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Novel mechanisms involving B cell receptor (BCR) and B cell activating factor (BAFF) signaling pathways… Rozmus, Jacob 2016

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NOVEL MECHANISMS INVOLVING B CELL RECEPTOR (BCR) AND B CELL ACTIVATING FACTOR (BAFF) SIGNALING PATHWAYS UNDERLYING HUMAN PRIMARY IMMUNODEFICIENCIES AND MALIGNANCY  by  JACOB ROZMUS  B. Sc., The University of Alberta, 2001 M.D., The University of Alberta, 2005   A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY   in   THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Pathology & Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)   June 2016               © Jacob Rozmus 2016 	   ii	  Abstract The proper differentiation and survival of human peripheral immature B cells relies on two critical signaling pathways: B cell receptor (BCR) signaling and the B cell activating factor (BAFF)/BAFF-receptor (BAFF-R) signaling axis. The quality of the BCR signal is regulated in a developmental manner. Self-reactive early immature B cells are eliminated in response to strong BCR-induced signals, while late immature B cells require BCR-induced signals for survival and further differentiation. Although components and events downstream of the BCR are well known, the mechanisms of BCR signaling and its role in the regulation of BAFF signaling are still poorly understood.  Through the use of transgenic and knockout murine models, the effects of BAFF on murine B cell maturation and survival are well characterized. There is a crucial need to better understand the functions of BAFF in humans. High levels of soluble BAFF, reduced expression of BAFF-R and BCR signaling abnormalities in B cells have been identified in a large group of clinically heterogeneous diseases including autoimmune and inflammatory conditions, allergy, viral infections and lymphoid cancers. In order to better understand BCR signaling mechanisms, functional properties of human BAFF and factors regulating BAFF-R expression, this thesis describes: 1) the phenotypic, molecular and functional characterization of rare unknown inherited monogenic immunodeficiencies involving defects in early B cell development and the BAFF/BAFF-R pathway and, 2) the functional characterization of dysregulated BAFF/BAFF-R signaling in B cell malignancy.  This led to the discovery of two novel primary immunodeficiencies involving MALT1 deficiency and gain-of-function PLCγ2 mutation. Our results indicate that MALT1 is essential for antigen-receptor mediated NF-κB activation and plays a role in the surface expression of BAFF-R and proper development of human B cells. The PLCγ2 mutation led to 	   iii	  hyper-reactive BCR signaling and increased apoptosis of transitional B cells. Work-up of this patient also allowed us to investigate how soluble BAFF down-modulates surface expression of it’s principal receptor, BAFF-R, through receptor internalization, in normal B cells. Further analysis of the BAFF/BAFF-R pathway in pre-B acute lymphoblastic leukemia provides evidence of different structural and functional BAFF isoforms.                    	   iv	  Preface  Chapter 3: Versions of this material have been published or submitted as: 1. *McKinnon ML, *Rozmus J, *Fung SY, Hirschfeld AF, Del Bel KL, Thomas L, Marr N, Martin SD, Marwaha AK, Priatel JJ, Tan R, Senger C, Tsang A, Prendiville J, Junker AK, Seear M, Schultz KR, Sly LM, Holt RA, Patel MS, Friedman JM, Turvey SE. 2014. Combined immunodeficiency associated with homozygous MALT1 mutations. J Allergy Clin Immunol;133(5):1458-62. * co-first authors 2. Turvey SE, Durandy A, Fischer A, Fung SY, Geha RS, Gewies A, Giese T, Greil J, Keller B, McKinnon ML, Neven B, Rozmus J, Ruland J, Snow AL, Stepensky P, Warnatz K. 2014. The CARD11-BCL10-MALT1 (CBM) signalosome complex: Stepping into the limelight of human primary immunodeficiency; J Allergy Clin Immunol;134(2):276-84 3. Rozmus J, McDonald R, Fung SY, Del Bel KL, Roden J, Senger C, Schultz KR, McKinnon ML, Davis J. 2016. Successful clinical treatment and functional immunological normalization of human MALT1 deficiency following hematopoietic stem cell transplantation; Clin Immunol;168:1-5 Dr. Shane Fung provided data for Figures 3.8, 3.9, 3.10 and 3.14. I performed all additional experiments. Work done on human samples reported in this chapter was covered by the Child & Family Research Institute Research Ethics Board Certificate number H09-01228. Chapter 4: This chapter is original, independent work by the author, Jacob Rozmus, except for whole exome sequencing and preliminary bioinformatics analyses, which were performed at the Michael Smith Genome Sciences Centre. Work done on human samples reported in this chapter 	   v	  was covered by the Child & Family Research Institute Research Ethics Board Certificate number H10-01954.  Chapter 5: This chapter is original, unpublished, independent work by the author, Jacob Rozmus. Work done on human samples reported in this chapter is covered by the Child & Family Research Institute Research Ethics Board Certificate numbers H10-01954, H09-01141 and H09-01228. Chapter 6: Data related to the phase III study ASCT0431 were provided by the Children’s Oncology Group. Jacob Rozmus performed all additional experiments. Work done on human samples reported in this chapter is covered by the Child & Family Research Institute Research Ethics Board Certificate numbers H10-01954, H09-01141 and H09-01228.             	   vi	  Table of Contents  Abstract ............................................................................................................................................ ii Preface ............................................................................................................................................ iv Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................... x List of Figures ................................................................................................................................. xi List of Abbreviations .................................................................................................................... xiii Acknowledgements ...................................................................................................................... xvi  Introduction ..................................................................................................................................... 1 1. Background .................................................................................................................................. 6 1.1. Overview .............................................................................................................................. 6 1.2. Early B Cell Development .................................................................................................... 6 1.3. V(D)J Recombination ........................................................................................................... 8 1.4. Immune Tolerance ................................................................................................................ 9 1.4.1. B Cell Self-Tolerance .................................................................................................. 11 1.4.2. Central B Cell Tolerance ............................................................................................. 11 1.4.3. Peripheral B Cell Tolerance ........................................................................................ 12 1.5. Differentiation into Mature B Cell Subsets ........................................................................ 12 1.5.1. B-1 Cells ...................................................................................................................... 12 1.5.2. B-2 Cell Subsets .......................................................................................................... 13 1.5.2.1. Follicular (FO) B Cells ......................................................................................... 13 1.5.2.2. Marginal Zone (MZ) B Cells ................................................................................ 15 1.5.3. Regulatory (B10) Cells ................................................................................................ 15 1.6. The Functional Diversity of B Cells ................................................................................... 16 1.6.1. Antibody Production ................................................................................................... 16 1.6.1.1. Neutralization ....................................................................................................... 16 1.6.1.2. Complement Activation ........................................................................................ 17 1.6.1.3. Binding to Fc Receptors ....................................................................................... 17 1.6.2. Antigen Presentation ................................................................................................... 19 1.6.3. Cytokine Production .................................................................................................... 20 1.7. B Cell Disorders in Human Disease ................................................................................... 20 1.8. The Role of B Cell Receptor Signaling .............................................................................. 23 1.9. PLCγ2 is Critical for BCR Signal Transduction  ............................................................... 24 1.9.1. PLCγ2 Deficient Mice ................................................................................................. 26 1.9.2. Murine Models of Gain-of-Function Mutations in PLCγ2 .......................................... 27 1.9.3. Human Disease Models Associated with Mutations in PLCγ2 ................................... 28 1.9.3.1. PLCγ2-Associated Antibody Deficiency and Immune Dysregulation (PLAID)/Familial Cold Autoinflammatory Syndrome-3 (FCAS3) ...................................... 28 1.9.3.2. Autoinflammation and PLCγ2-Associated Antibody Deficiency and Immune Dysregulation (APLAID) .............................................................................................................. 28 1.10. The Role of MALT1 in BCR Signaling ........................................................................... 29 1.11. B Cell Activating Factor (BAFF) ..................................................................................... 31 1.11.1. Crucial Role for BAFF/BAFF-R Pathway in the Maintenance of Mature B Cells ... 34 1.11.2. Role of TACI ............................................................................................................. 37 1.11.3. Role of BCMA .......................................................................................................... 39 	   vii	  1.11.4. The Functional Complexity of BAFF ........................................................................ 40 1.11.4.1. Structure of Human BAFF ..................................................................................... 40 1.11.4.2. Structural and Functional Isoforms of BAFF ......................................................... 41 1.11.4.3. Cellular Origin of BAFF ........................................................................................ 48 1.11.4.4. Transcriptional Regulation of BAFF ...................................................................... 48 1.11.4.5. Cytokine Regulation of BAFF Production ............................................................. 49 1.11.6. The Role of Elevated BAFF in Disease .................................................................... 50 1.12. Primary Hypothesis and Aims .......................................................................................... 53  2. Materials and Methods  ............................................................................................................. 54 2.1. Patient Samples .................................................................................................................. 54 2.2. Cell Lines and Reagents ..................................................................................................... 55 2.3. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) .............................................. 56 2.4. Isolation of CD19+ B Cells ................................................................................................ 57 2.5. Isolation of Neutrophils ...................................................................................................... 57 2.6. Cell Sorting of Cord Blood ................................................................................................. 58 2.7. Exome Library Construction and Sequencing of MALT1 Deficient Patient  .................... 58 2.8. Bioinformatic Analysis and Identification of the MALT1 Variant  ................................... 59 2.9. Quantification of MALT1 Gene and Protein Expression  .................................................. 