes, counting of apoptosis bodies and mitosis in tissue of the small intestine and colon were performed on 36

Leica DM2500 microscope in cooperation with Dr. Maximilian Seidl (CCI and department of pathology, University clinic Freiburg).

2.2.9. Statistical analysis The obtained data was analyzed using GraphPad Prism 6 software. For calculation of the significance by p-value, Kruskal-Wallis test was performed, which is a nonparametric approach for the evaluation of more than two unpaired groups. For comparisons in between the groups, Dunn-Bonferroni test was applied as post-hoc test.

3. Results

3.1. Genotyping of Lrba-/- mice In order to determine the genotype of the mice for the planned experiment, PCR on genomic DNA (gDNA) was performed as described in the methods section (see chapter 2.2.1). Since Lrba-/- mice have a deletion of exon 4, yet intron 4 is still present, only the primers complementary to a sequence in exon 3 and intron 4 anneal, resulting in a fragment of 377 bp, whereas a fragment of 528 bp, corresponding to the wild type sequence with expression of exon 4, is observed in Lrba+/+ mice. Lrba+/- mice show both fragments of 528 and 377 bp due to the presence of a wildtype and a knockout allele. A representative gel electrophoresis of the genotyping is shown in Figure 10.

Figure 10: Gel electrophoresis after genotyping PCR to distinguish Lrba+/+, Lrba+/-, and Lrba-/- mice. Through a PCR with a forward primer aligning in exon 3, a reverse primer in intron 4 and a reverse primer in exon 4, PCR products of 528 bp, and 377 bp were generated depending on the genotype. Water was used as a negative control. Bp, base pair; ctrl, control.

3.2. Sequencing of Lrba-/- mice To further confirm effective knock out of exon 4 in Lrba-/- mice, sequencing of the genotyping PCR product was performed (by the company GATC). Sequence analysis of the amplified fragment showed the absence of exon 4. The fragment contains intron 3, a remaining LoxP site from the construct used for knock out, and intron 4. Figure 11 shows representative sequencing data of a Lrba-/- mouse.

Figure 11: Lrba-/- mice lack exon 4 in sequencing analysis. A fragment of 377 bp was obtained by PCR, as described in chapter 3.1. The amplified fragment contains intron 3, a remaining LoxP site from the construct used to achieve knock out, and intron 4, where the reverse primer binds. Exon 4 is effectively knocked out. We performed digestion of the PCR product. Sequencing was performed by the company GATC.

3.3. Lrba-/- mice do not express LRBA in the spleen To confirm the abolishment of LRBA protein expression after deletion of exon 4 in Lrba, a Western Blot protein analysis was performed in LPS-stimulated splenocytes from all three murine genotypes. LPS stimulation has been reported to induce LRBA expression (Wang et al., 2001), therefore providing higher LRBA protein quantities and facilitating detection. Single cell solutions from spleen were obtained with a 70 µm cell strainer and then stimulated with LPS at 1 ng/ml overnight. On the next day, protein extraction and Western Blot were performed. Tubulin was used as a loading control. Figure 12 shows, that Lrba+/+ and Lrba+/- mice express LRBA. Wang et al. have reported expression of different LRBA isoforms in murine splenocytes (Wang et al., 2001). With regard to these findings, the two bands present in Lrba+/+ and Lrba+/- mice can be interpreted as a 317 kDa protein corresponding to transcript variant 1 and a 309 kDa protein corresponding to transcript variant 4 (as registered on the database www.ensembl.org). Lrba-/- mice lack these bands, demonstrating an effective LRBA knockout on the protein level. Since the knockout was constructed as a so-called conventional knockout, affecting protein expression in all tissues, verified lack of LRBA in spleen tissue allows to conclude successful suppression of LRBA in all tissues.

Figure 12: Splenocytes (stimulated with 1ng/ml LPS overnight) of Lrba-/- mice do not express LRBA compared to Lrba+/+ and Lrba+/- mice. Using the murine anti-LRBA antiserum provided by Prof. M. Kilimann, two bands at 317 kDa and 309 kDa corresponding to transcript variant 1 and 4, respectively, were detected. Effective loading was proven with Tubulin control.

3.4. Lrba-/- mice present low body weight and signs of splenomegaly Patients suffering from LRBA deficiency present with symptoms such as recurrent infections or immune dysregulation early in life, with a median age of onset of 4 years and 2 years, respectively. Furthermore, failure to thrive has been reported in 24% and 6% of patients (Gámez-Díaz et al., 2016; Azizi et al., 2017). To explore, whether Lrba-/- mice present signs of illness or reduced body weight compared to Lrba+/+ mice, overall health was clinically evaluated, and body weight was measured on day 52 of the immunization protocol in mice of all three genotypes.

Lrba-/- mice presented a trend towards lower bodyweight in comparison to Lrba+/- and Lrba+/+ mice. Whilst LRBA-deficient patients develop symptoms early in life (Gámez-Díaz et al., 2016), Lrba-/- mice lived up to two years without signs of infections, possibly due to specific- pathogen-free (SPF) conditions (Figure 13).

Figure 13: Lrba-/- mice presented with lower body weight compared to Lrba+/+ (p=0.1261) and Lrba+/- mice (p=0.8626) under SPF conditions. Mice of all three genotypes were weighed on day 52 of the immunization experiment, revealing lower body weight in Lrba-/- mice compared to Lrba+/+ and Lrba+/- mice. This data was collected in: Lrba+/+ n=8; Lrba+/- n=4; Lrba-/- n=5. Almost 80% of LRBA-deficient-patients develop organomegaly, including splenomegaly in 64% and 77%, respectively (Gámez-Díaz et al., 2016; Azizi et al., 2017). When weighing spleens of Lrba-/- mice, a tendency towards higher spleen weight compared to Lrba+/+ and Lrba+/- mice was observed. A spleen weight to body weight ratio analysis revealed significantly higher ratios in Lrba-/- mice compared to Lrba+/+ mice (Figure 14).

Figure 14: Spleen weight of Lrba-/- mice was higher compared to Lrba +/+ (p=0.9694) and Lrba+/- (p>0.9999) mice. The ratio of spleen weight to body weight was significantly higher in Lrba-/- mice compared to Lrba +/+ (p=0.0181) and Lrba+/- mice (p=0.7023). Spleen of mice of all three genotypes were weighed upon organ extraction on day 52 of the immunization experiment. A ratio to the measured body weight was calculated, showing significantly higher ratios in Lrba-/- compared to Lrba +/+ mice, suggestive of splenomegaly. This data was collected in: Lrba +/+ n=7; Lrba+/- n=4; Lrba-/- n=5. Data are depicted as mean + S.D. These results were consistent to our macroscopic analysis of the spleen after extraction. Lrba-/- mice showed signs of splenomegaly after immunization with NP-Ficoll or NP-CGG. Figure 15 exemplarily shows spleens of wildtype and knockout mice:

Figure 15: Macroscopic aspect of spleens of Lrba-/- and Lrba +/+ mice suggests splenomegaly in Lrba-/- mice after vaccination with a TI antigen (NP-Ficoll) and a TD antigen (NP-CGG). Spleen sizes of Lrba-/- and Lrba+/+ mice were measured upon organ extraction on day 52 of the immunization protocol. To search for the correlates of splenomegaly on a microscopic level, histopathology analysis of haematoxylin and eosin-stained cryo-fixed spleen slices was performed. No lymphocytic infiltration was found, and the morphology and organ architecture were normal in mice under steady state conditions (Figure 16). Also, after vaccination with NP-CGG, which leads to a thymus-dependent immune response, no anomalies in spleen histology were found (Figure 17).

Figure 16: Lrba+/+ and Lrba-/- mice presented normal spleen histology under steady state conditions. HE staining was performed in 10 µm slices of cryo fixated spleens of seven months old Lrba+/+ and Lrba-/- mice. The histological organ architecture was normal, and no signs of lymphocytic infiltrations were found.

Figure 17: Lrba+/+ and Lrba-/- mice presented normal spleen histology after TD vaccination with NP-CGG. HE staining was performed in 10 µm slices of cryo fixated spleens of Lrba+/+ and Lrba-/- mice, extracted on day 52 of the immunization experiment. The histological organ architecture was normal, and no signs of lymphocytic infiltrations were found. 43

3.5. Lrba-/- mice have a normal B cell compartment, except for low percentages of B-1a B cells LRBA-deficient patients present with hypogammaglobulinemia and recurrent infections, as well as reduced or absent titers of specific antibodies upon vaccination (Gámez-Díaz et al., 2016, Kostel Bal et al., 2017) suggesting an affected B cell and plasma cell compartment. To investigate a possible defect in B cell development in Lrba-/- mice, which could be the cause of a defective humoral immune response, as found in LRBA-deficient patients, the subsets of different B cell developmental stages were analyzed, using flow cytometry. Lrba+/+, Lrba+/-, and Lrba-/- mice were vaccinated with either a TI antigen (NP-Ficoll) or a TD antigen (NP- CGG) or injected with PBS as a control group, to trigger an immune response and allow study of B cell subsets in immune reactive conditions. In order to collect enough data to perform statistical analysis, data from two series of experiments using the immunization protocol were pooled, resulting in at least two mice per condition (injection with PBS as control, vaccination with NP-Ficoll or NP-CGG) and genotype (Lrba+/+, Lrba+/-, Lrba -/-).

In flow cytometric analysis, first all samples were gated on lymphocytes by size and granularity, followed by gating on singlets and exclusion of dead cells by DAPI staining. In the following, the analysis of B cell subsets in the bone marrow, spleen, and peritoneal cavity will be described.

Stages of B cell differentiation were investigated in cells of the bone marrow of Lrba+/+, Lrba+/, and Lrba-/- mice. The gating strategy is depicted in Figure 18. Pre and Pro B cells express B220- an isoform of CD45 expressed in mice, which serves as a pan-B cell marker (Rodig et al., 2005) and is a protein tyrosine phosphatase involved in lymphocyte activation and development (Trowbridge, 1994), yet no IgM. To distinguish both subgroups, CD43, a cell surface protein involved in T cell activation (Fuhlbrigge et al., 2006) was used. Pre B cells are CD43- whereas Pro B cells are CD43+. Expression intensity of IgM and B220 discriminates immature and mature B cells. Transitional B cells are B220+ and IgMhigh. Finally, plasmablasts are characterized by low B220 and high CD138 expression. CD138 (syndecan-1) is a member of the transmembrane heparan sulfate proteoglycan family and is involved in cell-cell adhesion and cell-matrix adhesion (O’Connell et al., 2004). A comparison of B220+, pro-, pre-, transitional-, mature-, immature- and marginal-B cells, as well as plasma cells in Lrba-/- mice, Lrba+/+ and Lrba+/- mice, showed no statistically relevant differences. Immunization with a TI and TD vaccine did not change this finding (Figure 19).

Figure 18 Gating strategy for bone marrow B cells. First gating on lymphocytes by size and granularity, followed by exclusion of dead cells by DAPI staining. Amongst B220+ cells, cells can be discriminated in Pro and Pre B cells through CD43 expression. Immature and mature B cells express IgM, yet can be distinguished through B220 expression. Transitional B cells form a population characterized by B220+ and IgMhigh expression. Plasmablasts are B220low and CD138high.

Figure 19: Normal repartition of B cell subsets on the bone marrow in Lrba-/- mice. B220+ B cells, Pro-, Pre-, transitional, immature, and mature B cells, as well as plasma cells show similar percentages in all three genotypes in the control group vaccinated with PBS, as well as upon vaccination with the TI antigen NP-Ficoll or the TD antigen NP-CGG. This data was collected in: Lrba+/+: PBS n=10, NP-Ficoll n=6, NP-CGG n=8; Lrba+/-: PBS n=4, NP-Ficoll n=6, NP-CGG n=5; Lrba-/- PBS n=10, NP-Ficoll n=5, NP-CGG n=6. for B220+ cells, Pro-, Pre-, Mature, and Immature B cells. Lrba +/+: PBS n=7, NP-Ficoll n=4, NP-CGG n=5; Lrba+/-: PBS n=4, NP-Ficoll n=3, NP-CGG n=4; Lrba-/- : PBS n=7, NP-Ficoll n=4, NP-CGG n=4 for Transitional B cells and Plasma cells. Data are depicted as mean + S.D. 45

Further, B cell phenotyping was performed in spleen cells, using the following gating strategy (Figure 20): CD23, a cell surface marker acting as a low affinity receptor for IgE (Henningson et al., 2011) was used to discriminate T1 and T2 cells in the spleen (Carsetti et al., 2004). CD21, a part of the CD19-CD21-CD81 coreceptor complex, involved in the inclusion of innate and humoral immune response (Caroll and Prodeus, 1998), was used for differentiation of the following subsets: In CD23+ cells, T2 B cells express CD21high and IgMhigh, whereas follicular B cells are CD21int and IgMint. Amongst CD23- cells, T1 are IgMhigh and CD23-, and MZ B cells CD21high and IgMhigh. Statistical analysis revealed no relevant differences in the percentages of the splenic B cell subsets between Lrba-/-, Lrba+/-, and Lrba+/+ mice. Immunization with a TI or TD antigen did not lead to significantly relevant change (Figure 21).

Figure 20: Gating strategy for B cells in the spleen. After gating on lymphocytes, using size and granularity, and living cells, using DAPI (as seen in Figure 18), amongst the B220+ subgroup, CD23 was used to discriminate T1 and T2 cells. CD23+ cells include CD21high IgMhigh T2 B cells, and CD21int IgMint follicular B cells. CD23- cells, include IgMhigh CD23- T1 B cells and CD21high IgMhigh MZ B cells.