59 2.10. Analysis of MALT1 Paracaspase Activity and Molecular Scaffold Function  ................ 60 2.11. Immunologic Phenotyping and Analysis of NF-κB Activation by Flow Cytometry  ...... 61 2.12. Cloning and Transfection Studies .................................................................................... 63 2.13. Whole Exome Sequencing of Patient With PLCγ2 Mutation  ......................................... 64 2.14. B Cell Immunophenotyping ............................................................................................. 64 2.15. Quantification of BAFF and BAFF-R Levels in Plasma and Media  ............................... 65 2.16. Calcium Flux Assay ......................................................................................................... 66 2.17. Intracellular ERK Phosphorylation .................................................................................. 66 2.18. Collagen Stimulation of Platelets ..................................................................................... 67 2.19. RNA Extraction and cDNA Synthesis ............................................................................. 67 2.20. Quantitative Real-Time PCR  ........................................................................................... 67 2.21. Cell Lysis, Immunoprecipitation, SDS-PAGE and Western Blotting  ............................. 68 2.22. Mass Spectrometry  .......................................................................................................... 69 2.23. Enzymatic Removal of N-Glycans  .................................................................................. 70 2.24. Statistical Analysis  .......................................................................................................... 70   3. Combined Immunodeficiency Due to MALT1 Deficiency ....................................................... 71 3.1. Overview ............................................................................................................................ 71 3.2. Case History ....................................................................................................................... 71 3.3. Laboratory Investigations ................................................................................................... 74 3.3.1. Lymphocyte Cell Counts ............................................................................................. 74 3.3.2. B Cell Function ............................................................................................................ 74 3.3.3. Flow Cytometric Immunophenotyping of B Cell Subsets .......................................... 74 3.3.4. Other Immune Cell Subsets ......................................................................................... 77 3.4. Bioinformatic Analysis and Identification of the MALT1 Variant .................................... 80 3.5. Functional Evaluation of MALT1 Mutation in Patient ...................................................... 83 	   viii	  3.5.1. Quantification of MALT1 Gene and Protein Expression ............................................ 83 3.5.2. Effect of Mutation on MALT1 Activity ...................................................................... 85 3.5.2.1. Scaffold Function in CBM Complex Formation .................................................. 85 3.5.2.2. Paracaspase Activity ............................................................................................. 87 3.5.2.3. Activation of the Canonical NF-κB Pathway ....................................................... 88 3.6. Establishing Causality of MALT1 Mutation ...................................................................... 93 3.6.1. MALT1 Transfection of Primary T Cells Restores NF-κB Signaling ........................ 93 3.6.2. Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) of MALT1 Deficiency Patient .................................................................................................................................... 95     3.7. Discussion and Future Directions ..................................................................................... 100  4. Primary Humoral Immunodeficiency Due to Gain-of-Function PLCγ2 Mutation ................. 105 4.1. Overview .......................................................................................................................... 105 4.2. Case History  .................................................................................................................... 105 4.3. Laboratory Investigations  ................................................................................................ 106 4.3.1. B Cell Function  ......................................................................................................... 107 4.3.2. Flow Cytometric Immunophenotyping of B Cell Subsets ........................................ 108 4.4. Abnormal Surface Expression of BAFF-R Not Associated With a BAFF-R Genetic Mutation .................................................................................................................................. 110 4.5. Whole Exome Sequencing and Identification of PLCγ2 Gene Variant ........................... 113 4.6. Assessment of PLCγ2 Activity in Patients Primary Cells ................................................ 115 4.6.1. Calcium Flux in B Cells ............................................................................................ 115 4.6.2. Downstream Signaling of Activated PLCγ2 ............................................................. 115 4.6.3. Platelet Hyperreactivity ............................................................................................. 118 4.7. Confirmation of Increased External Calcium Flux Due to PLCγ2 Mutation ................... 120 4.8. Increased Apoptosis of Immature B Cell Subsets ............................................................ 122 4.9. Discussion and Future Directions  .................................................................................... 125      5. Soluble BAFF Regulates Surface BAFF-R Expression .......................................................... 127 5.1. Introduction  ..................................................................................................................... 127 5.2. Transient Suppression of BAFF-R Surface Expression in (PLCγ2mut/+) Patient  ............. 129 5.3. Overnight Incubation in Patient (PLCγ2mut/+) Serum Reduces Surface BAFF-R Expression on Healthy Donor B Cells  .......................................................................................................... 130 5.4. Soluble BAFF in the Patients Serum Down-Modulates Surface Expression of BAFF-R 135 5.5. G-CSF and IFNγ Stimulated Neutrophils Secrete sBAFF That Suppresses BAFF-R Expression ................................................................................................................................... 138 5.6. The Effect of Recombinant Soluble BAFF on Surface Expression of BAFF-R .............. 141 5.6.1. Recombinant Soluble BAFF Down-Modulates Surface BAFF-R Expression .......... 141 5.6.2. Recombinant Soluble BAFF is Biologically Active and Acts on Early Human Transitional B Cells ................................................................................................................. 143 5.7. Mechanism of Down-Modulation of Surface BAFF-R Expression by sBAFF ............... 150 5.7.1. BAFF-R Gene Transcription ..................................................................................... 150  5.7.2. Release of Soluble BAFF-R ...................................................................................... 150 5.7.3. Surface BAFF-R Internalization ................................................................................ 150  5.8. Discussion and Future Directions ..................................................................................... 152  	   ix	  6. Alternative Isoforms of BAFF: Relevance to Pre-B Acute Lymphoblastic Leukemia (pre-B ALL) ............................................................................................................................................ 157 6.1. Introduction ...................................................................................................................... 157 6.2. Pre-B Acute Lymphoblastic Leukemia as a Model System for Aberrant BAFF-BAFF-R Signaling .................................................................................................................................. 158  6.2.1. Pre-B ALL Cells Express BAFF Receptors .............................................................. 158  6.2.2. Pre-B ALL Cells Express and Release Glycosylated BAFF ..................................... 160  6.2.3. BAFF is Functional Active in Pre-B ALL ................................................................ 164 6.3. Potential Differences Between sBAFF Isoforms ............................................................. 166 6.3.1. Belimumab Does Not Bind Leukemic BAFF ........................................................... 166 6.3.2. Leukemic sBAFF Inhibits the Ability of Normal sBAFF to Down-Modulate Surface Expression of BAFF-R ........................................................................................................ 167  6.3.3. The Effect of Normal sBAFF on Pre-B ALL is Anti-Proliferative ........................... 168  6.4. The Presence of Soluble BAFF Post-HSCT ..................................................................... 170 6.4.1. The Role of Soluble BAFF Post-HSCT on the Surface Expression of BAFF-R in Pre-B ALL Cells  ....................................................................................................................... 171  6.4.2.  High Levels of Soluble BAFF are Associated with Relapse of Pre-B ALL and Increased Disease-Related Mortality Post-Hematopoietic Stem Cell Transplantation (HSCT) ................................................................................................................................ 173  6.5. Discussion and Future Directions ..................................................................................... 176  7. Discussion ................................................................................................................................ 179  Bibliography ................................................................................................................................ 187  Appendices .................................................................................................................................. 229 Appendix 1: Candidate genes with novel variants identified by exome sequencing  ............. 229                  	   x	  	  List of Tables  Chapter 1 1.1. Diseases associated with elevated soluble BAFF  .................................................................. 50  Chapter 2 2.1. Flow cytometry immunophenotyping of peripheral blood circulating B cell subsets ............ 63  Chapter 3 3.1. Variant prioritization based on autosomal recessive inheritance  .......................................... 81	  	  Chapter	  4	  4.1. Immunological parameters  .................................................................................................. 107   	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	     	   xi	  List of Figures  Chapter 1 1.1. Schematic overview highlighting the central roles of PLCγ2 and the CBM complex in transmitting BCR signals  .............................................................................................................. 25 1.2. BAFF and its receptors  .......................................................................................................... 33 1.3. Schematic representation of BAFF ........................................................................................ 42  Chapter 3 3.1. Family pedigree, growth chart and histopathology ................................................................ 73 3.2. B cell subset immunophenotyping of CD19+ B cells from patient (MALT1mut/mut), sibling (MALT1mut/+) and healthy donor ................................................................................................... 75 3.3. CD25+CD127lowFoxP3+ regulatory T cells as a percentage of CD4+ T cells in the patient (MALT1mut/mut), sibling (MALT1mut/+), and healthy controls  ...................................................... 78 3.4. Change in cell size of MALT1-mutated CD3+ T cells (MALT1mut/mut) after stimulation with phytohaemagglutinin (PHA) ......................................................................................................... 79 3.5. Secretion of IL-2 by primary T cells after PHA-dependent T cell receptor stimulation  ....... 80 3.6. Site of mutation in MALT1 protein  ....................................................................................... 82 3.7. Quantification of MALT1 gene expression  ........................................................................... 83 3.8. Quantification of MALT1 protein expression  ....................................................................... 84 3.9. Scaffold function of MALT1 as determined by ability to bind BCL10 ................................. 86 3.10. Paracaspase activity as visualized by cleavage of BCL10 ................................................... 88 3.11. IκBα degradation and NF-κB p65 subunit phosphorylation in primary CD3+ T cells ........ 90 3.12. IκBα degradation and NF-κB p65 subunit phosphorylation in primary CD19+ B cells  ..... 92 3.13. Artificial expression of the normal MALT1 protein in patient’s primary CD3+ T cells  ..... 94 3.14. MALT1 protein expression and NF-κB signaling in primary lymphocytes post-HSCT  .... 95 3.15. IκBα degradation and NF-κB p65 subunit phosphorylation in primary CD19+ B cells and CD3+ T cells post-HSCT  ............................................................................................................. 96 3.16. Primary B Cell Immunophenotyping Post-HSCT  ............................................................... 98  Chapter 4 4.1. B cell subset immunophenotyping of CD19+ B cells from patient (PLCγ2mut/+) and healthy donor ............................................................................................................................................ 109 4.2. BAFF-R expression by flow cytometry  .............................................................................. 112 4.3. Domain structure of PLCγ2 .................................................................................................. 114 4.4. Calcium flux in primary CD19+ B cells after BCR stimulation .......................................... 115 4.5. ERK1/2 phosphorylation after BCR stimulation  ................................................................. 117 4.6. Collagen-induced platelet alpha granule release .................................................................. 119 4.7. BCR stimulation of PLCγ2 KO DT40 cells transfected with PLCγ2 constructs  ................ 121 4.8. Cell death in immature primary B cells  ............................................................................... 124  Chapter 5 5.1. Surface BAFF-R expression on patients (PLCγ2mut/+) primary CD19 B cells after overnight incubation with various serums  .................................................................................................. 129 5.2. Suppression of surface BAFF-R expression on healthy control CD19+ B cells  ................. 131 	   xii	  5.3. Dose response relationship between patient (PLCγ2mut/+) serum and surface BAFF-R expression  ................................................................................................................................... 132 5.4. Hindrance due to receptor occupancy .................................................................................. 134 5.5. Effect of blocking sBAFF on ability of patient (PLCγ2mut/+) serum to suppress surface BAFF-R expression ..................................................................................................................... 136 5.6. Soluble BAFF in the patients’ serum increases the cellular size of healthy donor B cells .. 137 5.7. Effect of G-CSF and IFNγ stimulated neutrophil conditioned media on surface BAFF-R expression in CD19+ B cells  ...................................................................................................... 140 5.8. Effect of recombinant sBAFF on surface BAFF-R expression in healthy donor CD19+ B cells  ............................................................................................................................................. 142 5.9. Gating strategy used to sort 1) CD27-CD10high and 2) CD27-CD10dim CD19+ B cell populations from cord blood  ...................................................................................................... 145 5.10. CD21 and BAFF-R expression on human transitional B cell subsets ................................ 146 5.11. Effect of sBAFF on surface expression of BAFF-R in human transitional B cell subsets  147 5.12. Expression of CD62L in transitional B cell subsets ........................................................... 148 5.13. Effect of sBAFF on CD62L expression in transitional B cell subsets ............................... 149 5.14. Surface and total expression of BAFF-R on normal primary B cells after incubation with recombinant sBAFF  .................................................................................................................... 151  Chapter 6 6.1. Surface expression of BAFF receptors (BAFF-R, TACI and BCMA) on relapse pre-B ALL cell lines  ...................................................................................................................................... 159 6.2. BAFF-R surface expression on primary pre-B ALL cells  ................................................... 160 6.3. Expression of glycosylated full-length and soluble BAFF in pre-B ALL cell lines ............ 162 6.4. Primary pre-B ALL cells release sBAFF ............................................................................. 164 6.5. Gene expression of Pim-2 after blocking sBAFF in 697 cells  ............................................ 165 6.6. Western blot of belimumab immobilized column eluate for sBAFF using polyclonal anti-BAFF antibody  ........................................................................................................................... 166 6.7. Effect of normal sBAFF on 697 pre-B ALL cells ................................................................ 169 6.8. Plasma from patients post-HSCT down-modulates surface expression of BAFF-R on pre-B ALL 697 cells .............................................................................................................................. 172 6.9. Soluble BAFF levels at 6 months post-HSCT for relapsed pre-B ALL  .............................. 175 6.10. Event free survival in GVHD negative patients based on sBAFF concentrations in blood at 6 months from patients on the COG ASCT0431 study  .............................................................. 176	    	        	   xiii	  List of Abbreviations ADCC – Antibody-dependent cell-mediated cytotoxicity AID – Activation-induced cytidine deaminase ALL – Acute lymphoblastic leukemia ANC – Absolute neutrophil count ANCA – Anti-neutrophil cytoplasmic antibody APC – Antigen presenting cell APLAID – Autoinflammation and PLCG2-associated antibody deficiency and immune dysregulation APRIL – A proliferation-inducing ligand BAFF – B cell activating factor BAFF-R – B cell activating factor receptor Bcl-xL – B-cell lymphoma-extra large BCMA – B cell maturation antigen BCR – B cell receptor BLNK – B-cell linker BM – Bone marrow Btk – Bruton’s tyrosine kinase CAML – Calcium-modulating cyclophilin ligand CBM complex - caspase recruitment domain family, member 11 (CARD11)–B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)–mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) CCE - Capacitative Ca2+ entry cIAP – Cellular inhibitor of apoptosis CLL – Chronic lymphocytic leukemia CLP – Common lymphoid progenitor CRD – Cysteine rich domain CSR – Class switch recombination CTCL – Cutaneous T cell lymphoma CVID – Common variable immune deficiency CXCR – C-X-C chemokine receptor DAG - Diacylglycerol DAMPs – Damage-associated molecular patterns DC – Dendritic cell EDTA – Ethylenediaminetetraacetic acid EGTA – Ethylene glycol tetraacetic acid ELISA – Enzyme-linked immunosorbent assay ENU - N-ethyl-N-nitrosourea ER – Endoplasmic reticulum ERK – Extracellular signal-regulated kinase FACS – Fluorescence-activated cell sorting FBS – Fetal bovine serum FCAS3 – Familial cold autoinflammatory syndrome-3 FcRL4 – Fc receptor-like 4 FISH – Fluorescence in-situ hybridization 	   xiv	  FOXP3 – Forkhead box P3 FSC – Forward scatter FVC – Forced vital capacity G-CSF – Granulocyte-colony stimulating factor GVHD – Graft-versus-host disease GVL – Graft-versus-leukemia GVT – Graft-versus-tumor HD – Healthy donor HEV – High endothelial venule HIV – Human immunodeficiency virus HSC – Hematopoietic stem cell HSCT – Hematopoietic stem cell transplantation ICOS – Inducible T-cell costimulator Ig – Immunoglobulin IL-2 – Interleukin-2 IL-4 – Interleukin-4 IL-6 – Interleukin-6 IL-8 – Interleukin-8 IL-10 – Interleukin-10 IL-12 – Interleukin-12 IFN-α – Interferon alpha IFN-β – Interferon beta IFN-γ – Interferon gamma IP3 – Inositol trisphosphate IP3R - IP3 receptor ITAMs – Immunoreceptor tyrosine-based activation motifs ITP – Idiopathic thrombocytopenic purpura IVIG – Intravenous immunoglobulin kDa - Kilodalton KIR – Killer cell immunoglobulin-like receptors KREC – Kappa recombination excision circle LPS – Lipopolysaccharide LUBAC – Linear ubiquitin chain assembly complex MALT1 – Mucosa-associated lymphoid tissue lymphoma translocation gene 1 MAPK – Mitogen-activate protein kinase MFI – Mean fluorescence intensity MHC – Major histocompatibility complex mTOR – Mammalian target of rapamycin MZ – Marginal zone NEMO - NF- B essential modifier NF-AT – Nuclear factor of activated T cells NHEJ – Non-homologous end joining NIK - NF- B-inducing kinase NK – Natural killer NOD-like receptors – Nucleotide-binding oligomerization domain receptors NSIP – Nonspecific interstitial pneumonitis 	   xv	  ODN - Oligodeoxynucleotide OMIM – Online Mendelian Inheritance in Man PAMPs – Pathogen-associated molecular patterns PBMCs – Peripheral blood mononuclear cells PBS – Phosphate-buffered saline PDC – Plasmacytoid dendritic cell PHA - Phytohemagglutinin PI3K – Phosphoinositide 3-kinase  PIB – phorbol ester and ionomycin plus brefeldin-A PIP2 - phosphatidylinositol 4,5-bisphosphate PLCγ1 – Phospholipase C gamma 1 PLCγ2 – Phospholipase C gamma 2 PMA/I- Phorbol 12-myristate 13-acetate PNGaseF – N-glycosidase F PTK – Protein tyrosine kinase PWM – Pokeweed mitogen RACC – Receptor activated calcium channel RAG1 – Recombination activating gene 1 RAG2 – Recombination activating gene 2 RIPA – Radioimmunoprecipitation assay buffer RSS – Recombination signal sequence RSV – Respiratory syncytial virus  RT-PCR – Reverse transcription polymerase chain reaction SAC – Staphylococcus aureus Cowan I SCID – Severe combined immune deficiency SDS-PAGE – Sodium dodecyl sulfate polyacrylamide gel electrophoresis SH2 – Src homology 2 SHM – Somatic hypermutation SHP – Src homology region 2 domain-containing phosphatase SLE – Systemic lupus erythematosus SNP – Single nucleotide polymorphism SOC - Store-operated Ca2+ channel SSC – Side scatter TACI – Transmembrane activator-calcium modulator and cyclophilin ligand interactor TCR – T cell receptor TdT – Terminal deoxynucleotidyl transferase TGFβ-1 – Transforming growth factor beta-1 TLR – Toll-like receptors TNF-α – Tumor necrosis factor alpha TRAF1 – TNF receptor-associated factor 1 TRAF2 – TNF receptor-associated factor 2 TRAF3 – TNF receptor-associated factor 3 TRAF6 – TNF receptor-associated factor 6 TRPC – Transient receptor potential cation WT – Wild-type XLA – X-linked agammaglobulinemia 	   xvi	  Acknowledgements I would like to sincerely thank Dr. Kirk R. Schultz for his guidance, understanding, patience, and most importantly, his friendship during my graduate studies. His mentorship as a clinician scientist was essential in providing me a experience consistent with my long-term career goals. I would also like to thank all the members of his laboratory, especially Dr. Amina Karaminia for all their teaching and support.  I would also like to thank all the members of my PhD supervisory committee including Dr. Janet Chantler, Dr. James Lim, Dr. Michael Gold, Dr. Poul Sorensen and Dr. Jan Dutz for their expertise and valuable discussions. I would especially like to thank my graduate studies advisor, Dr. Haydn Pritchard, for all his support in navigating the last steps of completing my thesis.  I would like to thank all the members in our collaborating laboratories, especially Dr. Stuart Turvey, for their support and expertise. I would like to thank the Division of Pediatric Oncology, Hematology and Bone Marrow Transplant at BC Children’s Hospital, especially our Division Head Dr. Caron Strahlendorf, for their mentorship and clinical support.   Finally, and most importantly, I would like to thank my children, Abigail and William and my wife Julia. Trying to balance my graduate studies, clinical work and family life proved almost impossible but she made sure that I did not miss any important events in our always hectic family life. I could not have done this without her unwavering support and patience and I will forever be grateful.  	   