Figure 21: Analysis of the repartition of B cell subsets in the spleen showed no statistically relevant differences in Lrba-/- mice compared to Lrba+/+ and Lrba+/- mice. T1, T2, FO and MZ B cells showed similar percentages in all three genotypes in the control group vaccinated with PBS, as well as upon vaccination with the TI antigen NP-Ficoll or the TD antigen NP-CGG. This data was collected in: Lrba +/+: PBS n=10, NP-Ficoll n=6, NP-CGG n=8; Lrba+/-: PBS n=4, NP-Ficoll n=6, NP-CGG n=5; Lrba-/- : PBS n=10, NP-Ficoll n=5, NP-CGG n=6. Data are depicted as mean + S.D. 46

Additional B cell phenotyping was performed in peritoneal cavity cells (gating strategy seen in Figure 22). B lymphocytes were recognized as B220+ and IgM+ cells. B1 cells consisting of B- 1a and B-1b subsets were distinguished by expression of CD5, exclusively found in B-1a cells. CD5 has been described as a negative regulator of BCR signaling (Gary-Gouy et al., 2000).

Figure 22: Gating strategy in B cells from the peritoneal cavity. After gating on lymphocytes, by size and granularity, and living cells, by DAPI (as seen in Figure 18), B220+ were gated. Amongst those cells, B-1a and B-1b can be distinguished through CD5 expression, both exhibiting IgM. Conventional B2 cells are CD5+ IgMint. As observed in Figure 23, Lrba-/- mice had a significantly reduced percentage of B-1a cells at steady state, as well as after TI immunization with NP-Ficoll. Vaccination with NP-CGG did not lead to this effect. Percentages of total B cells from the peritoneal cavity showed no significant differences in Lrba-/- mice, compared to Lrba+/+ and Lrba+/- mice. However, a tendency towards higher B cell percentages after vaccination regardless of the antigen type was observed in all three murine subgroups.

Figure 23: B-1a cells in the peritoneal cavity were statistically relevant reduced in Lrba-/- compared to Lrba+/+ mice under steady state conditions (p=0.0238) and after vaccination with the TI antigen NP-Ficoll (p=0.0286). B and B-1b cell percentages showed no differences between the groups. This data was collected in: Lrba +/+: PBS n=9, NP-Ficoll n=6, NP- CGG n=8; Lrba+/- PBS n=4, NP-Ficoll n=6, NP-CGG n=6; Lrba-/- PBS n=9, NP-Ficoll n=5, NP-CGG n=6 for B cells. Lrba +/+: PBS n=7, NP-Ficoll n=4, NP-CGG n=5; Lrba+/- PBS n=4, NP-Ficoll n=3, NP-CGG n=3; Lrba-/- PBS n=7, NP-Ficoll n=5, NP-CGG n=6 for B-1a cells. Lrba +/+: PBS n=3, NP-Ficoll n=4, NP-CGG n=5; Lrba+/-: PBS n=4, NP-Ficoll n=3, NP-CGG n=3; Lrba-/- : PBS n=3, NP-Ficoll n=4, NP-CGG n=4 for B-1b cells Data are depicted as mean + S.D.

3.6. Lrba-/- exhibit normal IgM and IgG titers in steady state conditions and increased IgA levels LRBA-deficient patients (57% and 82.4%) suffer from hypogammaglobulinemia (Gámez-Díaz et al., 2016; Azizi et al., 2017) leading to a higher susceptibility to recurrent infections. To investigate whether antibody titers are impaired in Lrba-/- mice, immunoglobulin titers of IgM, IgG (IgG1, IgG2b, IgG2c, and IgG3 subclass), and IgA isotype were evaluated at steady state conditions in serum of mice of all three genotypes, using ELISA as explained in the Methods section.

Interestingly, Lrba-/- mice showed significantly more IgA, compared to Lrba+/+ and Lrba+/-. Amounts of IgM and IgG subsets produced in Lrba-/- mice were comparable to Lrba+/+ and Lrba+/- mice, suggesting a normal antibody-mediated immune response (Figure 24).

Figure 24: Lrba-/- mice produced comparable amounts of IgM and IgG subtypes, but significantly more IgA compared to Lrba+/+ (p=0.0012) and Lrba+/- mice (p=0.0399) under steady state conditions. IgM, IgG1, IgG2b, IgG2c, IgG3, and IgA titers were assessed by ELISA in serum withdrawn in mice of all three genotypes. This data was collected in: Lrba +/+ n=9; Lrba+/- n=9; Lrba-/- n=10. Data are depicted as mean + S.D. In order to determine, whether the described increase in IgA production can be explained through enhanced IgA production in the intestine, which has been formerly associated with inflammatory bowel disease (Wang et al., 2004), commonly found in LRBA-deficient patients (Gámez-Díaz et al., 2016; Azizi et al., 2017), analysis of IgA levels in the lavage from the small intestine was performed. However, IgA titers from intestinal washing were at comparable levels in Lrba-/-, Lrba+/-, and Lrba+/+ mice, as shown in Figure 25.

Figure 25: IgA titers from intestinal washing showed no statistically relevant differences in Lrba-/- mice compared to Lrba+/+ and Lrba+/- mice under steady state conditions. IgA titers were assessed in lavage of the small intestine. This data was collected in: Lrba +/+ n=9; Lrba+/- n=9; Lrba-/- n=10. Data are depicted as mean + S.D.

3.7. Lrba-/- mice have a normal T cell compartment Investigation of the T cell compartment in LRBA-deficient patients revealed normal cell counts + + in total T cells, CD4 , and CD8 cells, but reduced Treg counts in 70% of patients (Gámez-Díaz et al., 2016; Charbonnier et al., 2015). Furthermore, impaired suppression by Treg and increased

Treg apoptosis have been described. The impairment of Treg function was linked to immune dysregulation found in patients suffering from LRBA deficiency (Charbonnier et al., 2015). To analyze in what manner lack of LRBA affects the T cell compartment, first development of T cells in the thymus of Lrba-/- mice under steady state conditions was analyzed using flow cytometry. Hereby, maturation stages from double negative T cells to double positive T cells and finally CD4+ or CD8+ T cells (as described in chapter 1.2.) were examined. Additionally, T cell subsets in the spleen were investigated under steady state conditions. Furthermore, T cell subsets were analyzed in splenocytes and in cells of the peritoneal cavity under immune response conditions, using vaccinations with either a TI (NP-Ficoll) or a TD antigen (NP-CGG). In order to collect enough data to perform statistical analysis, data from two series of experiments using the immunization protocol were pooled, resulting in at least two mice per condition (injection with PBS as control, vaccination with NP-Ficoll or NP-CGG) and genotype (Lrba+/+, Lrba+/-, Lrba -/-).

Stages of T cell differentiation were investigated in cells from the thymus of Lrba+/+, Lrba+/- and Lrba-/- mice under steady state conditions. The gating strategy is depicted in Figure 26. CD4 is a cell surface glycoprotein involved in antigen recognition together with the T cell receptor through major histocompatibility complex (MHC) class II, and plays a role in T helper cell development and activation (Zeitlman et al., 2001). CD8 is a cell surface protein enabling antigen recognition by binding MHC class I and activating cytotoxic T cells (Gao and Jakobsen, 2000). Using these two markers, single positive CD4+ or CD8+ cells were gated, as well as double negative (DN) CD4- CD8- cells and double positive (DP) CD4+ CD8+ cells.

Figure 26: Gating strategy for T cells in the thymus. First, gating on lymphocytes, by size and granularity, and living cells, by DAPI, is performed as seen in Figure 18. T cells are identified as CD3+. Using cell surface marker CD4 and CD8,

50 different subsets of T cell development can be discriminated. Double negative (DN) T cells do not express either CD4 or CD8, while double positive (DP) T cells are CD4+ CD8+. Furthermore, CD4+, as well as CD8+ T cells can be distinguished. Statistical analysis of T cell subsets revealed no statistical differences between Lrba-/- and Lrba+/+ mice, regarding T cells, CD4+ cells, CD8+ cells, double positive and double negative cells, as seen in Figure 27.

Figure 27: Analysis of the repartition of T cell subsets in the thymus under steady state conditions showed no statistically relevant differences in Lrba-/- mice compared to Lrba+/+ mice. T cells, CD4+, CD8+, double positive CD4+ CD8+, and double negative CD4- CD8- T cells show similar percentages in both genotypes under steady state conditions. This data was collected in: n=3 for T cells and CD8+ cells, n=2 for CD4+, double positive and double negative T cells. Data are depicted as mean + S.D. Next, T cells subsets in the spleen of Lrba+/+, Lrba+/-, and Lrba-/- mice were investigated after immunization with NP-Ficoll or NP-CGG, and in a control group injected with PBS. T cells express CD3, a pan T cell marker forming a complex with the TCR, enabling T cell activation (Clevers et al., 1988). Further, CD4+ T helper cells and CD8+ cytotoxic T cells were gated. For analysis of Treg, staining with forkhead box P3 (FoxP3), which acts as a regulator of the development and function of Treg, was performed (Fontenot et al., 2003). The gating strategy is depicted in Figure 28.

Figure 28: Gating strategy in spleen cells for T cell subsets. After gating on lymphocytes, by size and granularity, and living cells, by DAPI, as seen in Figure 18, T cells were gated due to CD3 expression. Amongst CD3+ T cells, CD4+ and + CD8 T cells are discriminated. Treg are gated through FoxP3 expression. Under steady state conditions, no statistical differences between Lrba-/- and Lrba+/+ mice, + + regarding T cells, CD4 cells, CD8 cells, double positive and double negative cells, and Treg were found, as displayed in Figure 29.

Figure 29: Analysis of the repartition of T cell subsets in the spleen under steady state conditions showed no statistically relevant differences in Lrba-/- mice compared to Lrba+/+ mice. T cells, CD4+, CD8+, double positive CD4+ CD8+, and double negative CD4- CD8- T cells showed similar percentages in both genotypes under steady state conditions. This data was collected in: n=10 for T cells, n=8 for CD4+ cells, n=3 for CD8+, n=2 for double positive and double negative T cells, n=6 for Treg. Data are depicted as mean + S.D Upon immunization with TI antigen NP-Ficoll and TD antigen NP-CGG no relevant differences + -/- +/+ +/- in percentages of T cells, CD4 cells or Treg in Lrba mice compared to Lrba and Lrba mice were revealed (Figure 30).

Figure 30: Analysis of the repartition of T cell subsets in the spleen showed no statistically relevant differences in -/- +/+ +/- + -/- +/+ Lrba mice compared to Lrba and Lrba mice. Percentages of T cells, CD4 , and Treg are similar in Lrba , Lrba and Lrba+/- mice. This data was collected in: Lrba +/+: PBS n=10, NP-Ficoll n=6, NP-CGG n=8; Lrba+/-: PBS n=4, NP-Ficoll n=6, NP-CGG n=5; Lrba-/- : PBS n=10, NP-Ficoll n=5, NP-CGG n=6 in T cells and CD4+ cells. Lrba +/+: PBS n=7, NP- Ficoll n=4, NP-CGG n=5; Lrba+/-: PBS n=4, NP-Ficoll n=4, NP-CGG n=4; Lrba-/- : PBS n=6, NP-Ficoll n=4, NP-CGG n=4 for Treg. Data are depicted as mean + S.D. Cells collected from the peritoneal cavity were analyzed regarding T cells, as well. T cells were discriminated through CD5 expression (Figure 31). CD5 is found in T cells and some B cells, and is considered to play a role in modulation of antigen receptor signaling, lymphocyte survival, and the process of tolerance (Soldevila et al., 2011).

Figure 31: Gating strategy in cells from the peritoneal cavity for T cells. After gating on lymphocytes, by size and granularity, and living cells by DAPI, as seen in Figure 18, T cells were gated due to CD5 expression. No statistical differences in peritoneal T cell percentages between Lrba-/-, Lrba+/+, and Lrba+/- mice were found. Immunization with TI antigen NP-Ficoll or TD antigen NP-CGG did not lead to relevant changes (Figure 32).

Figure 32: T cells in the peritoneal cavity are similar in Lrba-/- compared to Lrba+/+ and Lrba+/- mice. T cells percentages show no differences between the groups and are not affected by vaccination. This data was collected in: Lrba +/+: PBS n=8, NP-Ficoll n=5, NP-CGG n=7; Lrba+/- : PBS n=4, NP-Ficoll n=4, NP-CGG n=5; Lrba-/-:PBS n=8, NP-Ficoll n=5, NP-CGG n=5. Data are depicted as mean + S.D.

3.8. Intestinal histopathology of Lrba-/- mice showed no sign of inflammation, but young Lrba-/- mice had elevated plasma cell counts in the colon Enteropathy is present in about 60% and 80% of LRBA-deficient patients, thus being one of the major clinical presentation (Gámez-Díaz et al., 2016; Azizi et al., 2017). In order to investigate whether the absence of LRBA causes enteropathy in mice, colon and ileal sections of young (below four months of age) and old (above eight months of age) mice under steady state conditions were evaluated using periodic acid-Schiff (PAS) staining, which is used to stain polysaccharides and mucosubstances (Baum, 2008). In the colon, a total of ten crypts were systematically analyzed for lymphocyte aggregates, signs of inflammation such as cryptitis or abscess, and erosion or ulcer. Goblet cells, intraepithelial lymphocytes (IEL) and plasma cells, as well as mitotic figures and apoptotic bodies were also counted. The same parameters were evaluated in a total of ten villi in ileum. All histopathological analyses were performed blinded of the genotype of the mice.