1	  Introduction B cells play a fundamental role in our adaptive immune system, which is defined by two key characteristics: specificity and memory. The principal function of B cells is to make antibodies in response to foreign antigens. Each B cell expresses a unique immunoglobulin (Ig) receptor as part of the B cell receptor (BCR) that recognizes a specific antigenic epitope. When a naïve B cell encounters its corresponding antigen in the presence of a co-stimulatory signal the B cell can proceed along two interrelated pathways. It can proliferate and differentiate into a plasma cell that secretes antibodies, which then act to eliminate pathogens and their toxins. It can also form a memory B cell generating an immunological memory. In a subsequent response to the same antigen, memory B cells are rapidly activated and differentiate into highly specific plasma cells. Aside from antibody production, B cells can also present antigen and regulate immune responses through the production of cytokines.  Our immune system is capable of generating an almost limitless repertoire of unique B cells through an error prone process of combinatorial genetic rearrangement. The trade off for achieving this diversity is the generation of self-reactive B cells that must be silenced by tolerance mechanisms. The generation and maintenance of the peripheral B cell pool involves three key steps: 1) the step-wise development of Ig expressing immature B cells from hematopoietic stem cells in the bone marrow followed by export to the periphery as transitional immature B cells; 2) the surveillance and removal of self-reactive immature B cells and 3) differentiation of immature B cells into effector cells depending on cytokine signals, exposure to antigen and interactions between antigen-specific T cells and dendritic cells. Perturbations in this tightly regulated 	   2	  developmental program give rise to a wide variety of human diseases including congenital immunodeficiency, malignancy and autoimmune disease.   The strength of the BCR signal and B cell activating factor (BAFF), a member of the tumor necrosis family (TNF) ligand superfamily, have emerged as critical factors in the development and maintenance of the peripheral B cell compartment. BAFF signals through three different receptors: BAFF receptor (BAFF-R), transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI) and B cell maturation antigen (BCMA), which are nearly exclusively expressed on B cells at various stages of maturation.  Through the use of transgenic and knockout murine models, the effects of the BCR and BAFF signaling pathways on murine B cell maturation and survival are well characterized. For example in mice, BAFF is critical for the survival of B cells beyond the immature transitional stage. BAFF improves the survival of responsive B cells by altering the ratio between pro-survival and pro-apoptotic molecules. In contrast, the effects of BAFF on the survival of resting immature and mature human B cells are less clear and there are strong phenotypic differences between mice and humans. Furthermore, human BAFF can be modified by glycosylation, multimerization and expression of splice variants. The biological significance of these modifications remains unclear.  There is a crucial need to better understand the functions of BAFF and BCR signaling in humans.  High levels of soluble BAFF, dysregulated expression of its receptors on B cells and dysfunctional BCR signaling have been identified in a large group of clinically heterogeneous diseases, including autoimmune and inflammatory conditions, allergy, viral infections and lymphoid cancers. There are several anti-BAFF therapies currently in phase II and III clinical trials for autoimmune diseases, however the overall results so far have only been modest or not 	   3	  effective at all, suggesting that we still lack crucial insights into many aspects of human BAFF biology.  A cornerstone of modern biomedical research is the use of mutant murine models to study the role of proteins in biological systems and disease. However, murine models are limited in their ability to help us understand the human BAFF system, necessitating alternative translational approaches. Revealing the genetic basis of rare inherited monogenic diseases involving defects in the immune system have been an essential tool in advancing our understanding of both disease mechanisms and fundamental elements of immunity. Animal models are inherently experimental and have limited correlation with human disease, while humans with monogenic diseases allow us to study the function of a particular gene in its natural setting that includes all the complex host environmental interactions [1]. The next-generation sequencing revolution has greatly accelerated our progress as it has allowed the rapid generation of lists of candidate genes in an increasing number of genetically undefined cases [2]. The mutations identified in rare monogenic diseases that share phenotypes with common diseases have exposed new biological pathways involved in the molecular pathogenesis of common health problems [3]. Much of what we know about the molecular background of common diseases such as atherosclerosis [4] and Alzheimer disease [5] is in fact based on what we have learned from rare familial forms of these diseases. Understanding the pathogenesis of these diseases has paved the way for immunological interventions demonstrating proof-of-principle for new treatments such as hematopoietic stem cell transplantation and gene therapy [6]. Lastly, information about the causal effects of a single genetic variant are invaluable to drug development, because mutations that alter the level of activity of gene products can be thought of 	   4	  as surrogates for perfectly targeted drugs, the job of which is to antagonize or agonize a given gene product [7]. In this thesis we demonstrate that the discovery of unknown rare disease pediatric patients with severe defects in peripheral B cell development and study of childhood precursor B cell acute lymphoblastic leukemia (ALL) will provide novel insights into the function of BAFF and the regulation of BAFF receptor expression.  The first chapter summarizes normal B cell development with a focus on the role of BCR and BAFF-mediated signaling pathways. In the third chapter we describe the discovery of a loss-of-function mutation in mucosa-associated lymphoid tissue lymphoma translocation gene 1 or MALT1 as a novel cause of combined immunodeficiency in a 15-year old girl. This patient had an arrest in B cell development at the transitional stage and severe reduction in mature B cells that mimicked B cell defects seen in patients with mutations in the BAFF-R. Our findings indicate that MALT1 is essential for antigen receptor induced regulation of NF-κB signaling in lymphocytes and that MALT1 is involved in the surface expression of BAFF-R. In the fourth chapter we describe the discovery of a novel gain-of-function mutation in phospholipase C gamma 2 or PLCγ2 in an 8-year old girl with hypogammaglobulinemia and reduced surface BAFF-R expression that was associated with hyper-reactive BCR signaling. This mutation appears to be associated with increased apoptosis of transitional B cells. Furthermore, the reduction in BAFF-R surface expression was mediated by its main ligand, soluble BAFF, which triggered receptor internalization.  	   5	  This phenomenon is studied in the fifth chapter where we demonstrate that sBAFF from our patient, sBAFF secreted by neutrophils and recombinant sBAFF are capable of down-modulating the surface expression of BAFF-R through a mechanism of receptor internalization.  In the sixth chapter we investigate the autocrine/paracrine BAFF signaling pathway in pre-B ALL and demonstrate the presence of a biological active leukemic BAFF isoform that is N-glycosylated. This is suggested by differential binding to the clinical anti-BAFF monoclonal antibody, belimumab and its ability to inhibit the down-modulation of surface BAFF-R caused by unmodified sBAFF. Most significantly we demonstrate that unmodified sBAFF has an anti-proliferative effect on pre-B ALL cells.  This thesis highlights the discovery of two novel primary immunodeficiencies involving mutations in MALT1 and PLCγ2, reveals mechanisms responsible for BAFF-R expression and provides evidence of different BAFF isoforms in human disease.            	   6	  Chapter 1: Background 1.1. Overview In this section, I will first summarize the steps of normal B-cell development and its functional outcomes. I will concentrate on two signaling pathways critical for the early development of B cells in the periphery: B cell receptor (BCR) and B cell activating factor (BAFF) signaling. With regards to BCR signaling, I will focus on 2 key molecules, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and phospholipase C-gamma 2 (PLCγ2), essential in transmitting BCR signals. This will be followed by a summary of what is known about the role of B cell activating factor (BAFF) in normal B cell development and how dysregulated BAFF signaling contributes to B cell disorders.  1.2. Early B Cell Development In 1965, Max Cooper and Robert Good, working with chickens, showed that cells that develop in the bursa of Fabricius (‘B cells’) were responsible for the production of antibody or immunoglobulin (Ig) [8]. This relationship was further reinforced by studies showing surface Ig expression on normal B cells [9]. B cells are distinguished from all other immune cells by the synthesis, display and secretion of an antibody. The binding of membrane Ig, a component of the B cell receptor (BCR) to its corresponding epitope on an antigen is the primary step in B cell activation. The Ig molecule is the central element of the BCR complex, which transduces the signal triggered by binding of antigen to the Ig. Mammalian B cell development is a tightly regulated sequence of stages that begins in primary lymphoid tissue; human fetal liver followed by fetal/adult bone marrow, with subsequent functional maturation in secondary lymphoid organs such as spleen and lymph nodes. 	   7	  The generation of a specific membrane-bound Ig molecule unique to each B cell is the primary aim of early B cell development.  An Ig is a Y-shaped molecule consisting of four polypeptide chains: two identical heavy chains and two identical light chains. There are five types of immunoglobulin heavy chain: γ, δ, α, µ and ε which define classes/isotypes of immunoglobulins: IgG, IgD, IgA, IgM, and IgE respectively. The isotype determines the functional activity of the antibody molecule. There are two types of immunoglobulin light chain, which are called lambda (λ) and kappa (κ). Each heavy and light chain contains a pre-determined constant region and a variable region that is generated by an error-prone combinatorial somatic genetic rearrangement process.  Each chain is composed of discrete, compactly folded domains, each about 110 amino acids long; four or five in the heavy chain and two in the light chain. It is only the first domain on the N terminus that contains the variable region, whereas the remaining domains are constant between Ig chains of the same isotype.  The two Ig heavy chains are linked by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. Each arm of the Y-shaped structure is formed by the association of the 2 domains of the light chain with the 2 domains of the amino-terminal half of the heavy chain. This pairing forms two identical antigen-binding sites that are referred to as the fragment antigen-binding (Fab fragment) region. The trunk of the Y is formed by the pairing of the two remaining C-terminal domains of the heavy chain and is called the fragment crystallizable region (Fc region).  The early stages of B cell development are defined by the step-by-step generation of heavy and light chains containing unique variable domains. The variable region of each heavy and light chain is generated by the combination of gene segments called variable (V), diversity 	   8	  (D) and joining (J) segments. The variable region in the heavy chain is a combination of V, D and J segments on human chromosome 14, while the light chain only combines V and J segments from human chromosome 2 for the κ light chain and human chromosome 22 for the λ light chain.  Each step in V(D)J recombination is linked to a specific stage in B cell development. During normal hematopoiesis, the earliest lineage restricted lymphoid progenitor is the common lymphoid progenitors (CLP). These CD34+/CD10+/CD45RA+ progenitors are capable of developing into B, T, NK, and lymphoid dendritic cell (DC) lineages, but not myeloid/erythroid lineages [10]. It has been suggested that CLPs are inherently biased toward B cell development [11]. The next stage is a pro-B cell which is characterized by the rearrangement of the V(D)J heavy chain [12]. Pro-B cells become pre-B cells when they express a pre-B cell receptor (BCR) composed of a membrane-bound µ heavy chain and surrogate light chains (VpreB and λ14.1) [13]. Signaling through the pre-BCR promotes light chain rearrangements in the IgK (κ) locus, leading to V to J coupling. If IgK light chain rearrangements are not successful, V to J gene rearrangements occur in the IgL (λ) light chain locus. Once VJ light chain rearrangements are successful, the light chains combine with the heavy chains as well as Igα/Igβ to form a functional BCR expressed on the surface of an immature B cell.   1.3. V(D)J Recombination In order to couple the V, D and J gene segments together to form a functional variable region in the heavy and light chains, double strand DNA breaks are made by recombination activating genes, RAG1 and RAG2 at recombination signal sequences flanking the gene 	   9	  segments [14]. The resulting ends at the side of the gene segments are ligated together forming a coding joint, by proteins of the non-homologous end joining repair pathway [15].  