No significant differences in the ileum of young (below four months of age) Lrba-/- mice, compared to Lrba+/- and Lrba+/+ mice were found, indicating an absence of inflammation as shown in Figure 33.

Figure 33: Absence of signs of inflammation in the ileum of young Lrba-/-mice (below four months of age). Ileum samples were fixated in Formalin, paraffin sections of 3.5 µm were stained with periodic acid-Schiff (PAS). One entire section per mouse was screened for lymphocytic aggregates, signs of inflammation, and erosion or ulcer. This data was collected in: Lrba+/+: n=2; Lrba+/- n=2; Lrba-/- n=3. 55

This finding was reproduced in the ileum of old (above twelve months of age) mice, showing no differences or signs of inflammation in Lrba-/- mice, compared to Lrba+/- and Lrba+/+ mice (Figure 34).

Figure 34: Absence of signs of inflammation in the ileum of young Lrba-/-mice (above twelve months of age). Ileum samples were fixated in Formalin, paraffin sections of 3.5 µm were stained with periodic acid-Schiff (PAS). One entire section per mouse was screened for lymphocytic aggregates, signs of inflammation, and erosion or ulcer. This data was collected in n=2. In addition, the cell counting revealed no difference in the numbers of goblet cells, IEL, plasma cells, mitotic figures or apoptotic bodies. These findings were independent of the age of the mice (Figure 35).

Figure 35: Cell counts of goblet cells, IEL and plasma cells as well as counts of mitotic figures and apoptotic bodies in ileum of Lrba-/- mice were similar to Lrba+/+ and Lrba+/- mice. In PAS stained slices of ilea of mice of all three genotypes, in ten vili per slide goblet cells, IEL, plasma cells, mitosis figures, and apoptotic bodies were counted. This data was collected in: (young/old) Lrba +/+: n=2/2; Lrba+/- n=2/2; Lrba-/- n=3/2. Data are depicted as mean + S.D. Similarly, the histopathological analysis of the colon of young Lrba-/- knockout mice, showed no difference regarding inflammation and erosion or ulcers (Figure 36).

Figure 36: Absence of signs of inflammation in the colon of young Lrba-/- mice (below four months of age). Colon samples were fixated in Formalin, paraffin sections of 3.5 µm were stained with periodic acid-Schiff (PAS). One entire section per mouse was screened for lymphocytic aggregates, signs of inflammation, such as cryptitis, and erosion or ulcer. This data was collected in: Lrba +/+: n=2; Lrba+/- n=2; Lrba-/- n=3.

Absence of anomalies and inflammation was also observed in the colon of old Lrba-/- mice, as depicted below in Figure 37.

Figure 37: Absence of signs of inflammation in the colon of old Lrba-/- mice (above twelve months of age). Colon samples were fixated in Formalin, paraffin sections of 3.5 µm were stained with periodic acid-Schiff (PAS). One entire section per mouse was screened for lymphocytic aggregates, signs of inflammation, such as cryptitis, and erosion or ulcer. This data was collected in: n=2. Cell counting in the colon revealed significant increased numbers of plasma cells in the crypts of Lrba-/- mice in comparison to Lrba+/+ and Lrba+/- mice aged below four months. This result however was not found in old mice. Other parameters such as goblet cells, IEL, mitotic figures and apoptotic bodies showed no statistical differences, as shown in Figure 38.

Figure 38: Young Lrba-/- mice exhibited higher counts of plasma cells in the colon compared to Lrba+/+ mice (p = 0.0454). In old mice, the finding could not be reproduced. Furthermore, young Lrba-/- mice exhibited higher counts of goblet cells in the colon compared to Lrba+/- mice (p= 0.0004), yet similar counts in old mice. Cell counts of IEL as well as counts of mitosic figures and apoptotic bodies in colon of Lrba-/- mice showed no relevant difference compared to Lrba+/+ and Lrba+/- mice; age of the mice had no impact on this finding. In PAS stained slices of colons of mice of all three genotypes, in ten crypts per slide goblet cells, IEL, plasma cells, mitosis figures, and apoptotic bodies were counted. This data was collected in: (young/old) Lrba +/+: n=2/2; Lrba+/- n=2/2; Lrba-/- n=3/2. Data are depicted as mean + S.D.

4. Discussion The gene encoding murine LPS-responsive beige-like anchor protein (mLRBA) was first described in 1996 as a LPS-responsive gene highly expressed in murine B cells (Kerr et al., 1996). mLRBA was further characterized in 2001, as a protein with features of kinase anchor proteins and Beige and Chediak Higashi (BEACH) proteins. Subcellular colocalization with the endoplasmic reticulum, trans-Golgi network, lysosomes and endocytosis vesicles was shown in mice (Wang et al., 2001). Later in 2012, Lopez-Herrera et al. described a human clinical phenotype of immunodeficiency and autoimmunity caused by homozygous mutations in LRBA, leading to a lack of LRBA expression – so called LRBA deficiency. Patients were found to suffer from hypogammaglobulinemia and autoimmunity, presenting with defective immunoglobulin secretion and B cell activation. LRBA-deficient B cells were shown to have impaired proliferation and differentiation into antibody-secreting cells, when being cultured under conditions enabling class-switch recombination and plasmablast development, explaining impaired immunoglobulin secretion through disrupted generation of plasma cells (Lopez-Herrera et al., 2012). In the following years, more patients suffering from LRBA deficiency were described, revealing the broad clinical spectrum of this disease, including autoimmunity, enteropathy, organomegaly, recurrent infections, described in bigger patient cohorts, (Gámez-Díaz et al., 2016; Alkhairy et al., 2016), as well as cases of erosive polyarthritis and type 1 diabetes (Lévy et al., 2016), autoimmune endocrine disorders (Bakhtiar et al., 2016), and bronchiolitis obliterans organizing pneumonia (Shokri et al., 2016). In addition, a possible explanation for autoimmune manifestations in LRBA-deficient patients was found, when Lo et al. unveiled the essential role of LRBA in trafficking and turnover of CTLA4 through a direct protein-protein interaction (Lo et al., 2015). Yet, the exact function of LRBA in immune cells, as well as its possible roles in other tissues, explaining the phenotype of LRBA deficiency remains unclear. As LRBA deficiency is a very rare disease with about 70 patients reported to date, sample acquisition is difficult. Therefore, studying a mouse model that offers high accessibility to samples of all organs, and allows analysis of different organs under distinct conditions is advantageous. With 99% of their encoded sequence shared with humans, and the possibility to manipulate it through knocking strategies, mice have become an attractive model to study human immunology (Nguyen and Xu, 2008). In addition, a mouse model allows exclusion of confounders such as other genetic mutations interacting with the functionality of LRBA, or infections triggering the immune system and leading to non-comparable outcome, as it happens in patients. As a next step it even permits deliberate manipulation of the conditions such as the induction of an immune response or usage of infectious models. Murine and human

LRBA share about 90% identity gene alignment and protein homology (calculated on UniProt Blast; Q9ESE1-1 and P50851-2). Therefore, the study of a LRBA-deficient mouse model allows drawing conclusions to the human counterpart.

In this thesis, we investigated the phenotype of a Lrba-/- mouse model, in order to gain further insight on the molecular and cellular mechanisms, impaired in LRBA deficiency.

4.1. Overall development and organomegaly in Lrba-/- mice Despite a trend towards lower weight, Lrba-/- mice developed normally under SPF conditions, when followed up to the age of two years, without presentation of health issues found at regular clinical inspection. Consistently, Park et al. reported a normal and healthful life span in their LRBA-null mouse model (Park et al., 2016). In contrast, LRBA-deficient patients manifest with first symptoms in childhood, at an average age of onset of 4 (Gámez-Díaz et al., 2016) or 5 years (Alkhairy et al., 2016) and suffer from growth restriction in 24% due to severe symptoms, like recurrent infections in 71% of the cases (Gámez-Díaz et al., 2016). Possibly, the immune system needs trigger through infection in order to fail functionality, however the induction of an immune response by immunization with TI and TD antigens did not provoke any clinical sign of illness in Lrba-/- knockout mice. Inflammatory bowel disease is characterized as a chronic idiopathic disease, presenting with recurrent inflammation of the gastrointestinal tract git (Tegtmeyer et al., 2017), and is commonly found in LRBA-deficient patients (Gámez-Díaz et al., 2016). It is often associated with weight loss, which even is part of the criteria of severity for IBD (Baumgart and Sandborn, 2007). Even though Lrba-/- mice did not present with clinical manifestations, a tendency towards lower bodyweight compared to Lrba+/+ or Lrba+/- mice could be observed.

As major clinical feature, LRBA-deficient patients present organomegaly (86%), especially splenomegaly (64%) (Gámez-Díaz et al., 2016). Consistently, LRBA-deficient mice showed a skewing towards higher spleen weight and the ratio of spleen to body weight was significantly higher in Lrba-/- mice compared to Lrba+/+ mice. However, data from the Lrba-/- mouse model by Burnett et al. showed no signs of splenomegaly at either six weeks or six months of age (Burnett et al., 2017). Immune response of any kind and red blood cell destruction are the most common causes for splenomegaly, leading to work hypertrophy in the tissue (Eichner, 1979). However, on histopathology level, spleen morphology of Lrba-/- mice presented normally.

4.2. B cell development in Lrba-/- mice Immunologically, LRBA-deficient patients present with low switched and memory B cell counts and hypogammaglobulinemia (75% and 57%, respectively) suggesting an impairment of the B cell differentiation (Gámez-Díaz et al., 2016). In order to unveil these possible defects, B cell subsets in the bone marrow and in the spleen were analyzed in mice injected with TI antigen NP-Ficoll and TD antigen NP-CGG, as well as in a control group injected with PBS only. Thymus independent immune response can be induced by polysaccharides and leads to IgM production by MZ cells without T cell support (Obukhanych and Nussenzweig, 2006). In contrast, Thymus dependent immune response works through recognition of presented antigens on B cells, by the TCR of T helper cells, resulting in B cell differentiation into plasma cells and the production of specific antibodies (DeFranco, 1987). As opposed to the immunological phenotype in humans, normal numbers and repartition of B cell subsets were observed in the bone marrow of Lrba-/- mice in comparison to Lrba+/+ and Lrba+/- mice, regardless of the immunization. This finding indicates a functioning B cell differentiation in the bone marrow of Lrba-/- mice, even after triggering an immune response through immunization. Evaluation of the B cell development in the periphery showed no significant differences between Lrba-/-, Lrba+/+ and Lrba+/- mice, regardless of the treatment. Normal B cell frequencies in Lrba-/- mice have been confirmed in two further mouse models (Burnett et al., 2017; Park et al., 2016). Furthermore, our group showed unaffected B cell proliferation and survival upon stimulation with LPS and LPS+IL-4, as well as intact class switch recombination, and plasmablast differentiation in LRBA-deficient mice, suggesting no crucial involvement of LRBA in cell proliferation and survival, Ig class switching and plasmablast generation (Gámez-Díaz et al., 2017). Since patients present with symptoms early in life but not at birth, one might hypothesize that a recurring contact with pathogens is needed to stress immune cells and finally reveal the immunological and clinical phenotype. Therefore, infectious models might expose defects in B cell development in Lrba-/- mice. Burnett et al. hypothesized, that the reduction in switched memory B cells found in LRBA-deficient patients, but not in Lrba-/- mice, might be explained through severe immune dysregulation caused by CTLA-4 deficiency, secondarily damaging the B cell compartment (Burnett et al., 2017).

Interestingly, a significant reduction of B-1a cells in the peritoneal cavity of Lrba-/- mice injected with control PBS, and after NP-Ficoll immunization, compared to Lrba+/+ was observed. Consistently, Burnett et al. described the same decrease in peritoneal B-1a cells in their LRBA-deficient mouse model (Burnett et al., 2017). B-1a cells are generated in the fetal liver and reside in body cavities, such as the peritoneal or pleural cavity (Prieto and Felippe, 62

2017). They act as innate-like B cells, producing natural antibodies with low affinity and polyreactivity (Carsetti et al., 2004). In contrast, B-1b cells produce antigen-specific antibodies (Baumgarth, 2011). B1 cells in the peritoneal cavity have been linked to development of autoimmune diseases such as systemic lupus erythematosus (SLE), Sjoegren’s syndrome and rheumatoid arthritis, (Duan and Morel, 2006), as well as with inflammatory bowel disease (IBD) (Shimomura et al., 2008) and CVID (Kraljevic et al., 2013). Different possible mechanisms of how B1 cells favor autoimmunity have been suggested: autoantibody production, self-antigen presentation to autoreactive T cells, and cytokine secretion (Duan and Morel, 2006). In a mouse model for primary biliary cholangitis, an autoimmune disease of the liver, a reduction of B-1a cells in the peritoneal cavity has been shown. Additionally, higher levels of CTLA-4 were expressed on B-1a cells compared to B-1b and B2 cells in these mice. Moreover, B-1a cells in these mice secreted less levels the of anti-inflammatory cytokine IL- 10 (Yang et al., 2016), which has been associated to be essential in the tolerance to apoptotic cells, hereby preventing autoimmune mechanisms described in SLE and Sjoegren’s syndrome (Miles et al., 2018). Work performed by our group confirmed impaired IL-10 production in LRBA-deficient B-1a cells, upon stimulation with LPS, phorbol 12-myristate 13-acetate (PMA), and ionomycin (Gámez-Díaz et al., 2017). Therefore, decreased B-1a cells and defective IL-10 production in Lrba-/- mice might serve as an explanation for frequent immune deregulatory manifestations in LRBA-deficient patients. Further investigation on IL-10 secretion by B-1a B cells in Lrba-/- mice could allow deeper insight, on possible contribution to the pathogenesis of autoimmunity in LRBA-deficient patients. One hypothesis, of how LRBA interferes with B-1a B cell functionality might be, through impairment of autophagy. Autophagy is degradation of unnecessary cellular material in lysosomes, stabilizing the cell and preventing cell death (Lopez-Herrera et al., 2012). B-1a B cells are energetically highly active, hereby relying on autophagy. The deletion of autophagy gene Atg7 was found to lead to selective loss of B-1a B cells due to insufficiency of self-renewal (Clarke et al., 2018). Since LRBA deficiency has been found to impair autophagy in B cells, a participation in autophagy pathways seems likely (Lopez-Herrera et al., 2012). In this manner, lack of LRBA might lead to compromised autophagy, resulting in lower B-1a B cells counts and less IL-10 production eventually leading to an overactive immune system and autoimmunity. However, autoimmune disease markers, such as ANA and anti-thyroglobulin autoantibodies, were not detected in Lrba-/- mice, representing the absence of autoimmunity (Gámez-Díaz et al., 2017). Possibly, Lrba-/- mice establish regulatory mechanism, compensating for autoimmune manifestations (Gámez-Díaz et al., 2017).

Another interesting involvement of B-1a B cells has been described in lymphoid cancer development. Specifically, a mouse model with overexpression of Epstein-Barr virus induced gene 2, a mediator of follicular B cell migration, lead to B-1a B cell upregulation and chronic lymphocytic leukemia-like B cell malignancies (Niss Arfelt et al., 2017). This suggests a possible link between B-1a B cell increase found in Lrba-/- mice and overexpression of LRBA in renal, pancreas, colorectal and central nervous system cancers found in microarray analysis (Wang et al., 2004).

4.3. Antibody production in Lrba-/- mice Since LRBA-deficient patients suffer from hypogammaglobulinemia, presenting with low IgM (52%), IgA (38%) and/or IgM (29%) (Gámez-Díaz et al., 2016), we examined total immunoglobulin titers under basal conditions in Lrba-/- mice. Different Ig isotypes have been analyzed in this work. Amongst IgG, the major class of the isotypes, different subclasses can be distinguished. In humans, four subclasses with 90% identical amino acids, can be discriminated: IgG1, IgG2, IgG3 and IgG4. IgG1, the most numerous subclass, is mostly involved in response to soluble and membrane proteins and polypeptides. IgG2 mounts a response to bacterial capsular polysaccharide antigens. IgG3 induce effector functions and are directed against polypeptides. Lastly, IgG4 production is caused by allergens (Vidarsson et al., 2014). In mice IgG1 can react to allergens (Jönsson et al., 2011), IgG2a and IgG2b to polypeptides. IgG3, corresponding to IgG2 in humans, responds to polysaccharides (Sarvas et al., 1983). In contrast to LRBA-deficient patients, Lrba-/- mice produced comparable titers of IgM and IgG isotypes to Lrba+/+ and Lrba+/- mice indicating that in absence of LRBA, mice are able to mount a sufficient humoral immune response. In the Lrba-/- mouse model by Burnett et al., significantly higher levels of basal IgG2b were described, which we could not reproduce (Burnett et al., 2017). Interestingly, in our mice IgA production was significantly higher in sera of mice lacking LRBA. IgA is found in the serum, as well as in a secretory form on mucosal surfaces. In humans, IgA mostly occurs in a monomeric form, whereas polymeric IgA is predominant in mice. The main function of IgA is to neutralize antigens at mucosal surfaces. Furthermore, it has been found to decrease inflammation through inhibition of effector functions of inflammatory cells. Also, a protective role in allergic diseases such as asthma has been described (Gloudemans et al., 2013). Therefore, we hypothesized that the elevated IgA titers observed in Lrba-/- mice might explain the absence of inflammation or autoimmune manifestations. In contrast to LRBA-deficient patients, who present low IgA titers in 38% (Gámez-Díaz et al., 2016), mice lacking LRBA might be able to compensate through higher IgA production protecting them from clinical illness. Burnett et al. also found a trend towards 64 increased IgA in their LRBA-deficient mouse model. In conjunction with their finding of increased IgG2b titers, an involvement of transforming growth-factor β was suggested, for it induces both isotypes (Burnett et al., 2017).

In addition, we hypothesized that increased serum IgA titers might correlate with reduced B-1a B cells in the peritoneal cavity (Gámez-Díaz et al., 2017). A fraction of serum IgA in rodents has been suggested to derive from intestinal IgA (Vaerman et al., 1973). Since intestinal IgA+ plasma cells in the lamina propria have been shown to originate from B1 cells from the peritoneal cavity (Fagarasan and Honjo, 2000), an augmented migration of peritoneal B1 cells to the intestine and on-site differentiation into IgA secreting plasma cells, resulting in higher IgA serum titers could be possible (Gámez-Díaz et al., 2017). Analysis of characteristics of IgA producing cells in the intestine might reveal a connection between reduced B-1a cells and increased serum IgA titers.

Further work published by our group investigated the antibody production in Lrba-/- mice upon stimulation with a TD antigen (NP-CGG), as well as with TI antigens (NP-Ficoll, eliciting TI type 1, and LPS, eliciting TI type 2 response). Lrba-/- mice were found to produce comparable IgM and IgG antibody titers to Lrba+/+- mice and showed similar numbers of IgM and IgG1 antibody secreting cells in the bone marrow and the spleen, suggesting a functioning TI and TD immune response in Lrba-/- mice (Gámez-Díaz et al., 2017).

4.4. Investigation of the T cell compartment in Lrba-/- mice Whilst 80% of LRBA-deficient patients show normal T and CD4+ cell counts, regulatory T cells are reduced in over 70% of patients. In Lrba-/- mice, we found T and CD4+ T cells in +/+ similar percentages compared to Lrba mice. Unexpectedly, Treg percentages in the spleen also were normal in mice lacking LRBA. Our group further showed normal percentages of CD8+ cells in the spleen, and a skewing of splenic CD4+ and CD8+ T cells towards a memory phenotype (CD44high CD62low) in old Lrba-/- mice (Gámez-Díaz et al., 2017). Normal T cell frequencies were also found in the two other published Lrba-/- mouse models (Park et al., 2016; Burnett et al., 2017). Concerning functionality of the T cell compartment of Lrba-/- mice upon infection with LCMV, inducing an acute viral infection and triggering anti-viral response by CD8+ cytotoxic T cells, mice were found to generate similar amounts of specific CD8+ cells, CD8+ memory precursor effector cells, and CD8+ short-lived effector cells compared to Lrba+/+ mice. The lysis of LCMV infected cells was not impaired in Lrba-/- mice. Tumor necrosis factor- α (TNF-α) and interferon-γ (IFN-γ) levels, produced in CD8+ cells, were equal in Lrba-/- mice, compared to Lrba+/+ mice. These findings indicate a normal anti-viral cytotoxic T cell response 65 in the absence of LRBA (Gámez-Díaz et al., 2017). In LRBA-deficient patients, low Treg counts with reduced expression of canonical Treg cell markers including FOXP3 CD25, Helios and

CTLA-4 have been described, accompanied by defective Treg mediated suppression

(Charbonnier et al., 2014). Impaired Treg numbers and functionality are a possible cause for autoimmune manifestations in LRBA-deficient patients (Charbonnier et al., 2014). Lo et al. found a potential explanation on how LRBA interferes with Treg function, by showing that LRBA blocks CTLA-4 trafficking to the lysosome, thus inhibiting degradation and maintaining CTLA-4 surface levels. CTLA-4 binds the T cell ligands CD80 and CD86 on antigen presenting cells and removes them from the cell surface through transendocytosis (Querechi et al., 2012). In case of LRBA deficiency, higher lysosomal degradation of CTLA-4 lead to lower CTLA-4 expression, resulting in impaired regulatory T cell function (Lo et al., 2015). Our group found decreased intracellular CTLA-4 expression in murine Treg (Gámez-Díaz et al., 2017), and another recently published mouse model of LRBA deficiency showed low CTLA-4 expression in Treg in young and chimeric mice, and additionally decreased FOXP3 in old mice (Burnett et al., 2017). Besides this, immune dysregulation could not be observed, not even after immunization or infection with chronic lymphocytic choriomeningitis virus infection. While CD86 was increased on non-GC B cells, it was found to be reduced on GC B cells, possibly indicating a compensation in the light of low CTLA-4, which might explain the non-striking immunological phenotype (Burnett et al., 2017). Deletion of Ctla-4 in murine Treg from adult mice led to increased IL-10 production and therefore enhancement of immunoinhibitory functions. Possibly, affection of Treg early in life is crucial for a phenotype caused by CTLA-4 deficiency (Paterson et al., 2015). In regards of these findings, further analysis of Treg in Lrba-/- mice, especially the associated functions of LRBA and CTLA-4 and their impact on the development of autoimmunity, should be done, hereby focusing on Treg functionality through investigation of Treg cell suppression capacity. This might unveil defects in Treg function despite normal percentages in LRBA-deficient mice, which furthermore might offer explanations for the striking immune deregulatory phenotype in LRBA deficiency.

4.5. Investigation of intestinal histopathology in Lrba-/- mice The major clinical manifestation of LRBA-deficient patients is immune dysregulation, among which the most common is enteropathy (Gámez-Díaz et al., 2015; Azizi et al., 2017). Therefore, a histopathological analysis of the small and large intestine of Lrba-/- mice was performed. The digestive tract provides several lines of defense. The first line is composed by the epithelial barrier including intracellular junctions, the goblet cells that produce mucus and the Paneth cells that produce antimicrobial peptides. The second line consists of intraepithelial leukocytes, 66 as well as leukocytes residing in the lamina propria, including IgA producing plasma cells (Tegtmeyer et al., 2017). Patients suffering from inflammatory bowel disease present with chronic idiopathic and recurrent inflammation of the gastro intestinal tract (Fiocchi, 2015). In the context of primary immunodeficiencies, age of onset can be early in childhood and IBD can be the predominant clinical presentation (Uhlig et al., 2014). However, the discrimination of polygenic diseases, such as Crohn’s disease and ulcerative colitis, and monogenic causes in the context of PID, by means of symptomatology and histopathology is not feasible. The available case reports present heterogeneous histopathological data. Amongst these unspecific findings, patients with IBD in a context of PID present with villous atrophy (Tegtmeyer et al., 2017), lack of mucosal plasma cells (Malamut et al., 2010) and nodular lymphoid hyperplasia in the intestine (Khodadad et al., 2007). Histopathological findings in LRBA-deficient patients suffering from enteropathy, are villous atrophy (Alangari et al., 2012; Alkhairy et al., 2016; Tegtmeyer et al., 2017), increased intraepithelial lymphocytes (Alangari et al., 2012) and T cell mediated epithelial destruction (Serwas et al., 2015). In addition, samples from the colon displayed inflammation (Alangari et al., 2012; Bakhtiar et al., 2017; Serwas et al., 2015) with increased intraepithelial lymphocytes (Alangari et al., 2012), ulcerative colitis (Tegtmeyer et al., 2017), lymphofollicular hyperplasia and increased apoptosis (Bakhtiar et al., 2017), as well as decreased crypt numbers (Alangari et al., 2012). In contrast to these findings young and old LRBA-deficient mice presented no signs of inflammation or ulceration, no counting of increased or decreased goblet cells, intraepithelial lymphocytes, mitotic figures or apoptotic bodies in the ileum or colon under steady state conditions. Consistently, no clinical illness or significant reduction of the body weight in line with chronic inflammation was found. In the LRBA-null mouse model by Park et al. no significant abnormalities in histopathological analysis of the gastrointestinal tract were revealed, either (Park et al., 2017). One might hypothesize, that an antigenic trigger, leading to manifest infection might be needed to stress the immune system and unveil the phenotype of enteropathy. In our setup, mice lived under SPF conditions and received no immunizations. However, when Lrba-/- mice were infected with S. typhimurium, to induce an intestinal infection, they did not present higher morbidity or mortality, although they were not able to entirely eliminate S. typhimurium from mesenteric lymph nodes (Gámez-Díaz et al., 2017).

Interestingly, we found a significant increase of plasma cells in the colon of young mice lacking LRBA. These might be migrated peritoneal B-1a cells, that differentiated into IgA producing plasma cells, according to the hypothesis formulated in chapter 4.5. Further analysis of characteristics and secretory function of these plasma cells should be done (Gámez-Díaz et al., 67

2017). IgA secreting plasma cells are known to be located in the lamina propria of the intestine of mice (Fagarasan et al., 2001). Increased serum IgA titers, associated with augmentation of IgA producing cells in the intestine, have been linked to patients suffering from inflammatory bowel disease (Wang et al., 2004). In conjunction, B-1a B cells migrating into the intestine, then differentiating into IgA secreting plasma cells, leading to elevated IgA titers might contribute to the pathogenesis of immune modulatory phenomena in LRBA-deficient patients.

4.6. Summary, limitations and outlook In summary, Lrba-/- mice had a significantly increased ratio of spleen to body weight, demonstrating signs of splenomegaly despite normal histological analysis of the spleen. In addition, a significant reduction of peritoneal B-1a cells, increased basal serum IgA titers, and elevated plasma cell counts in the colon of young mice (< 4 months) were observed. Association between reduced B-1a cells and elevated IgA titers to autoimmunity in absence of LRBA should be further investigated. Development of the B cell lineage and composition of the T cell compartment showed no anomalies, enabling Lrba-/- mice to mount a sufficient humoral immune response (including IgM, IgG subsets and IgA). Finally, ileum and colon of young and old Lrba-/- mice showed no signs of inflammation or lymphocytic infiltration compared to wildtype mice. The cohort size investigated in this work is rather sized thus not allowing final conclusions. Yet in conjunction with the other two published models, valid statements can be made.