Receptor diversity is generated during V(D)J recombination at several levels: 1) Multiple V, D and J gene segments; for example, the human genome contains 51 functional VH segments, 25 DH segments and 6 JH segments on chromosome 14; 2) The coding ends, generated by RAG1/2 cleavage, are processed at several steps prior to ligation. Firstly, the initial cleavage may be off center resulting in an overhang of bases on one of the strands. DNA repair enzymes add palindromic (P) nucleotides to resolve the overhang. Secondly, DNA polymerases may also add non-templated (N) nucleotides to the ends. Thirdly, exonucleases can remove bases from the coding ends, including any added P or N nucleotides. Lastly, other polymerases will then insert additional nucleotides as needed to make ends compatible for joining. All of these processing steps contribute to generating a highly variable region leading to an almost limitless repertoire of immunoglobulins that will be displayed on B cells. However, a consequence of these assorted genetic manipulations results in the generation of a significant portion of Igs that will bind self-antigens. Therefore, the first pool of B cells created in the bone marrow with a fully formed Ig will be very rich in autoreactive clones due to the very randomness of the combinatorial process. Analysis of the bone marrow early immature B-cell repertoire indicates that a staggering 50%–75% of these cells express BCRs that are specific for self-antigens in humans [16].    1.4. Immune Tolerance  The immune system must possess mechanism(s) to ensure that newly formed B cell lymphocytes are non-reactive to self-antigens. These self-tolerance mechanisms are not perfect, as diseases involving an autoimmune component now constitute the third leading cause of 	   10	  morbidity and mortality after cardiovascular disease and cancer [17]. Defects lead to emergence of self-reactive B cells that can either produce autoantibodies or serve as antigen-presenting cells that stimulate autoreactive T cells.  Tolerance can be acquired and specific. In 1945, Dr. Ray Owen observed in cattle that fraternal offspring who shared blood elements via vascular anastomoses did not form antibodies against their sibling’s red cell antigens [18]. Despite the twins’ having distinctly different blood groups, transfusions between them did not cause any transfusion reactions. It was subsequently shown that they also did not reject each other(s) skin grafts. In 1953, the Medawar group injected foreign lymphoid tissues into neonatal mice and found that these mice acquired immune tolerance to grafts from donors of the same strain but would reject unrelated grafts [19]. The classic definition of immunological tolerance is a state of non-reactivity towards an antigen that would normally be expected to trigger an immunological response in a normally functioning immune system [20].  All multicellular organisms contain massive quantities of foreign elements that do not trigger immune responses including commensal bacteria, protozoan parasites and parasitic worms, fetal tissue and chimeric states established during tissue transplantation [21]. Induction of immune tolerance to non-self antigens is a pre-requisite for successful allogeneic solid organ and bone marrow transplantation [22]; either a donor organ tolerated by the immune system of the recipient or a donor immune system tolerating the recipient’s tissue. A state of donor-specific unresponsiveness without a need for pharmacologic immunosuppression, termed operational immune tolerance, remains the holy grail of clinical transplantation.    	   11	  1.4.1. B Cell Self-Tolerance The suppression of self-reactive B cells involves two main processes. The first step is the elimination of high-affinity autoreactive B cells in central and peripheral lymphoid organs. Subsequently, there are processes that prevent the activation and differentiation of low-affinity autoreactive B cells that escaped elimination in lymphoid organs. In order for a tolerance mechanism to be effective it must consist of processes that recognize self-reactivity followed by a mechanism to eliminate or correct the self-reactivity.   1.4.2. Central B Cell Tolerance The newly formed Ig on the B cell surface, as part of the emerging BCR complex, is first tested for self-reactivity via interactions with bone marrow stromal cells. Central tolerance is the process by which newly generated immature B cells that react with a self-antigen in the bone marrow environment are negatively selected [23]. If stimulation of the BCR by a self-antigen is of sufficient strength, the immature B cell temporarily halts its maturation by internalizing its autoreactive BCR [24]. The persistent expression of RAG genes in autoreactive B cells enables further V(D)J recombination of Ig light chains to eliminate self-reactivity through receptor editing [25]. If the autoreactive B cell fails to correct its self-reactivity, cell death will occur in the bone marrow or in the periphery as an early transitional B cell. This is mediated through increased levels of pro-apoptotic factors belonging to the BCL2 protein family [26,27].       	   12	  1.4.3. Peripheral B Cell Tolerance  Immature autoreactive B cells that have avoided clonal deletion or receptor editing, but can still react with low avidity self-antigens can be rendered functional unresponsive, or anergic [28-30]. Anergic B cells fail to respond to their specific antigen. Anergy can be induced in both transitional and mature B cells via multiple regulatory pathways that increase the threshold of B cell activation such as decreased surface expression of BCR, reduced intracellular signaling from the BCR complex and inhibition of stimulatory nuclear factor kB signaling pathways [31-33].   There is also a limited population of autoreactive B cells that remain clonally ignorant because of a lack of physical access to antigen or competition for developmental growth factors and inflammatory mediators [34,35].   1.5. Differentiation into Mature B Cell Subsets  The transitional B cell stage is the critical juncture at which newly emerged B cells from the bone marrow can differentiate further into various mature B cell subsets in the lymphoid organs in response to different types of signal(s) through the BCR and other cell surface receptors such as Toll-like receptors [36].  Mature B cells can be separated into multiple lineages: B-1, B-2 and B-10 [37]. The phenotypic and functional characteristics of all these lineages have been well established in mice along with B-10 and B-2 in humans. The B-1 population is less well characterized in humans. Each lineage also consists of multiple subsets.   1.5.1. B-1 Cells In mice, B-1 cells predominate during fetal and neonatal development, are capable of self-renewal and localize primarily to peritoneal and pleural cavities [38,39]. They provide low-	   13	  affinity IgM-mediated polyreactive humoral immunity against a broad spectrum of infections [40]. The identity and existence of the human counterpart of murine B-1 cells is still strongly debated [41].    1.5.2. B-2 Cell Subsets Immature transitional B cells are continually generated from the bone marrow and transit to the spleen where they undergo further differentiation into either marginal zone or follicular B cells depending on signaling through the B cell receptor, Notch2, the receptor for B cell-activating factor and the canonical NF-κB pathway, as well as signals involved in the migration and anatomical retention within splenic compartments [42].  1.5.2.1. Follicular (FO) B Cells  Follicular B cells recirculate freely between the bloodstream and lymphoid tissues in which they undergo T cell dependent antigen-induced activation resulting in the formation of germinal centers within secondary lymphoid organs. In addition to binding antigen, FO B cells require a second activating signal that can come from T cells (T cell dependent). The B cell receives help from a T cell when antigen that was bound initially by the BCR is internalized, processed and returned to the surface as a peptide bound to a MHC class II molecule. A helper T cell specifically recognizes the peptide fragment and provides a second activating signal; an essential part of this co-signal is the interaction between CD40 ligand on the T cell and CD40 receptor on the B cell [43]. Some T cell activated B cells undergo clonal expansion and become short-lived IgM-secreting plasma cells. A plasma cell is a terminally differentiated, end-stage B cell that secretes soluble Ig [44]. Other B cells migrate into B cell rich areas within secondary 	   14	  lymphoid tissue called follicles to form germinal centers [45] where they undergo two more genetic recombination processes that further select for high affinity Ig: 1) somatic hypermutation and 2) class switch recombination.  1. Somatic Hypermutation  The initial Ig repertoire generated by V(D)J recombination will generally bind antigens with only modest affinity and specificity requiring a second mutational change to increase affinity. SHM occurs when surface Ig is engaged by antigen triggering the introduction of point mutations by activation-induced cytidine deaminase into the variable regions of the Ig genes [46]. Some of these mutations will generate higher affinity antibodies. Successive cycles of mutation and selection leads to the generation of a B cell with a higher affinity antibody.  2. Class Switch Recombination  The antibody isotype is determined by the heavy chain constant region and influences antibody function and determines which cells will be activated by the antibody-antigen complex. Class switch recombination is an intrachromosomal deletional recombination event that leads to Ig isotype switching [47].   Activated follicular B cells expressing mutated BCRs with enhanced affinities can then differentiate further into short and long-lived antibody-secreting plasma cells or memory B cells. Long-lived antigen-specific memory B cells provide immunological memory, which enables the immune system to respond rapidly and specifically upon a second exposure to an antigen.   	   15	  1.5.2.2. Marginal Zone (MZ) B Cells  These reside primarily in the marginal zone of the spleen, a unique environment designed to provide maximum exposure to open blood circulation [48]. Without the need for T cell help, MZ B cells can rapidly produce antibodies, mostly IgM, in response to either type 1 T-cell independent (TI) antigens such as LPS which bind the BCR and co-engage TLRs, or type 2 TI antigens such as polysaccharides which are capable of extensive BCR crosslinking [49,50]. MZ B cells can also undergo somatic hypermutation and produce IgG via class switch recombination [51]. IgM+IgD+CD27+ memory B cells correspond to circulating splenic marginal zone B cells [52]. They are considered a front-line defense of innate-like lymphocytes that express polyreactive BCRs that bind to microbial molecular patterns, similar to TLRs [53]. They also express high levels of TLRs, which allows them to bridge the innate and adaptive immune system. Along with B1 B cells, they mediate the initial wave of humoral immunity while follicular B cells undergo maturation.   1.5.3. Regulatory (B10) Cells There is a subset of B cells defined by their characteristic production of IL-10 (B10 cells) that can negatively regulate immune responses by suppressing T cell polarization, inhibiting antigen presentation, and proinflammatory cytokine production [54]. The absence of these regulatory B cells exacerbates disease symptoms in murine models of experimental autoimmune encephalomyelitis, colitis, arthritis and systemic lupus erythematosus [55-58].  IL-10-producing human B cells have been recently characterized in humans [59]. They normally represent <1% of peripheral blood B cells and were identified by their ability to express cytoplasmic IL-10 after in vitro stimulation with phorbol ester and ionomycin and negatively regulate monocyte cytokine 	   16	  production. They express high levels of CD27, CD48 and CD148, indicative of activation/memory and do not express the immature B cell marker CD10. It appears that they do not express unique markers other than their ability to secrete IL-10, but are enriched within the CD24hiCD27+ B cell subset.  1.6. The Functional Diversity of B Cells B cells perform several roles critical to normal immune system function including producing antibodies, antigen presentation and regulatory cytokine production.   