Unexpectedly, the clinical phenotype of LRBA-deficient patients could not be reproduced in our mouse model. While murine models represent an important tool for investigating primary immunodeficiencies, for they often mirror the clinical findings in humans, some knockout mouse models show different findings compared to their corresponding genotype in humans (Gámez-Díaz et al., 2017). The beige mouse represents the human phenotype of Chediak- Higashi syndrome (Roder, 1979). In contrast, a model of heterozygous Ctla-4 knockout mice does not mimic the immunodeficiency observed in humans (Waterhouse et al., 1995). Further data from LRBA-deficient mouse models has been published to date: One model of Lrba-null mice showed impaired rejection of allogenic, xenogenic and missing self-bone-marrow grafts explained by defective signaling through NKp46 and NKG2D in NK cells, indicating bone marrow transplant as important treatment tool for LRBA-deficient patients (Park et al., 2016). -/- Burnett et al. reported on CTLA-4 deficiency in Treg of Lrba mice without signs of immune dysregulation (Burnett et al., 2017). Furthermore, non-immunologic phenotypes in Lrba-/- mice have been described: Kurtenbach et al. found a reduction of the heterotrimeric G-protein Golf

68 in the sensory cilia of olfactory neurons of Lrba-/- mice, resulting in disturbed olfaction. Additionally, high LRBA expression in photo receptor cells was detected, yet visual function was not affected in Lrba-/- mice (Kurtenbach et al., 2017). Moreover, LRBA deficiency in mice was linked to sensorineural hearing loss, due to degradation of stereociliary bundles. Loss of LRBA lead to reduction of two adaptor proteins, radixin and Nherf2, involved in stabilization of the basal taper region of stereocilia. Similar, yet less severe clinical presentation in humans was reported as well (Vogl et al., 2017). Murine models allow easy sample access and manipulation of the conditions and therefore remain a valuable instrument. An explanation for the discrepancy between LRBA-deficient mice and human could be, that the clinical phenotype of LRBA deficiency is revealed through a trigger, i.e. further mutations found in modifier genes in patients or recurrent infections resulting in an exhaustion mechanism or an autoimmune predisposition. Through exposure of possible triggers, a new therapy target could be found, resulting in better treatment options for LRBA-deficient patients.

Abstract Biallelic mutations in human LPS-responsive beige-like anchor protein (LRBA) lead to an early-onset primary immunodeficiency. LRBA, as one of the BEACH domain-containing proteins, is an ubiquitously expressed protein, inducible by LPS in lymphocytes, and has been linked to vesicle trafficking, autophagy, and apoptosis. Homozygous or compound heterozygous mutations in LRBA cause a phenotype of autoimmune manifestations and recurrent infections. Patients suffer from hypogammaglobulinemia, reduced numbers of switched memory B cells, and regulatory T cell deficiency. Since LRBA deficiency is a rare and so far not entirely understood disease, we used a Lrba-/- mouse model in order to investigate the B and T cell compartment upon immunization with T-dependent (TD) and T-independent (TI) antigens, antibody production, as well as immune dysregulation through histopathological analysis of spleen and the intestine.

We found that Lrba-/- mice do not present any severe clinical or immunological phenotype at steady state or upon vaccination with TD or TI antigens. A trend towards increased splenic weight, and a significantly increased spleen/body weight ratio were observed, suggesting splenomegaly in Lrba-/- mice. Yet, histopathological spleen analysis revealed no abnormalities, neither at steady state, nor upon immunization. The investigation of the B cell compartment by flow cytometry revealed reduced B-1a B cells in the peritoneal cavity under basal conditions, as well as upon vaccination with a TD antigen. Besides that, the repartition of B cell subsets in the bone marrow and spleen of Lrba-/- mice was normal. Investigation of the capacity of antibody secretion was assessed by ELISA. Under basal conditions, Lrba-/- mice were found to produce increased IgA titers, while IgM and IgG subclasses showed comparable levels to Lrba+/+ mice. Analysis of the T cell compartment in the thymus in steady state conditions, as well as in the spleen upon immunization with TI and TD antigens were normal. Apart from increased plasma cell counts in the colon of young Lrba-/- mice, histopathological analysis of the ileum and colon showed no signs of inflammation or aberrant cell counts of goblet cells, intraepithelial lymphocytes, mitosis figures, or apoptotic bodies.

In conjunction, reduced B-1a B cells and elevated IgA titers might point towards autoimmune presentations found in LRBA-deficient patients, for they have been previously linked to autoimmunity. However, Lrba-/- mice had a mild clinical phenotype compared to humans, raising the question on modifier genes or environmental factors contributing to disease pathogenesis.

Zusammenfassung Biallelische Mutationen in LPS-responsive beige-like anchor Protein (LRBA) führen zu einem primären Immundefizienzsyndrom. LRBA, welches Teil einer Familie BEACH Domäne enthaltender Proteine ist, wird ubiquitär exprimiert, wobei die Expression in Lymphozyten durch LPS induziert wird. Beteiligungen an Vesikeltransport, Autophagie und Apoptose wurden beschrieben. Homozygote oder kombiniert heterozygote Mutationen sind Auslöser eines Immundefizienzphänotyps mit Autoimmunität und rezidivierenden Infektionen. Die Patienten leiden unter Hypogammaglobulinämie, Verringerung unterschiedlicher B Zell Subtypen und einer gestörten Funktion regulatorischer T Zellen. Da LRBA Defizienz eine sehr seltene, bisher nur unvollständig verstanden Erkrankung darstellt, wurde in dieser Arbeit anhand eines Lrba-/- Mausmodells die B und T Zell Zusammensetzung vor sowie nach Immunisierung mit einem Thymus-unabhängigen und einem Thymus-abhängigen Antigen untersucht. Weiterhin wurden die Antikörperproduktion und Zeichen für Immundysregulation anhand histologischer Untersuchung von Milz und Dünn- und Dickdarm analysiert.

Lrba-/- Mäuse waren klinisch und immunologisch unauffällig. Es zeigte sich ein erhöhtes Gewicht der Milz sowie ein signifikant erhöhter Milz-/Körpergewicht-Quotient im Sinne einer Splenomegalie. Histologische Untersuchungen des Milzgewebes vor sowie nach Immunisierung ergaben jedoch keine Auffälligkeiten. Die Analyse der B Zell Zusammensetzung mittels Durchflusszytometrie zeigte verringerte B-1a B Zellen in der Peritonealhöhle sowie nach Impfung mit dem Thymus-abhängigen Antigen NP-Ficoll. Daneben ergaben sich keine Auffälligkeiten der B Zell Subkategorien in Knochenmark und Milz von Lrba-/- Mäusen. Die Ermittlung der Antikörperproduktion zeigte erhöhte IgA Titer bei normalen IgM und IgG Titern im Serum von Lrba-/- Mäusen. Die Untersuchung der T Zell Zusammensetzung im Thymus sowie in der Milz nach Immunisierung ergab keine Auffälligkeiten. Neben erhöhter Plasmazellzahlen im Kolon junger Lrba-/- Mäuse zeigte die histologische Analyse von Ileum- und Kolongewebe keine Hinweise auf Inflammation, normale Zahlen an Becherzellen und intraepithelialer Lymphozyten sowie das Auftreten von Apoptosekörperchen und Mitosefiguren in regulärer Frequenz.

Verringerte B-1a B Zellen sowie erhöhte IgA Produktion könnten an der Entstehung der autoimmunologischen Symptomatik bei LRBA-defizienten Patienten beteiligt sein. Der diskrete Phänotyp von Lrba-/- Mäusen wirft jedoch die Frage nach Modifier-Genen oder die Krankheit beeinflussenden Umweltfaktoren auf.

References

Alangari, A., Alsultan, A., Adly, N., Massaad, M.J., Kiani, I.S., Aljebreen, A., Raddaoui, E., Almomen, A.-K., Al- Muhsen, S., Geha, R.S. and Alkuraya, F.S. (2012), “LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency”, The Journal of allergy and clinical immunology, Vol. 130 No. 2, pp. 481-8.e2. Alkhairy, O.K., Abolhassani, H., Rezaei, N., Fang, M., Andersen, K.K., Chavoshzadeh, Z., Mohammadzadeh, I., El-Rajab, M.A., Massaad, M., Chou, J., Aghamohammadi, A., Geha, R.S. and Hammarström, L. (2016), “Spectrum of Phenotypes Associated with Mutations in LRBA”, Journal of Clinical Immunology, Vol. 36 No. 1, pp. 33–45. Anderson, G., Moore, N.C., Owen J.J.T., and Jenkinson, E.J. (1996), "Cellular interactions in thymocyte development", Annual Review of Immunology, Vol. 14, pp. 73-99. Atzeni, F., Sarzi-Puttini, P., Mutti, A., Bugatti, S., Cavagna, L., and Caporali, R. (2013), "Long-term safety of abatacept in patients with rheumatoid arthritis", Autoimmunity Reviews, Vol. 12, pp. 1115 - 1117. Azizi, G., Abolhassani, H., Mahadaviani, S.A., Chavoshzadeh, Z., Eshgi, P., Yazdani, R., Kiaee, F., Shaghagi, M., Mohammadi, J., Rezaei, N., Hammarström, L., and Aghamohammadi, A. (2017), "Clinical, immunologic, molecular analyses and outcomes of iranian patients with LRBA deficiency: A longitudinal study", Pediatric Allergy and Immunology, Vol. 28, pp. 478- 484. Bakhtiar, S., Gámez-Díaz, L., Jarisch, A., Soerensen, J., Grimbacher, B., Belohradsky, B., Keller, K.M., Rietschel, C., Klingebiel, T., Koletzko, S., Albert, M.H. and Bader, P. (2017), „Treatment of Infantile Inflammatory Bowel Disease and Autoimmunity by Allogenic Stem Cell Transplantation in LPS-Responsive Beige-Like Anchor Deficiency”, Frontiers in Immunology, Vol. 8, No. 52. Bakhtiar, S., Ruemmele, F., Charbit-Henrion, F., Lévi, E., Rieux-Laucat, F., Cerf-Bensussan, N., Bader, P., and Paetow, U. (2016), "Atypical manifestations of LPS-Responsive Beige-like Anchor Deficency Syndrome as an Autoimmune Endocrine Disorder without Enteropathy and Immunodeficiency", Frontiers in Pediatrics, Vol.14, No. 98. Baum, S. (2008) "The PAS reaction for staining cell walls", CHS Protocols, Vol. 3 Baumgart D.C. and Sansborn, W.J. (2007), "Inflammatory bowel disease: clinical aspects and established and evolving therapies" The Lancet, Vol. 369, pp. 1641-1657. Baumgarth, N. (2011), "The double life of a B-1 cell: self-reactivity selects for protective effector functions", Nature Reviews, Vol. 11, pp. 34-46 Borowski, C., Martin, C., Gounari, F., Haughn, L., Aifantis, I., Grassi, F., and von Boehmer, H. (2002), "On the brink of becoming a T cell", Current opinion in Immunology, Vol. 14, pp. 200-206. Burgess, A., Mornon, J.-P., Saint-Basile, G. de and Callebaut, I. (2009), “A Concavanalin A-like lectin domain in the CHS1/LYST protein, shared by members of the BEACH family”, Bioinformatics, Vol. 25, pp. 1219–1222. Burnett, D.L., Parish, I.A., Masle-Farquhar, E., Brink, R. and Goodnow, C.C. (2017), "Murin LRBA deficiency causes CTLA-4 deficiency in Tregs without progression to immune dysregulation", Immunology and Cell Biology, Vol. 95, pp.775 - 788. Burns, S.O., Zenner, H.L., Plagnol, V., Curtis, J., Mok, K., Eisenhut, M., Kumararatne, D., Doffinger, R., Thrasher, A.J. and Nejentsev, S. (2012), “LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia”, Journal of Allergy and Clinical Immunology, Vol. 130, pp. 1423–1432. Caroll, M.C. and Prodeus A.P. (1998), "Linkages of innate and adaptive immune response", Current Opinion in Immuology, Vol. 10, pp. 36 - 40. Carsetti R., Rosado M. and Wardemann H. (2004), "Peripheral development of B cells in mouse and men", Immunological Reviews, Vol. 197, pp. 179-191 Charbonnier, L.-M., Janssen, E., Chou, J., Ohsumi, T.K., Keles, S., Hsu, J.T., Massaad, M.J., Garcia-Lloret, M., Hanna-Wakim, R., Dbaibo, G., Alangari, A.A., Alsultan, A., Al-Zahrani, D., Geha, R.S. and Chatila, T.A. (2015), “Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked- like disorder caused by loss-of-function mutations in LRBA”, The Journal of allergy and clinical immunology, Vol. 135 No. 1, pp. 217–227. Chen, J., Lu, Y., Xu, J., Huang, Y., Cheng, H., Hu, G., Luo, C., Lou, M., Cao, G., Xie, Y. and Ying, K. (2004), “Identification and characterization of NBEAL1, a novel human neurobeachin-like 1 protein gene from fetal

brain, which is up regulated in glioma”, Brain research. Molecular brain research, Vol. 125 No. 1-2, pp. 147– 155. Clarke, A. J. Riffelmacher, T., Braas, D., Cornall, R.J. and Simon, A.K.. (2018) "B1a B cells require autophagy for metabolic homeostasis and self-renewal", The Journal of Experimental Medicine, 215(2), pp. 399–413. Clevers, H., Alarcon, B., Wileman, T., and Terhorst, C. (1988), "The T cell receptor/ CD3 complex: A dynamic protein ensemble", Annual Reviews of Immunology, Vol. 6, pp. 629 - 662. Colledge, M. and Scott, J.D. (1999), “AKAPs: from structure to function”, Trends in Cell Biology, Vol. 9 No. 6, pp. 216–221. Cullinane, A.R., Schäffer, A.A. and Huizing, M. (2013), “The BEACH is hot: a LYST of emerging roles for BEACH-domain containing proteins in human disease”, Traffic (Copenhagen, Denmark), Vol. 14 No. 7, pp. 749–766. DeFranco, A.L. (1987) "Molecular Aspects of B-Lymphocyte Activation", Annual Review of Cell Biology, Vol. 3, pp. 143 - 178. Duan, B. and Morel, L. (2006) ‘Role of B-1a cells in autoimmunity’, Autoimmunity Reviews, Vol. 5, pp. 403–408. doi: 10.1016/j.autrev.2005.10.007. Eibel, H., Kraus H., Sic H., Kienzler A. and Rizzi, M. (2014), "B cell Biology: An Overview", Current Allergy and Asthma Reports, Vol. 14, No. 434 Eichner, E.R. (1979), "Splenic function: Normal, Too much and Too little", The American Journal of Medecine, Vol. 66, pp.311-320. Fagarasan, S. and Honjo, T. (2000), "T-Independent Immune Response: New Aspects of B Cell Biology", Science, Vol. 290, pp. 89–92. Fagarasan, S., Kinoshita, K., Muramtsu, M., Ikuta, K. and Honjo, R. (2001), "In situ class switching and differentiationto IgA producing cells in the gut lamina propria", Nature, Vol. 413, pp. 639 - 643. Fairfax, K., Kallies, A., Nutt, S.L., and Tarlinton, D.M. (2008), "Plasma cell development: From B cell subsets to long-term survival niches", Seminars in Immunology, Vol. 20, pp. 49-58. Fiocchi, C. (2015), "Inflammatory bowel disease pathogenesis: where are we?", Journal of Gastroenterology and Hepatology, Vol. 30, pp. 12-18. Fontenot, J.D., Gavin, M.A., and Rudensky, A.Y. (2003), "Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells", Nature immunology, Vol. 4, pp. 330 - 336. Fuhlbrigge, R.C., King, S.L., Sackstein, R., and Kupper, T.S. (2006), "CD43 is a ligand for E-selectin on CLA+ human T cells", Blood, Vol. 107, pp. 1421 - 1426. Gámez-Díaz, L., August, D., Stepensky, P., Revel-Vilk, S., Seidel, M.G., Noriko, M., Morio, T., Worth, Austen J J, Blessing, J., Van de Veerdonk, Frank, Feuchtinger, T., Kanariou, M., Schmitt-Graeff, A., Jung, S., Seneviratne, S., Burns, S., Belohradsky, B.H., Rezaei, N., Bakhtiar, S., Speckmann, C., Jordan, M. and Grimbacher, B. (2016), “The extended phenotype of LPS-responsive beige-like anchor protein (LRBA) deficiency”, The Journal of allergy and clinical immunology, Vol. 137 No. 1, pp. 223–230. Gámez-Díaz, L., Neumann, J., Jäger, F., Proietti, M., Felber, F, Soulas-Sprauel, P., Peruzza, L., Grassi, F., Kögl, T., Aichele, P., Kilimann, M., Grimbacher, B. and Jung, S. (2017), "The immunological phenotype of the murine Lrba knockout", Immunology and Cell biology, Vol. 95, No. 9, pp. 789-802. Gao, G.F., and Jakobsen, B.K. (2000), "Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-cell receptor", Immunology Today, Vol.21, pp. 630 - 636. Gary-Gouy, H., Bruhns, P., Schmitt, C., Dalloul, A., Daëron, M., and Bismuth, G. (2000), "The Pseudo- immunereceptor Tyrosin-based Activation Motif od CD5 Mediates its Inhibitory Action on B-cell Receptor Signaling", The Journal of Biological Chemistry, Vo. 275, pp. 548 - 556. Gebauer, D., Li, J., Jogl, G., Shen, Y., Myszka, D.G. and Tong, L. (2004), “Crystal structure of the PH-BEACH domains of human LRBA/BGL”, Biochemistry, Vol. 43 No. 47, pp. 14873–14880. Gloudemans, Anouk K., Bart N. Lambrecht, and Hermelijn H. Smits (2013), „Potential of Immunoglobulin A to Prevent Allergic Asthma“. Clinical and Developmental Immunology, Vol.2013, pp. 1–12. Gulsuner, S., Tekinay, A.B., Doerschner, K., Boyaci, H., Bilguvar, K., Unal, H., Ors, A., Onat, O.E., Atalar, E., Basak, A.N., Topaloglu, H., Kansu, T., Tan, M., Tan, U., Gunel, M. and Ozcelik, T. (2011), “Homozygosity mapping and targeted genomic sequencing reveal the gene responsible for cerebellar hypoplasia and quadrupedal locomotion in a consanguineous kindred”, Genome research, Vol. 21 No. 12, pp. 1995–2003.

Gunay-Aygun, M., Falik-Zaccai, T.C., Vilboux, T., Zivony-Elboum, Y., Gumruk, F., Cetin, M., Khayat, M., Boerkoel, C.F., Kfir, N., Huang, Y., Maynard, D., Dorward, H., Berger, K., Kleta, R., Anikster, Y., Arat, M., Freiberg, A.S., Kehrel, B.E., Jurk, K., Cruz, P., Mullikin, J.C., White, J.G., Huizing, M. and Gahl, W.A. (2011), “NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet α-granules”, Nature genetics, Vol. 43 No. 8, pp. 732–734. Henningsson, F., Ding, Z., Dahlin, J.S., Linkevicius, M., Carlsson, F., Grönvik, K.O., Hallgren, J., and Heyman, B. (2011), "IgE-Mediated Enhancement of CD4+ T Cell Response in Mice Requires Antigen Presentation by CD11c+ Cells and not by B cells", PLoS One, Vol. 6, e21760. Hogquist, K.A., Tomlinson, A.J., Kieper, W.C., McGargill, M.A., Hart, M.C., Naylor, S., and Jameson, S.C. (1997), "Identification of a naturally occuring ligand for thymic positive selection", Immunity, Vol. 6, pp. 389- 399. www.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000198589;r=4:150264531-151015727 (accessed 21 January 2018). Jogl, G., Shen, Y., Gebauer, D., Li, J., Wiegmann, K., Kashkar, H., Krönke, M. and Tong, L. (2002), “Crystal structure of the BEACH domain reveals an unusual fold and extensive association with a novel PH domain”, The EMBO journal, Vol. 21, pp. 4785–4795. Jönsson, F., Mancardi, D.A., Kita, Y., Karasuyama, H., Iannascoli, B., Van Roojien, N., Shimizo, T., Daëron, M. and Bruhns, P. (2011), "Mouse and human neutrophils induce anaphlaxis", Journal of Clinical Investigation, Vol. 121, No.4, pp. 1484-1496. Kerr, W.G., Heller, M., Herzenberger, L.A. (1996), "Analysis of Lipopolysaccharide-Response Genes in B-lineage Cells Demonstrates that they can have Differntiation Stage-restricted Expression and Contain SH2 domains", Proceedings of the Nationall Academy of Syciences USA, Vol. 93, pp. 3947 - 52. Khodadad, A., Aghamohammadi, A., Parvaneh, N., Rezaei, N., Mahjoob, F., Bashashti, M., Movahedi, M., Fazlollahi, M.R., Zandieh, F., Roohi, Z., Abdollahzade, S., Salavati, A., Kouhi, A., Talebpour, B. and Daryani, N.E. (2007), "Gastrointestinal manifestations in patients with common variable immunodeficiency", Digestive Diseases and Sciences, Vol. 52, pp. 2977-2983. Kishimoto, H., and Sprent, J. (1997), "Negative selection in the thymus includes semi-mature T cells", Journal of Experimental Medecine, Vol. 185, pp. 263-271. Kostel Bal, S., Haskologlu, S., Serwas, N.K., Islamoglu, C., Aytekin, C., Kendirli, T., Kuloglu, Z., Yavuz, G., Dalgic, B., Siklar, Z., Kansu, A., Ensari, A., Boztug, K., Dogu, F., and Ikinciogullari, A. (2017), "Multiple presentations of LRBA Deficiency: a Single-Center Experience", Journal of Clinical Immunology, Vol. 37, pp. 790 - 800. Kraljevic, K., Wong, S. and Fulcher, D. A. (2013), "Circulating phenotypic B-1 cells are decreased in common variable immunodeficiency and correlate with immunoglobulin M levels: B-1 cells in CVID", Clinical & Experimental Immunology, 171(3), pp. 278–282. Kurtenbach, S., Gießl, A., Strömberg, S., Kremers, J., Atorf, J., Rasche, S., Neuhaus, E.M., Hervé, D., Brandstätter, J.H., Asan, E., Hatt, H., and Kilimann, M.W. (2017), "The BEACH protein LRBA promotes the localozation

of the heterotrimeric G-protein Golf to olfactory cilia", Scientific Reports, Vol.7, No. 8409. LeBien, T.W., and Todder, T.F. (2008), "B lymphocytes: how they develop and function", Blood, Vol. 112, pp. 1570 - 1580. Levine, B. and Kroemer, G. (2008), “Autophagy in the pathogenesis of disease”, Cell, Vol. 132 No. 1, pp. 27–42. Lévy, E., Stolzenberg, M.-C., Bruneau, J., Breton, S., Neven, B., Sauvion, S., Zarhrate, M., Nitschké, P., Fischer, A., Magérus-Chatinet, A., Quartier, P. and Rieux-Laucat, F. (2016), “LRBA deficiency with autoimmunity and early onset chronic erosive polyarthritis”, Clinical immunology (Orlando, Fla.). Lim, M.S. and Elenitoba-Johnson, K.S.J. (2004), "The Molecular Pathology of Primary Immunodeficiencies", Journal of Molecular Diagnostics, Vol.6, pp. 59 - 83. Lo, B., Fritz, J.M., Su, H.C., Uzel, G,, Jordan, M.B., and Lenardo, M.J. (2016), "CHAI and LATAIE: new genetic diseases of CTLA-4 checkpoint insufficiency", Blood, Vol. 128, pp. 1037 - 1042. Lo, B., Zhang, K., Lu, W., Zheng, L., Zhang, Q., Kanellopoulou, C., Zhang, Y., Liu, Z., Fritz, J.M., Marsh, R., Husami, A., Kissel, D., Nortman, S., Chaturvedi, V., Haines, H., Young, L.R., Mo, J., Filipovich, A.H., Bleesing, J.J., Mustillo, P., Stephens, M., Rueda, C.M., Chougnet, C.A., Hoebe, K., McElwee, J., Hughes, J.D., Karakoc-Aydiner, E., Matthews, H.F., Price, S., Su, H.C., Rao, V.K., Lenardo, M.J. and Jordan, M.B.