1.6.1. Antibody Production  There are two types of adaptive immune responses: cell-mediated immunity, mediated by T cells, and humoral immunity, mediated by B cells. Humoral immunity is the portion of the immune system mediated by macromolecules found in extracellular fluids such as antibodies and complement proteins. B cells specialize in the production of antigen specific antibodies, which provide a protective immune defense against bacteria, viruses, and harmful protein antigens such as toxins. After binding to their target antigens, soluble antibodies are capable of triggering several immune mechanisms:   1.6.1.1. Neutralization Antibodies can prevent pathogens such as viruses from entering host cells by binding to the proteins on the pathogen surface that are necessary for entry into host cells.     	   17	  1.6.1.2. Complement Activation   Complement consists of a group of serum proteins that activate inflammation, destroy cells and enhance the opsonization/phagocytosis of antigens. Complement proteins respond in a sequential manner resulting in a cascade of reactions. The major components are named C1 through C9 (not in the order that they function but in the order that they were discovered). The complement cascade can become activated via three different pathways: the classical, alternative or lectin pathway. Antibodies exclusively trigger the classical pathway in which the binding of an antibody (IgG or IgM) to an antigen on the cell surface exposes the Fc region of the antibody in a way that allows the first complement protein (C1) to bind and become activated. This triggers a cascade leading to the production of several principal effector molecules: 1) C3a – an anaphylatoxic peptide that mediates local inflammatory processes such as smooth muscle contraction, increased vascular permeability and histamine release from mast cells and basophils; 2) C3b – an opsonin that covalently binds to cell-surface glycoproteins and promotes phagocytosis; 3) C5a – a chemoattractant for phagocytes and 4) membrane attack complex (consisting of C5b, C6, C7, C8 and C9) that forms a pore in the cell, causing the cell to lyse.  1.6.1.3. Binding to Fc Receptors  Effector cells, including natural killer cells, monocytes, macrophages, neutrophils, eosinophils and dendritic cells, bind to the antibody-antigen complexes via Fc receptors that recognize the Fc region of immunoglobulins. There are several classes of Fc receptors grouped according to which Ig isotype they bind; Fc-gamma receptors (FcγR), Fc-alpha (FcαR), Fc-epsilon (FcεR) bind IgG, IgA and IgE respectively. Therefore, the isotype of the antibody determines which effector cell will be engaged in a given response such as: 1) phagocytosis, 2) induction of antibody-dependent cell-mediated cytotoxicity (ADCC), and 3) degranulation. 	   18	  1. Phagocytosis Phagocytes are activated by IgG antibodies that bind to specific Fcγ receptors on the phagocyte surface. Opsonisation is the process by which a foreign particle is coated with plasma antibodies that are specific for antigenic determinants on that particle to enable the attachment and internalization of that particle by a professional phagocyte. When an antibody-coated pathogen binds to Fcγ receptors on the surface of the phagocyte, the particle is endocytosed in an acidified cytoplasmic vesicle called a phagosome, which subsequently fuses with a lysosome containing enzymes that destroy the pathogen.  2. Antibody Mediated Cellular Cytotoxicity (ADCC)   ADCC is the non-phagocytic killing of an antibody-coated target cell by a natural killer (NK) cell [60]. NK cells express FcγRIII (CD16), which binds the Fc portion of IgG triggering the release of pore-forming proteins called perforins and proteolytic enzymes called granzymes. The granzymes pass through the pores of the target cell and activate enzymatic cascades leading to apoptosis of the target cell.  3. Degranulation  Mast cells, basophils and eosinophils release cytoplasmic granules containing lipid inflammatory mediators such as leukotrienes and prostaglandins, cytokines such as TNF-α and chemical mediators via antibody bound to Fc receptors specific for IgE (FcεRI) [61]. One of the best-known chemical mediators released is histamine, which rapidly increases vascular permeability leading to an accumulation of antibodies, complement proteins and influx of immune cells in inflammatory sites.  	   19	  1.6.2. Antigen Presentation  The antigen presenting cell (APC) function of B cells is often overlooked, but B cells possess a special ability to bind intact antigens via the BCR and subsequently process them via the class II pathway for presentation to T cells [62]. B cells are capable of inducing antigen-specific naïve CD4+ T cell priming [63,64].  B cells, unlike macrophages, are relatively ineffective at antigen uptake by fluid-phase pinocytosis, a process by which a cell takes up liquid by invagination of the plasma membrane. Antigen uptake by B cells occurs by receptor-mediated endocytosis through molecules on the B cell surface such as Fc receptors, transferrin receptors, MHC class II and CD19/CD21/CD81 complex [65]. What makes them unique APCs is their ability to engulf intact antigen via their unique membrane bound immunoglobulin. This specificity allows B cells to concentrate very small quantities of a specific antigen recognized by that particular B cell and present it efficiently. It has been shown that the affinity of the BCR for a specific antigen is directly proportional to the capacity of the B cells to present antigen to CD4 T cells [66]. B cells bearing a high-affinity BCR can induce CD4+ T cell proliferation at extremely low concentrations making them extremely effective APCs. In addition to this quantitative effect, the high affinity of the BCR-antigen interaction selectively blocks the processing of regions of the antigen bound to the BCR resulting in preferential presentation of certain fragments [67,68]. Therefore antigen-specific B cells possess a highly efficient mechanism for concentrating small amounts of antigen that results in an enhancement of its presentation and can shape the immune response against it. Not only does the BCR bind to a specific antigen with high affinity, the act of binding to the BCR itself triggers B cell activation signals that facilitate the trafficking, generation and presentation of processed antigen-MHCII complexes by B cells [69]. 	   20	  1.6.3. Cytokine Production  B cells produce cytokines and can be subdivided into discrete subsets based on their cytokine production. This means that they can both amplify and suppress immune responses. Regulatory or B10 cells secrete IL-10 or TGFβ-1, while effector B cell populations can differentiate into two distinct cytokine-secreting subsets, Be1 and Be2. Be-1 cells produce cytokines such as IFNγ, IL-12 and TNFα while Be-2 cells produce IL-2, IL-4, TNFα and IL-6 [70].  1.7. B Cell Disorders in Human Disease B cell disorders in humans can be broadly divided into three categories: 1) immunodeficiencies associated with defects in B cell development leading primarily to an absence of immunoglobulin production [71]; 2) disruption of normal B-cell differentiation and activation leading to benign and malignant B cell lymphoproliferative disorders [72] and 3) breakdown in B cell tolerance leading to autoimmune diseases. X-linked agammaglobulinemia (XLA) resulting from mutations in Bruton’s tyrosine kinase (Btk) was one of the first primary immunodeficiencies described and lead to the first use of intravenous immunoglobulin therapy [73,74]. Btk is a member of the Tec family of cytoplasmic protein tyrosine kinases that transduces signals originating from the pre-B cell receptor (pre-BCR) and BCR resulting in PLCγ tyrosine phosphorylation, inositol triphosphate production and calcium mobilization [75]. Mutations in Btk, components of the pre-BCR and BCR (λ14.1, Igα and Igβ), or the scaffold protein BLNK account for approximately 90% of patients with absence of circulating B cells with severe reduction in all serum immunoglobulin levels [76]. Affected patients typically present with early onset of recurrent bacterial infections.  	   21	  Hypogammaglobulinemia with normal or low number of B cells is the prototype of common variable immunodeficiency (CVID). Although most patients have normal numbers of B cells, their B cells fail to differentiate into immunoglobulin-secreting plasma cells. These patients have defective specific antibody production, increased susceptibility to recurrent infections and an increased incidence of cancers and autoimmune disorders [77]. Molecular defects in inducible T cell co-stimulator (ICOS), transmembrane activator and CAML interactor (TACI), CD19 and B cell activating factor receptor (BAFF-R) have been discovered in a small subset of these patients. Finally, there are patients with immunoglobulin class switch recombination deficiencies due to molecular defects in CD40, CD40L, inhibitor of nuclear factor kappa-B kinase subunit gamma/NF-kappa-B essential modulator (IKKγ/NEMO), activation-induced deaminase (AID), and uracil-DNA glycosylase (UNG) [78]. These disorders are clinically characterized by normal or elevated IgM, low or absent IgA, IgE and IgG levels. Clinical manifestations include recurrent and chronic bacterial infections, lymphoid hyperplasia, and autoimmune disorders. On the opposite side, germline and somatic gain-of-function mutations in the same genes resulting in constitutive activity of the mutated protein are the basis of B cell lymphoproliferative disorders. For example, Btk shows constitutive activity in chronic lymphocytic leukemia (CLL) and is the target of irreversible inhibition by ibrutinib, an orally bioavailable Btk inhibitor that has shown strong activity in CLL [79].  Each stage of B cell development can give rise to B cell malignancies. These are frequently caused by chromosomal translocations and gene mutations that confer hallmark cancer cell characteristics including self-sufficiency in growth signals, evading apoptosis and insensitivity to antigrowth signals [80]. For example, B cell acute lymphoblastic leukemias 	   22	  (ALL) arise from pre-B cells and involve mutations in runt-related transcription factor 1 (RUNX1), pre-B cell leukaemia homeobox 1 (PBX1), mixed-lineage leukaemia (MLL), protein tyrosine phosphatase non-receptor type 11 (PTPN11) and RAS genes. Several types of lymphoma arise from differentiated subsets of mature B cells in the secondary lymphoid organs. Splenic marginal zone lymphoma (SMZL) and mucosa-associated lymphoid tissue (MALT) lymphoma are indolent malignancies derived from marginal zone B cells. Follicular lymphoma, diffuse large B cell lymphoma (DLBCL) and Burkitt's lymphoma are all derived from germinal centre (GC) B cells [81]. The production of autoantibodies by self-reactive B cells is a cardinal feature of systemic autoimmune diseases. When an adaptive immune response develops against self-antigens, it is usually impossible for immune effector mechanisms to completely eliminate the antigen and so a chronic inflammatory response persists. The mechanisms of tissue damage in autoimmune diseases are the same as those that operate in normal immunity and hypersensitivity reactions [82].  These self-reactive B cells are able to persist due to defects in both central and peripheral tolerance checkpoints. For example, alterations in BCR signaling pathways result in defective central tolerance and failure to remove autoreactive immature B cells in the bone marrow of patients with type 1 diabetes, rheumatoid arthritis and systemic lupus erythematosus [83]. Therefore specific mutations that result in defects in B cell tolerance can clinically present as both immunodeficiencies and autoimmune diseases. The advent of next generation sequencing technology has rapidly accelerated the discovery of previously unknown Mendelian or monogenic disorders associated with B cell defects [84]. The identification of rare-disease-causing genes in humans along with genetically 	   23	  modified animals provides vital insight into biological pathways responsible for B cell development.   I will now concentrate on two critical signaling pathways required for proper peripheral B cell development: B cell receptor (BCR) and B cell activating factor (BAFF) signaling. The first section is on BCR signaling with a particular focus on the role of phospholipase C gamma 2 (PLCγ2) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1).   1.8. The Role of B Cell Receptor Signaling Mechanisms that establish immune tolerance highlight the importance of the BCR complex in transmitting signals that regulate the fate of a B cell at various developmental checkpoints. The BCR is a complex made up of the heavy and light immunoglobulin chains as described previously, associated with two signaling components, Igα (CD79a) and Igβ (CD79b) [85]. Antigen binding induces the recruitment of the protein tyrosine kinases (PTKs) Lyn and Syk to immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic tails of CD79a and CD79b [86]. Their phosphorylation results in the recruitment of a signalosome, which includes many kinases and adaptor proteins that leads to the activation of 3 main pathways via Bruton’s tyrosine kinase (Btk), phospholipase C gamma2 (PLCγ2) and phosphoinositide 3-kinase (PI3K) [87,88]. This in turn triggers further downstream pathways such as the Ras/ERK and Akt pathways and protein kinase C-dependent NF-κB activation through the CARMA-Bcl10-MALT1 (CBM) complex via production of second messengers such as diacylglycerol (DAG) and inositol trisphosphate (IP3) (Figure 1.1). These downstream pathways respond differentially to BCR stimulation depending on the stage of B cell development leading to different biological outcomes and subsequent production of certain mature B cell subsets. 	   24	  There are several signaling pathways that regulate the BCR signal. CD19 is a co-stimulatory surface protein that serves to reduce the threshold for signalling via the B-cell receptor (BCR). Co-ligation of CD19 with the BCR synergistically enhances mitogen-activated protein (MAP) kinase activity, calcium release and proliferation through the recruitment of PI3K [89]. In order to prevent excessive BCR signaling, the BCR signal transduction threshold is regulated by CD22, a sialic-acid-binding immunoglobulin-type lectin. CD22 is an inhibitory coreceptor that can attenuate BCR signaling by concominantly binding, in trans, sialylated moieties of multivalent antigens that engage the BCR [90]. Antigen-mediated BCR crosslinking causes rapid tyrosine phosphorylation of CD22 by the tyrosine kinase Lyn, thereby recruiting and activating the SH2-domain-containing tyrosine phosphatase, SHP-1 [91]. SHP-1 subsequently dephosphorylates multiple components of the BCR signaling cascade, resulting in suppression of BCR-mediated signaling [92]. Signaling through FCRL2 has also been shown to recruit SHP-1 leading to inhibition of BCR-triggered calcium mobilization [93]. Another inhibitory signal occurs when FcγRIIb binds to the Fc portion of secreted IgG Abs in complex with Ag. FcγRIIb engagement results in phosphorylation of its ITIM tyrosine, recruitment of the SHIP inositol-phosphatase, and the consequent dampening of BCR-mediated signal transduction events [94].  1.9. PLCγ2 is Critical for BCR Signal Transduction Enzymes of the phospholipase C family play a key role in cell signaling by generating the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from hydrolysis of plasma membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) [95]. These second messengers then trigger numerous cell-specific downstream signaling cascades involved in a 	   25	  variety of cellular functions. The PLCγ subfamily consists of two family members, PLCγ1 and PLCγ2. PLCγ1 is ubiquitously expressed and is mainly activated downstream of growth factor stimulation. PLCγ2 is predominantly expressed in hematopoietic cells and is activated by immune cell receptors such as B cell and Fc receptors. The only exception is T cell receptor activation, which is linked to PLCγ1. PLCγ enzymes are mainly activated through tyrosine phosphorylation by receptor kinases. This interaction is mediated by binding through SH2 domains.      Figure 1.1. Schematic overview highlighting the central roles of PLCγ2 and the CBM complex in transmitting BCR signals. BCR, B-cell receptor; BLNK, B-cell linker protein; DAG, diacylglycerol; IP3, inositol-1,4,5-trisphosphate; ITK, IL-2–inducible T-cell kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; SRC, Src family kinase. (Figure taken from Turvey et al. JACI. 2014 Aug; 134(2): 276-284 of which I am co-author). 	   26	   Engagement of the BCR and activation of Syk triggers phosphorylation of the adaptor protein BLNK. BLNK in turn simultaneously recruits PLCγ2 and Bruton’s tyrosine kinase (Btk) to phosphotyrosine residues in SH2 domains. This enables Btk to phosphorylate and activate PLCγ2. PLCγ2 hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) [95]. DAG activates various signaling pathways, including those of PKC and Ras leading to downstream activation of the Raf/Mek/Erk pathway [96]. IP3 triggers rapid Ca2+ release from internal Ca2+ stores of the endoplasmic reticulum (ER) by activating IP3 receptors (IP3R) causing a transient increase in cytosolic Ca2+ concentration. This initial phase of receptor-activated Ca2+ signaling is followed by a sustained influx of Ca2+ across the plasma membrane [97].  The 10,000-fold concentration gradient for Ca2+ across the plasma membrane of resting cells coupled with a hyperpolarized resting membrane potential results in a huge electrochemical driving force in favor of Ca2+ influx. It is clear that ER Ca2+ content falls after initial stimulation, however to maintain the integrity of the ER, these Ca2+ stores must be replenished. The second phase of Ca2+ signaling is primarily driven by capacitative Ca2+ entry (CCE) mediated by store-operated Ca2+ channels (SOCs) in the plasma membrane in response to a fall in ER Ca2+ content [98]. It has been shown that PLCγ2, in addition to its role in initiating the first phase of ER Ca2+ release upon BCR stimulation also mediates the second phase of external calcium influx [99].   1.9.1. PLCγ2 Deficient Mice PLCγ2-deficient mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. Ig levels are reduced and T cell-independent antibody production is 	   27	  absent. FcγR signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcεR function, and NK cell FcγRIII and 2B4 function [100]. Targeted deletion of PLCγ2 in germinal center B cells demonstrated an additional essential role in the maintenance of memory B cells [101].   1.9.2. Murine Models of Gain-of-Function Mutations in PLCγ2 Gain of function mutations in PLCγ2 were first studied in murine models established in large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screens. Two mouse strains with limb defects, named abnormal limb 5 and 14 (Ali5 and Ali14), were found to have single amino acid replacements in PLCγ2 leading to severe autoinflammatory disease characterized by inflammatory skin lesions, arthritis, keratitis, glomerulonephritis, autoantibodies and male infertility [102-105]. Abnormalities identified in hematopoietic cells included:  i. B cells: 1) increased and sustained external calcium entry after anti-IgM stimulation; 2) decreased number of mature B cells ii. Myeloid Populations: Expansion of innate immune cells in the bone marrow. There were no functional differences in granulocytes, macrophages or dendritic cells except that stimulation of Gr1-positive granulocytes from peripheral blood via the FcγR resulted in sustained Ca2+ mobilization similar to that observed in B cells iii. Platelets: Platelets showed enhanced Ca2+ mobilization, integrin activation, granule secretion and phosphatidylserine exposure upon GPVI (collagen receptor) or C-type lectin-like receptor-2 stimulation   	   28	  1.9.3. Human Disease Associated With Mutations in PLCγ2 There are no reported cases of humans with PLCγ2 deficiency, however there are 2 reports of germline gain-of-function mutations in human PLCγ2.  1.9.3.1. PLCγ2-Associated Antibody Deficiency and Immune Dysregulation (PLAID)/Familial Cold Autoinflammatory Syndrome-3 (FCAS3) Ombrello et al. reported a cohort of 27 patients from 3 families with a dominantly inherited complex of cold-induced urticaria, atopy, granulomatous rash, autoimmune thyroiditis, presence of antinuclear antibodies, sinopulmonary infections and common variable immunodeficiency [106]. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. The molecular mechanism of dysfunction is deletions (Δ(646-685) in cSH2, Δ(686-806) in cSH2/SH3), located within a region encoding an autoinhibitory domain, resulting in protein products with constitutive phospholipase activity.   1.9.3.2. Autoinflammation and PLCγ2-Associated Antibody Deficiency and Immune Dysregulation (APLAID)  Zhou et al. reported a father and daughter with a systemic autoinflammatory disorder characterized by early-onset recurrent blistering skin lesions, nonspecific interstitial pneumonitis with respiratory bronchiolitis (NSIP), arthralgia, eye inflammation, enterocolitis, cellulitis, and recurrent sinopulmonary infections [107]. Laboratory studies showed a decrease in circulating IgM and IgA antibodies, decreased numbers of class-switched memory B cells, and decreased numbers of NK T cells. The mutation is a single amino acid substitution (S707Y) in the cSH2 domain. Overexpression of the altered p.Ser707Tyr protein and ex vivo experiments using 	   29	  affected individuals’ leukocytes showed clearly enhanced PLCγ2 activity, suggesting increased intracellular signaling in the PLCγ2-mediated pathway.  1.10. The Role of MALT1 in BCR Signaling Mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) is one of the three proteins that make up the CARMA1/CARD11 (CARD [caspase recruitment domain]-containing MAGUK protein 1)-BCL10 (B-cell chronic lymphocytic leukemia/lymphoma 10)-MALT1 (CBM) signalosome complex. The CBM complex is essential in linking antigen receptor activation to canonical NF-κB activation in lymphoid immune cells [108].   As part of the CBM signaling complex, the association of MALT1 with TNF receptor–associated factor 6 (TRAF6)–containing ubiquitin ligase complexes results in the addition of K63-linked ubiquitin chains to a multitude of proteins, including TRAF6, BCL10, MALT1, and the IKK regulator NF-κB essential modulator (NEMO) [109]. Linear ubiquitin chains are also conjugated to NEMO through the linear ubiquitin chain assembly complex (LUBAC), which was recently shown to interact with the CBM complex in B-cell lymphomas [110,111]. Both types of ubiquitination events are essential for the recruitment and activation of the IKK complex, which can then phosphorylate the NF-κB inhibitor nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor α (IκBα) [112]. In resting cells IκBα is mainly bound to NF-κB dimers, keeping those transcription factors inactive in the cytoplasm. Upon phosphorylation by the IKK complex, IκBα is modified by K48-linked ubiquitin chains and subsequently degraded by the proteasome, resulting in the translocation of NF-κB dimers from the cytoplasm to the nucleus, where they can mediate transcription of a large set of immunity-relevant target genes [113]. Within the CBM signaling complex, the paracaspase MALT1 serves also as a caspase-like 	   30	  protease that shares structural homology with the family of caspase-like proteins known as metacaspases found in yeast, plants, and parasites [114]. The list of substrates includes BCL10, A20, CYLD, RelB, and Regnase-1.  B cell receptor (BCR)-mediated NF-κB signaling through the CBM complex is initiated when immunoreceptor tyrosine-based activation motifs (ITAMs) within Igα/β signaling chains of the BCR are phosphorylated by activated protein tyrosine kinases of the Src family [115]. This recruits and activates SYK, leading to the recruitment of the adaptor protein BLNK and the TEC kinase BTK. This complex activates PLCγ2, triggering the release of intracellular calcium and activation of PKC-β in B cells. In turn, PKC-β phosphorylates CARD11 inducing a conformational change that allows the recruitment of BCL10, which is already constitutively associated with MALT1. As described, MALT1 acts as both a scaffold protein and paracaspase. The exact mechanism by which BCL10 and MALT1 regulate IKK-mediated NF-κB activation is not completely understood.   Based on murine models, MALT1 is strictly required for the development of marginal zone and B-1 B cells, but is largely dispensable for antigen receptor-mediated activation of conventional B-2 B cells [116,117]. Although MALT1 is largely dispensable for canonical NF-κB signaling downstream of the BCR, the absence of MALT1 results in impaired BAFF-induced phosphorylation of the non-canonical NF-κB2 (p100), p100 degradation and RelB nuclear translocation in murine B cells [118]. This corresponds with impaired survival of marginal zone but not follicular B cells in response to BAFF stimulation in vitro. The role of MALT1 in human B cell development is poorly understood.   	   31	  1.11. B Cell Activating Factor (BAFF) The replenishment and maintenance of the mature B cell pool is essential for effector B cells to fulfill their various functions. The cytokine, B cell activating factor (BAFF) plays a fundamental role in the survival and differentiation of mature B cells. BAFF (also termed BLyS, CD257, DTL, TALL-1, TALL1, THANK, TNFSF20 or ZTNF4) is a member of the TNF ligand family and binds to three receptors: two high-affinity receptors, BAFF receptor (BAFF-R), transmembrane activator-calcium modulator and cyclophilin ligand interactor (TACI), and a low-affinity receptor, B-cell maturation antigen (BCMA). These receptors are differentially expressed on B cell subsets during development contributing to the pleiotropic effects of BAFF [119]. Studies of genetically altered mice demonstrate that BAFF-R deletion results in greater than 90% loss of mature B cells, revealing it as an essential mediator of B cell survival beyond the immature stage. In contrast, TACI and BCMA perform niche roles in B cell Ig isotype switching and plasma cell maintenance respectively.   