(2015), “Patients with LRBA deficiency show CTAL4 loss and immune dysregulation responsive to abatacept therapy”, Science (New York, N.Y.), Vol. 349, pp. 436–440. Lopez-Herrera, G., Tampella, G., Pan-Hammarström, Q., Herholz, P., Trujillo-Vargas, C.M., Phadwal, K., Simon, A.K., Moutschen, M., Etzioni, A., Mory, A., Srugo, I., Melamed, D., Hultenby, K., Liu, C., Baronio, M., Vitali, M., Philippet, P., Dideberg, V., Aghamohammadi, A., Rezaei, N., Enright, V., Du, L., Salzer, U., Eibel, H., Pfeifer, D., Veelken, H., Stauss, H., Lougaris, V., Plebani, A., Gertz, E.M., Schäffer, A.A., Hammarström, L. and Grimbacher, B. (2012), “Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity”, American journal of human genetics, Vol. 90 No. 6, pp. 986–1001. Malamut, G., Verkarre, V., Suarez, F., Viallard, J.F., Lascaux, A.S., Cosnes, J., Bouhnik, Y., Lambotte, O., Béchade, D., Ziol, M., Lavergne, A., Hermine, O., Cerf-Bensussan, N., and Cellier, C. (2010), "The enteropathy associated with common variable immunodeficiency: the delineated frontiers with celiac disease", American Journal of Gastroenterology, Vol. 105, pp. 2262-2275. Manjarrez-Orduño, N., Quách, T.D., and Sanz, I. (2009), "B cells and immunological tolerance", Journal of Investigative Dermatology, Vol.129, pp.278 - 288. Melchers, F. (2005), "The pre-B-cell receptor: selector of fitting immunoglobulin heavy chains for the B-cell repertoire", Nature Reviews Immunology, Vol. 5, pp. 578 - 584. Miles, K., Simpson, J., Brown, S., Cowan, G., Gray, D. and Gray, M. (2018) "Immune Tolerance to Apoptotic Self Is Mediated Primarily by Regulatory B1a Cells", Frontiers in Immunology, 8. Montfort, A., Badts, B. de, Douin-Echinard, V., Martin, Pascal G P, Iacovoni, J., Nevoit, C., Therville, N., Garcia, V., Bertrand, M.-A., Bessières, M.-H., Trombe, M.-C., Levade, T., Benoist, H. and Ségui, B. (2009), “FAN stimulates TNF(alpha)-induced gene expression, leukocyte recruitment, and humoral response”, Journal of immunology (Baltimore, Md. 1950), Vol. 183 No. 8, pp. 5369–5378. Nguyen, D., and Xu, T. (2008), "The expending role of mouse genetics for understanding human biology and diesease", Diesease Models & Mechanisms, Vol. 1, pp. 56 - 66. Niss Arfelt, K., Barington, L., Benned-Jensen, T., Kubale, V., Kovalchuck, A.L., Daugvilaite, V., Pravsgaard Christensen, J., Randrup Thomsen, A., Egerod, K.L., Bassi, M.R., Spiess, K., Schwartz, T.W., Wang, H., Morse III, H.C., Holst, P.J.and Rosenkilde, M.M. (2017) "EBI2 overexpression in mice leads to B1 B-cell expansion and chronic lymphocytic leukemia–like B-cell malignancies", Blood, 129(7), pp. 866–878. Neer, E.J., Schmidt, C.J., Nambudripad, R. and Smith, T.F. (1994), “The ancient regulatory-protein family of WD- repeat proteins”, Nature, Vol. 371, pp. 297–300. Newlon, M.G., Roy, M., Morikis, D., Hausken, Z.E., Coghlan, V., Scott, J.D. and Jennings, P.A. (1999), “The molecular basis for protein kinase A anchoring revealed by solution NMR”, Nature Structural Biology, Vol. 6, pp. 222–227. Obukhanych, T. and Nussenzweig M. (2006), "T-independent Type II immune responses generate memory B- cells", The Journal of Experimental Medecine, Vol. 203, No. 2, pp.305-310. O'Connell, F.P., Pinkus, J.L., and Pinkus, G.S. (2004), "CD138 (Syndecan-1), a Plasma Cell Marker. Immunohistochemical Profile in Hematopoietic and Non-hematopoietic Neoplasms", American Journal of Clinical Pathology, Vol. 121, pp. 254 - 263. Oliveira, J.B., Bleesing, J.J., Dianzani, U., Fleisher, T.A., Jaffe, E.S., Lenardo, M.J., Rieux-Laucat, F., Siegel, R.M., Su, H.C., Teachey, D.T. and Rao, V.K. (2010), “Revised diagnostic criteria and classification for the autoimmune lymphoproliferative syndrome (ALPS): report from the 2009 NIH International Workshop”, Blood, Vol. 116 No. 14, pp. e35-40. Park, J.H., Resnick, E.S. and Cunningham-Rundles, C. (2011), “Perspectives on common variable immune deficiency”, Annals of the New York Academy of Sciences, Vol. 1246, pp. 41–49. Park, M.A., Li, J.T., Hagan, J.B., Maddox, D.E. and Abraham, R.S. (2008), “Common variable immunodeficiency: a new look at an old disease”, The Lancet, Vol. 372 No. 9637, pp. 489–502. Parks, M.Y., Sudan, R., Srivastava, N., Neelam, S., Youngs, C., Wang, J.W., Engelman, R.W. and Gerr, W.G. (2016), "LRBA is essential for Allogenic Responses in Bone Marrow Transplantation", Scientific Reports, Vol. 6, No.36568. Paterson, A.M., Lovitch, S.B., Sage, P.T., Juneja, V.R., Lee, Y., Trombley, J.D., Arancibia-Cárcamo, C.V., Sobel, R.A., Rudensky, A.Y., Kuchroo, V.K., Freeman, G.J. and Sharpe, A.H. (2015), "Deletion of CTLA-4 on regulatory T cells during adulthood leads to resistance to autoimmunity", Journal of Experimental Medecine, Vol.212, No.10, pp. 1603 - 1621.

Picard, C., Al-Herz, W., Bousfiha, A., Casanova, J.L., Chatila, L., Conley, M.E., Cunningham-Rundles, C., Etzioni, A., Holland, S.M., Klein, C., Nonoyama, S., Ochs, H.D., Oksenhendler, E., Puck, J.M., Sullivan, K.E., Tang, M.L., Franco, J.L., and Gaspar, H.P. (2015), "Primary Immunodeficiency diseases: an Update on the Classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015.", Journal of Clinical Immunology, Vol. 35, pp. 696 - 726. Pieper, K., Grimbacher, B., and Eibel, H. (2013), "B-cell biology and development", Journal of Allergy and Clinical Immunology, Vol. 131, pp. 959 - 971. Pengo, N., Scolari, M., Oliva, L., Milan, E., Mainoldi, F., Raimondi, A., Fagioli, C., Merlini, A., Mariani, E., Pasqualetto, E., Orfanelli, U., Ponzoni, M., Sitia, R., Casola, S. and Cenci, S. (2013), “Plasma cells require autophagy for sustainable immunoglobulin production”, Nature immunology, Vol. 14 No. 3, pp. 298–305. Perou, C.M., Moore, K.J., Nagle, D.L., Misumi, D.J., Woolf, E.A., McGrail, S.H., Holmgren, L., Brody, T.H., Dussault Jr., Barry J., Monroe, C.A., Duyk, G.M., Pryor, R.J., Li, L., Justice, M.J. and Kaplan, J. (1996), “Identification of the murine beige gene by YAC complementation and positional cloning”, Nature genetics, Vol. 13, pp. 303–308. Prieto, J.M.B., and Felippe, M.J.B (2017), "Development, phenotype and function of non-conventional B cells", Comparative Immunology, Microbiology and Infectious Diseases, Vol. 54, pp. 38 - 44. Qureshi, O.S., Zheng, Y., Nakamura, K., Attridge, K., Manzotti, C., Schmidt, E.M., Baker, J., Jeffrey, L.E., Kaur, S., Briggs, Z., Hou, T.Z., Futter, C.E., Anderson, G., Walker, L.S.K., and Sansom, D.M. (2011), "Trans- Endocytosis of CD80 and CD86: A Molecular Basis for the Cell-extrinsic Function of CTLA-4", Scienece, Vol. 332, pp. 600-603. Rebecchi, M.J. and Scarlata S. (1998), “PLECKSTRIN HOMOLOGY DOMAINS: A Common Fold with Diverse Functions”, Annual Reviews of Biophysics and Biomolecular Structure, No. 27, pp. 503–528. Revel-Vilk, S., Fischer, U., Keller, B., Nabhani, S., Gámez-Díaz, L., Rensing-Ehl, A., Gombert, M., Hönscheid, A., Saleh, H., Shaag, A., Borkhardt, A., Grimbacher, B., Warnatz, K., Elpeleg, O. and Stepensky, P. (2015), “Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation”, Clinical immunology (Orlando, Fla.), Vol. 159 No. 1, pp. 84–92. Roder, J.C. (1979) "The beige mutation in the mouse. I. A stem cell predetermined Impairment in Natural Killer cell function", The Journal of Immunology, Vol.123, pp.2168 - 2173. Rodig, S.J., Shahsafaei, A., Li, B., and Dorfman, D.M. (2005), "The CD45 isoform B220 identifies select subsets of human B cells and B-cell lymphoproliferative disorders", Human pathology, Vol. 36, pp. 51 - 57. Salzer, U., Warnatz, K., and Peter, H.H. (2012), "Common variable immunodeficiency - an update", Arthritis research & therapy, Vol. 14, p. 223 Samitas, K., Lötvall, J., and Bossois, A. (2010), "B cells: From early development to regulating allergic diseases", Archivum Immunulogiae et Therapiae Experimentalis, Vol. 58, pp. 209 - 225. Sari, S., Dogu, F., Hwa, V., Haskologlu, S., Dauber, A., Rosenfeld, R., Polat, M., Kuloglu, Z., Kansu, A., Dalgic, B. and Ikinciogullari, A. (2016), “A Successful HSCT in a Girl with Novel LRBA Mutation with Refractory Celiac Disease”, Journal of Clinical Immunology, Vol. 36 No. 1, pp. 8–11. Sarvas, H.O., Seppälä, I.J.T., Tähtinen, T., Péterfy, F. and Mäkelä, O. (1983), "Mouse IgG Antibodies have Subclass associated Affinity Differences", Molecular Immuology, Vol. 20, No.3, pp.239-246. Schreiner, F., Plamper, M., Dueker, G., Schoenberger, S., Gámez-Díaz, L., Grimbacher, B., Hilger, A.C., Gohlke, B., Reutter, H. and Woelfle, J. (2016), “Infancy-Onset T1DM, Short Stature, and Severe Immunodysregulation in Two Siblings With a Homozygous LRBA Mutation”, The Journal of clinical endocrinology and metabolism, Vol. 101 No. 3, pp. 898–904. Schubert, D., Bode, C., Kenefeck, R., Hou, T.Z., Wing, J.B., Kennedy, A., Bulashevska, A., Petersen, B.-S., Schäffer, A.A., Grüning, B.A., Unger, S., Frede, N., Baumann, U., Witte, T., Schmidt, R.E., Dueckers, G., Niehues, T., Seneviratne, S., Kanariou, M., Speckmann, C., Ehl, S., Rensing-Ehl, A., Warnatz, K., Rakhmanov, M., Thimme, R., Hasselblatt, P., Emmerich, F., Cathomen, T., Backofen, R., Fisch, P., Seidl, M., May, A., Schmitt-Graeff, A., Ikemizu, S., Salzer, U., Franke, A., Sakaguchi, S., Walker, Lucy S K, Sansom, D.M. and Grimbacher, B. (2014), “Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations”, Nature medicine, Vol. 20 No. 12, pp. 1410–1416. Ségui, B., Cuvillier, O., Adam-Klages, S., Garcia, V., Malagarie-Cazenave, S., Lévêque, S., Caspar-Bauguil, S., Coudert, J., Salvayre, R., Krönke, M. and Levade, T. (2001), “Involvement of FAN in TNF-induced apoptosis”, The Journal of clinical investigation, Vol. 108 No. 1, pp. 143–151.

Seidel, M.G. (2014), “Autoimmune and other cytopenias in primary immunodeficiencies: pathomechanisms, novel differential diagnoses, and treatment”, Blood, Vol. 124, pp. 2337–2344. Seidel, M.G., Hirschmugl, T., Gámez-Díaz, L., Schwinger, W., Serwas, N.K., Deutschmann, A., Gorkiewicz, G., Zenz, W., Windpassinger, C., Grimbacher, B., Urban, C. and Boztug, K. (2014), “Long-term remission after allogenic hematopoietic stem cell transplantation in LPS-responsive beige-like anchor (LRBA) deficiency”, Journal of Allergy and Clinical Immunology. Serwas, N.K., Kansu, A., Santos-Valente, E., Kuloğlu, Z., Demir, A., Yaman, A., Gámez-Díaz, Laura Yaneth, Artan, R., Sayar, E., Ensari, A., Grimbacher, B. and Boztug, K. (2015), “Atypical manifestation of LRBA deficiency with predominant IBD-like phenotype”, Inflammatory bowel diseases, Vol. 21 No. 1, pp. 40–47. Shields, A. and Patel, S. (2017) "Defence against infection: The primary immunodeficiency disorders", Medecine, Vol. 45, No. 10, pp. 597-604. Shimomura, Y., Mizoguchi E., Sugimoto, K., Kibe, R, Benno, Y., Mizoguchi, A. and Bhan, A.K. (2008) "Regulatory role of B-1 B cells in chronic colitis", International Immunology, 20(6), pp. 729–737. Shokri, S., Nabavi, M., Hirschmugl, T., Aghamohammadi, A, Arshi, S., Bemanian, M.H., Fallahpour, M, Molatefi, R., Rekabi, M., Eslami, N., Ahmadian, J., Darabi, K., Sedighi, G.R., Monajemzadeh, M., Parvaneh, N, Boztug, K., and Rezaei, N. (2016), "LPS-Responsive Beige-like Anchor Gene Mutation Assiciated With Possible Bronchiolitis Obliterans Organizing Pneumonia Associated With Hypogammaglobulinemia and Normal IgM Phenotype and Low Number of B Cells", Acta Medica Iranica, Vol. 54, pp. 620 - 623. Simonsen, A., Birkeland, Hanne C G, Gillooly, D.J., Mizushima, N., Kuma, A., Yoshimori, T., Slagsvold, T., Brech, A. and Stenmark, H. (2004), “Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes”, Journal of cell science, Vol. 117 No. Pt 18, pp. 4239–4251. Smith, T.F., Gaitatzes, C., Saxena, K. and Neer, E.J. (1999), “The WD repeat: a common architecture for diverse functions”, Trends in Biochemical Sciences, Vol. 24 No. 5, pp. 181–185. Soldevila, G, Raman, C., and Lozano, F. (2011), "The immunomodulatory properties of CD5 lymphocyte receptor in health and disease", Current Opinion in Immunology, Vol. 23, pp. 310 - 318. Souza, N. de, Vallier, L.G., Fares, H. and Greenwald, I. (2007), “SEL-2, the C. elegans neurobeachin/LRBA homolog, is a negative regulator of lin-12/Notch activity and affects endosomal traffic in polarized epithelial cells”, Development (Cambridge, England), Vol. 134 No. 4, pp. 691–702. Spritz, R.A. (1998), “Genetic defects in Chediak-Higashi syndrome and the beige mouse”, Journal of Clinical Immunology, Vol. 18, pp. 97–105. Stavnezer, J. (1996), "Immunoglobulin class switching", Current opinion in immunology, Vol.8, pp. 199-205. Su, Y., Balice-Gordon, R.J., Hess, D.M., Landsman, D.S., Minarcik, J., Golden, J., Hurwitz, I., Liebhaber, S.A. and Cooke, N.E. (2004), “Neurobeachin is essential for neuromuscular synaptic transmission”, The Journal of neuroscience the official journal of the Society for Neuroscience, Vol. 24 No. 14, pp. 3627–3636. Tarlinton, D. (2006), "B-cell memory: are subsets necessary?", Nature Reviews Immunology, Vol. 6, pp. 785 - 790. Tchernev, V.T., Mansfield, T.A., Giot, L., Kumar, A.L., Nandabalan, K., Li, Y., Mishra, V.S., Detter, J.C., Rothberg, J.M., Wallace, M.R., Southwick, F.S., and Kingsmore, S.F. (2002), "The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins.", Molecular Medicine, Vol. 8, pp. 56 - 64. Tegtmeyer, D., Seidl, M., Gerner, P., Baumann, U. and Klemann, C. (2017), "Inflammatory bowel disease caused by primary immunodeficiencies - Clinical presentations, review of literature, and proposal of a rational diagnostic algorithm", Pediatric Allergy and Immunology, Vol. 28, pp. 412-429. Tesi, B., Priftakis, P., Lindgren, F., Chiang, Samuel C C, Kartalis, N., Löfstedt, A., Lörinc, E., Henter, J.-I., Winiarski, J., Bryceson, Y.T. and Meeths, M. (2016), “Successful Hematopoietic Stem Cell Transplantation in a Patient with LPS-Responsive Beige-Like Anchor (LRBA) Gene Mutation”, Journal of Clinical Immunology, Vol. 36, No. 5, pp. 480-489. Trowbridge, I. S. (1994), "CD45: An Emerging Role as a Protein Tyrosin Phosphatase Required for Lymphocyte Activation and Development", Annual Review of Immunology, Vol. 12, pp. 85 - 116. Uhlig, H.H., Schwerd, T., Koletzko, S., Shah, N., Kammermeier, J., Elkardi, A., Ouahed, J., Wilson, D.C., Travis, S.P., Turner, D., Klein, C., Snapper, S.B. and Muise, A.M. (2014) "The diagnostic approach to monogenic very early onset inflammatory bowel disease", Gastroenterology, Vol. 147, pp. 990-1007. Vaerman, J. P., André, C. Bazin and H. Heremans, J.F. (1973) ‘Mesenteric lymph as a major source of serum IgA in guinea pigs and rats’, European Journal of Immunology, Vol. 3, pp. 580–584.

Vidarsson, G., Dekkers, G. and Rispens, T. (2014), "IgG subclasses and allotypes: from structure to effector functions", Frontiers in immunology, Vol. 5 No. 520. Vogl, C., Butola, T., Haag, N., Hausrat, T.J., Leitner, M.G., Moutschen, M., Lefèbvre, P.P., Speckmann, C., Garrett, L., Becker, L., Fuchs, H., Hrabe de Angelis, H., Nietzsche, S., Kessels, M.M., Oliver, D., Kneussel, M., Kilimann, M.W., and Strenzke, N. (2017) "The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing", EMBO reports, Vol. 18, pp. 2015 - 2029. Wang, J., Anders, R.A., Wu, Q., Peng, D., Cho, J.H., Sun, Y., Karaliukas, R., Kang, H.S., Turner, J.R. and Fu, Y.X. (2004), "Dysregulated LIGHT expression on T cells mediates intestinal inflammation and contributes to IgA nephropathy", Journal of Clinical Investigation, Vol.113, pp. 826 - 835. Wang, J.-W., Gamsby, J.J., Highfill, S.L., Mora, L.B., Bloom, G.C., Yeatman, T.J., Pan, T.-c., Ramne, A.L., Chodosh, L.A., Cress, W.D., Chen, J. and Kerr, W.G. (2004), “Deregulated expression of LRBA facilitates cancer cell growth”, Oncogene, Vol. 23 No. 23, pp. 4089–4097. Wang, J.-W., Howson, J., Haller, E. and Kerr, W.G. (2001), “Identification of a Novel Lipopolysaccharide- Inducible Gene with Key Features of Both a Kinase Anchor Proteins and chs1/beige Proteins”, The Journal of Immunology, Vol. 166 No. 7, pp. 4586–4595. Wang, X., Herberg, F.W., Laue, M.M., Wüllner, C., Hu, B., Petrasch-Parwez, E. and Kilimann, M.W. (2000), “Neurobeachin: A Protein Kinase A-Anchoring, beige/Chediak-Higashi Protein Homolog Implicated in Neuronal Membrane Traffic”, The Journal of Neuroscience, Vol. 20, pp. 8551–8565. Waterhouse, P., Penninger, J.M., Timms, E., Wakeham, A., Shahinian, A., Lee, K.P., Thompson, C.B., Griesser, H., Mak, T.W. (1995), "Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4", Science, Vol. 270, pp.985-988. www.hos.ufl.edu/meteng/HansonWebpagecontents/Proteinprotocols.html (accessed 03.01.2018) Yang, W., Shen, N., Ye, D.-Q., Liu, Q., Zhang, Y., Qian, X.-X., Hirankarn, N., Ying, D., Pan, H.-F., Mok, C.C., Chan, T.M., Wong, Raymond Woon Sing, Lee, K.W., Mok, M.Y., Wong, S.N., Leung, Alexander Moon Ho, Li, X.-P., Avihingsanon, Y., Wong, C.-M., Lee, T.L., Ho, Marco Hok Kung, Lee, Pamela Pui Wah, Chang, Y.K., Li, P.H., Li, R.-J., Zhang, L., Wong, Wilfred Hing Sang, Ng, Irene Oi Lin, Lau, C.S., Sham, P.C. and Lau, Y.L. (2010), “Genome-wide association study in Asian populations identifies variants in ETS1 and WDFY4 associated with systemic lupus erythematosus”, PLoS genetics, Vol. 6 No. 2, pp. e1000841. Yang, Y.-Q., Yang, W., Yao, Y., Ma, H., Wang, Y., Li, L., Wu, Q., Gershwin, M.E. and Lian, Z. (2016) “Dysregulation of peritoneal cavity B1a cells and murine primary biliary cholangitis”, Oncotarget, 7(19). Zeitlman, L., Sirim, P., Kremmer, E., and Kolanus, W. (2001), "Cloning of APC33 as a novel intracellular ligand of CD4", Journal of Biological Chemistry, Vol. 276, pp. 9123 - 9132.

List of publications Gámez-Díaz, L., Neumann, J., Jäger, F., Proietti, M., Felber, F., Soulas-Sprauel, P., Perruzza, L. Grassi, F., Kögl, T., Aichele, P., Kilimann, M., Grimbacher, B., and Jung, S. (2017), "The immunological phenotype of the murine Lrba knockout", Immunology and Cell biology, Vol. 95, No. 9, pp. 789-802.

Eidesstattliche Versicherung Gemäß § 8 Absatz 1 Nr. 3 der Promotionsordnung der Universität Freiburg für die Medizinische Fakultät

Bei der eingereichten Dissertation zu dem Thema „Immunological phenotype of LRBA-deficient mice“ handelt es sich um meine eigenständig erbrachte Leistung.

Ich habe nur die angegebenen Quellen und Hilfsmittel benutzt und mich keiner unzulässigen Hilfe Dritter bedient. Insbesondere habe ich wörtlich oder sinngemäß aus anderen Werken übernommene Inhalte als solche kenntlich gemacht. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.

Die Ordnung der Albert-Ludwigs-Universität zur Sicherung der Redlichkeit der Wissenschaft habe ich zur Kenntnis genommen und akzeptiert.

Die Dissertation oder Teile davon habe ich bislang nicht an einer Hochschule des In- oder Auslands als Bestandteil eine Prüfungs- oder Qualifikationsleistung vorgelegt.

Die Richtigkeit der vorstehenden Erklärungen bestätige ich.

Die Bedeutung der eidesstattlichen Versicherung und die strafrechtlichen Folgen einer unrichtigen oder unvollständigen eidesstattlichen Versicherung sind mit bekannt.

Ich versichere an Eides statt, dass ich nach bestem Wissen die reine Wahrheit erklärt und nichts verschwiegen habe.

______Ort und Datum Unterschrift

Erklärung zum Eigenanteil/ Declaration of own contribution Thesis: Immunological phenotype of LRBA-deficient mice

Involved persons:

CCI, University of Freiburg: Prof. Dr. Bodo Grimbacher, Dr. Laura Gámez-Díaz, Dr. Sophie Jung, Fiona Jäger

1. Supervision: Prof. Dr. Bodo Grimbacher, Dr. Laura Gámez-Díaz 2. Study design: Prof. Dr. Bodo Grimbacher, Dr. Laura Gámez-Díaz 3. Tierversuchsantrag: Prof. Dr. Bodo Grimbacher, Dr. Laura Gámez-Díaz 4. Mouse genotyping: Fiona Jäger 5. Sequencing: a. Sample acquisition and preparation: Fiona Jäger b. Sequencing: company GATC 6. LRBA expression by Western Blot: Fiona Jäger 7. Body and spleen weight analysis a. Weighing: Dr. Laura Gámez-Díaz, Fiona Jäger b. Statistical analysis: Fiona Jäger 8. B- and T-cell phenotyping by FACS: a. First round of experiments including 18 mice: Fiona Jäger, Dr. Laura Gámez-Díaz b. second round of experiments including 15 mice: Dr. Laura Gámez-Díaz, Dr. Sophie Jung, Julika Neumann c. Overall statistical analysis: Fiona Jäger 9. Antibody titer analysis by ELISA: a. ELISA of first 18 mice: Dr. Laura Gámez-Díaz, Fiona Jäger b. statistical analysis: Fiona Jäger 10. Histological analysis of ileum and colon: a. Sample acquisition and paraffin embedding: Dr. Sophie Jung, Dr. Laura Gámez-Díaz, Julika Neumann b. Hematoxilin/Eosin staining: Institute of pathology, University Freiburg c. Cell counting: Fiona Jäger d. Overall sample interpretation: Dr. Max Seidl, Fiona Jäger e. Statistical analysis: Fiona Jäger 11. Literature research: Fiona Jäger 12. Continuation of the study: Dr. Laura Gámez-Díaz, Dr. Sophie Jung

Publication: Immunological phenotype of LRBA-deficient mice

Involved persons:

a) CCI, University of Freiburg: Prof. Dr. Bodo Grimbacher, Dr. Laura Gámez-Díaz,Dr. Sophie Jung, Dr. Michele Proietti, Julika Neumann, Fiona Jäger, Felicitas Felber b) Institute of Research in Biomedicine, Università della Svizzera Italiana, Bellinzona: Lisa Peruzza, Fabio Grassi c) Institute of Molecular and Cellular Biology, Strasbourg: Pauline Soulas-Sprauel d) Deprartment of Immunology, Institute for Medical Microbiology and Hygiene, University of Freiburg: Tamara Kögl, Peter Aichele e) Department of Molecular Neurobiology, Max-Planck-Institute for Experimental Medecine, Göttingen: Prof. Dr. Manfred Kilimann 1. Supervision: Dr. Sophie Jung, Prof. Dr. Bodo Grimbacher, Dr. Laura Gámez-Díaz 2. Study design: Prof. Dr. Bodo Grimbacher, Dr. Laura Gámez-Díaz 3. Knockout mice generation: Prof. Dr. Manfred Kilimann 4. Tierversuchsantrag: Prof. Dr. Bodo Grimbacher, Dr. Laura Gámez-Díaz 5. Establishing of the mouse immunization protocol: Dr. Laura Gámez-Díaz, Fiona Jäger 6. B- and T-cell phenotyping by FACS: Dr. Sophie Jung, Dr. Laura Gámez-Díaz, Julika Neumann, Felicitas Felber, Fiona Jäger 7. B cell in vitro proliferation, survival and class switching: Dr. Sophie Jung, Dr. Laura Gámez- Díaz, Julika Neumann 8. Antibody titer analysis in serum and intestine, including autoantibodies by ELISA: Dr. Sophie Jung, Dr. Laura Gámez-Díaz, Julika Neumann, Dr. Pauline Soulas-Sprauel 9. LCMV infection model: Dr. Tamara Kögl, Prof. Dr. Peter Aichele 10. S. typhimurium colitis model: Dr. Sophie Jung, Dr. Laura Gámez-Díaz, Dr. Michele Poietti, Dr. Lisa Peruzza, Prof. Dr. Fabio Grassi 11. Histological analysis of ileum and colon: a. Sample acquisition and paraffin embedding: Dr. Sophie Jung, Dr. Laura Gámez-Díaz, Julika Neumann b. Slide staining: Institute of pathology, University Freiburg c. Cell counting: Fiona Jäger d. Overall sample interpretation: Dr. Max Seidl, Fiona Jäger e. Statistical analysis: Dr. Sophie Jung, Dr. Laura Gámez-Díaz 12. Writing: Dr. Laura Gámez-Díaz, Dr. Sophie Jung

Acknowledgements First of all, I would like to thank the entire team of AG Grimbacher for the continuous support and motivation. Their team spirit and expertise made my time and work with the group interesting, challenging and fun.

Special thanks goes to my supervisor Prof. Bodo Grimbacher for enabling me to conduct this interesting work in his laboratory. Furthermore, I want to thank Dr. Sophie Jung for the cooperation on this project. I am especially grateful for the mentoring and supervision by Dr. Laura Gámez-Díaz who provided me with excellent support along the entire way.