BAFF exists in a variety of forms; soluble, membrane-bound, spliced, glycosylated, homotrimerized, heterotrimerized and multimerized. The effects of each form remain to be well characterized [120]. Within the TNF superfamily of cytokines, BAFF shares the highest homology with APRIL (a proliferation-inducing ligand). BAFF shares with APRIL the ability to bind to BCMA and TACI and also binds to proteoglycans. Even though BAFF and APRIL share many characteristics including the ability to multimerize with each other, their functions and effects are not redundant. BAFF is considered a primordial B cell survival factor while APRIL contributes specifically to plasma cell survival, isotype switching and T independent antibody responses [121]. 	   32	  Most of our knowledge of the function of BAFF in B cell development is based on murine studies. Further study in human B cells have confirmed some of the mouse-based findings but also revealed key differences that require further investigation particularly regarding the role of BAFF in human disease. In the next three sections, I will summarize what is known about BAFF signaling pathways in mice, humans and unanswered questions regarding the role of BAFF in disease. This will primarily focus on the interaction between BAFF and its cognate receptors: BAFF-R, TACI and BCMA (Figure 1.2).     	   33	   Figure 1.2. BAFF and its receptors. BAFF exists in a variety of forms: soluble, membrane-bound, homotrimerized, heterotrimerized and multimerized. Within the TNF superfamily of cytokines, BAFF shares the highest homology with APRIL. Even though BAFF and APRIL share many characteristics including the ability to multimerize, their functions and effects are not redundant. BAFF is considered a primordial B cell survival factor while APRIL contributes specifically to plasma cell survival, isotype switching and T independent antibody responses. BAFF-R is activated by all forms of BAFF and is the only BAFF receptor that responds strongly 	   	   	  	   	   	  TACI BAFF-­‐R BCMA 	   	  	  	   	  	   sBAFF APRIL 	   	  	  	   	  	  	   	  	  	  	   	   	  	  	  	   	  	  	  	   	   	   	  	   	  	  	   	   	  	  	   	  	  	   	  	  	   	  	   	  	  	   	  	  	  	  	   	  	  	   	   	   	  	  	   	  	  	   	  	  60-­‐mer	  sBAFF 	   	   	  	  membrane-­‐bound BAFF 	   	  	   BAFF/APRIL	  heterotrimers Nanomolar	   Affinity Micromolar	   Affinity - Negative	  regulation	  of	  B	  cell	  maturation	  - Ig	  class	  switch	  recombination	  - B	  cell	  development	  beyond	  early	  transitional	  stage	  - Plasma	  cell	  survival	  - Antibody	  production	  	   34	  to BAFF homotrimers. TACI specifically requires oligomeric APRIL, membrane-bound BAFF, cross-linked BAFF, or BAFF multimers in order to signal, but is unresponsive to soluble BAFF homotrimer. In humans, BCMA is selectively induced during plasma cell differentiation and there is a 100-fold higher selectivity of APRIL binding to BCMA over BAFF.    1.11.1. Crucial Role for BAFF/BAFF-R Pathway in the Maintenance of Mature B Cells  In mice, BAFF plays a fundamental role in the maturation of transitional B cells and in vivo maintenance of the peripheral mature B cell pool. The necessity of BAFF for mature B cell survival and differentiation beyond the transitional stage is well demonstrated by an almost complete lack of follicular or marginal zone B cells (B-2 cells) in BAFF deficient mice [122-125]. Following injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. There is a decrease in the size of B cell follicles, an impairment of a T cell dependent humoral immune response, and a reduction in the formation of memory B cells [126]. The only mature B-2 lineage subset whose survival is independent of BAFF is long-lived memory B cells [127].  The mature B cell compartment in BAFF-deficient mice can be rescued by treatment with recombinant Fc-BAFF [128]. On the other hand, BAFF transgenic mice demonstrate B cell hyperplasia from the late transitional B cell stage onwards [129,130]. In contrast, the necessity of BAFF in human mature B cell development and maintenance is not well defined.  There are no published reports of a human with deletion of the BAFF gene. The only human data we can evaluate are patients treated with targeted anti-BAFF therapies in autoimmune diseases associated with high serum BAFF levels. This is not an ideal biological 	   35	  model to study the effect of BAFF on normal human mature B cell homeostasis but does provide some insight.   Patients with systemic lupus erythematosus (SLE) treated with an anti-BAFF monoclonal antibody, belimumab, had a preferential reduction of naïve and transitional B cells to less then 20% of their pre-treatment numbers. These studies did not examine whether the levels of these B cell subsets were elevated in the first place, as has been previously described in SLE [132]. In contrast, non-class switched memory cells and plasma cells decreased only after 18 months of treatment and conventional CD27+/IgD- class-switched memory cells were resistant to BAFF inhibition [133,134]. Therapy with belimumab in patients with primary Sjogren’s syndrome (pSS) induced a significant reduction in transitional and naïve B cell subsets to levels similar to those observed in healthy donors [135]. CD27- memory B cells and all subsets in the CD27+ compartment were not significantly affected by belimumab treatment. Furthermore, belimumab normalized BAFF-R expression in all B subsets that comprise the memory compartment. In contrast to murine models, inhibition of BAFF in both diseases does not lead to a significant developmental arrest in B cell development.  BAFF-R is the predominant BAFF receptor expressed on all murine peripheral B cells with the exception of long-lived bone marrow plasma cells and memory cells. This suggests that formation of memory B cells requires BAFF signaling, while the maintenance and survival of plasma does not have a unique requirement for BAFF as APRIL binding to BCMA can fulfill these functions. For plasma cell survival, BAFF is redundant with APRIL.   BAFF-R is first expressed by B cells during the transitional stages and its expression level increases with further differentiation. It has been shown that BAFF-R is a potential NF-κB target gene. BCR signaling induces activation of c-Rel, an active NF-κB transcription factor, in 	   36	  B cells leading to increased expression of BAFF-R [136]. It has been shown that c-Rel has affinity for the putative promoter sites upstream of the BAFF-R gene. It is hypothesized that early transitional (T1) cell clones that do not undergo apoptosis due to a strong BCR signal increase basal or tonic BCR signaling through specific developmentally regulated default pathways. This results in the gradual expression of BAFF-R, which increases the BAFF-dependent survival potential of late transitional (T2) B cells. [137].  Similar to mice, there is a developmental arrest of most B cells at the stage of CD10+ transitional cells in two siblings with BAFF-R deficiency resembling the phenotype found in BAFF-R mutant mice [131]. The dependence of memory B cells on BAFF-R function seems to be less obvious, as both siblings had a reduced but still detectable population of class-switching memory B cells correlating with a strong T-dependent immune response after immunization with tetanus toxoid. In contrast to mice, the deletion of the BAFF-R gene in humans does not necessarily lead to a clinically manifest immunodeficiency. BAFF-R is activated by all forms of BAFF and is the only BAFF receptor that responds strongly to BAFF homotrimers. BAFF-R mainly activates the non-canonical NF-κB signaling pathway [138]. The binding of BAFF to BAFF-R releases NF-κB-inducing kinase (NIK) which activates a downstream kinase, Iκβ kinase-α (IKKα), for triggering NF- B2/p100 phosphorylation and processing, leading to the liberation of a p52/RelB active heterodimer. This heterodimer translocates to the nucleus where it activates target genes involved in the peripheral B cell survival and maturation such as members of the Bcl-2 family [139,140]. In the absence of BAFF binding to BAFF-R, NIK forms a complex with TNF receptor-associated factor 2 (TRAF2) and TRAF3. TRAF2 recruits cellular inhibitor of apoptosis (cIAP), which targets NIK for degradation by ubiquitination, thereby inhibiting the alternative NF-κB signaling pathway 	   37	  [141]. When BAFF binds BAFF-R, TRAF3 is recruited to the receptor and TRAF2 promotes TRAF3 degradation, resulting in the release of NIK, which is then free to activate non-canonical NF-κB signaling. Consistent with this, deletion of NIK in adult mice results in decreases in B cell populations in lymph nodes and spleen, similar to what is observed upon blockade of BAFF. B cells from mice in which NIK is acutely deleted, fail to respond to BAFF stimulation in vitro and in vivo [142].    The binding of murine BAFF to its principal receptor, BAFF-R improves the survival of responsive B cells by altering the ratio between pro-survival and pro-apoptotic molecules. The signaling mechanisms include: 1) increased transcription of anti-apoptotic genes such as members of the Bcl-2 family [143], 2) inhibition of BCR-induced death by down-regulating Bim via sustained ERK activation [144], 3) promoting the cytoplasmic retention of protein kinase C δ (PKCδ), thus opposing the pro-apoptotic potential of PKCδ upon translocation to the nucleus [145]. 4) up-regulation of Pim-2, an anti-apoptotic serine/threonine kinase and proto-oncogene [146] and 5) phosphorylation of Akt, which promotes cell survival by increasing glucose uptake and glycolysis [147].  1.11.2. Role of TACI  In the murine model, TACI is believed to counteract BAFF signaling through BAFF-R thereby acting as a negative regulator. BAFF-R and TACI differ in their requirements for BAFF and APRIL oligomerization in order to transmit productive signals. TACI specifically requires oligomeric APRIL, membrane-bound BAFF, cross-linked BAFF, or BAFF multimers in order to signal, but is unresponsive to soluble BAFF homotrimer [148]. 	   38	  It limits non-canonical NF-κB signaling by inducing cIAP-mediated ubiquitination of NIK [149]. TACI-deficient mice demonstrate B cell hyperplasia from the T1 B cell stage onwards and have an increased rate of B cell proliferation [150,151]. There is also development of autoimmunity, glomerulonephritis, production of autoantibodies and B cell lymphomas. This indicates that TACI delivers negative signals suppressing B cell activation via BAFF-R mediated non-canonical NF-κB signaling.  On the other hand, TACI also provides positive signals driving T cell-independent B cell responses in innate B cells such as marginal zone (MZ) and B1 B cells. TACI expression is particularly high on MZ and B1 B cells [152]. These mature B cell subsets are also responders to Toll-like receptor (TLR) activation that in turn strongly up-regulate expression of TACI [130,153,154]. In turn, TACI promotes plasma cell differentiation in response to T-independent type II antigens (TI-2) [155,156]. Responses to TI-2 antigens were almost completely abolished in TACI knockout mice [157]. Therefore, TACI likely controls activation and survival of plasmablasts derived from TLR-activated innate B cells stimulated with TI-2 antigens [158].  TACI also mediates T cell-independent class switch recombination (CSR) [159,160]. TACI mediates CSR by binding to MyD88, a universal adaptor protein that is used by almost all TLRs to activate canonical NF-κB signaling [161]. Activation of canonical NF-κB signaling triggers the transcription of germ line Ig heavy chain constant region genes and induces the expression of AID.   Lastly, TACI also binds calcium-modulating cyclophilin ligand (CAML), which is expressed on the surface of cytoplasmic vesicles [162]. Via CAML, TACI activates the transcription factor nuclear factor of activated T cells (NF-AT), which controls the release of 	   39	  calcium from intracellular stores, as well as the activation of c-Jun NH2-terminal kinase and the transcription factor AP-1.  The primarily immunodeficient phenotype of human TACI deficiency is in contrast to the murine model. Homozygous and heterozygous TACI mutations are present in 8-10% of common variable immune deficiency (CVID) patients but also in about 1% of the normal population [163]. CVID is a frequently diagnosed (1:25,000) immunodeficiency characterized by hypogammaglobulinemia and associated with susceptibility to infections as well as autoimmunity, granulomatous disease and cancer. Mutations in TACI have only been found to be significantly associated with CVID when both alleles are mutated or when one TACI allele carries either the A181E or C104R mutation [164,165].  In contrast to TACI deficient mice, TACI deficient patients have hypogammaglobulinemia and the hypothesis that TACI plays a direct role in T cell-independent responses is also challenged by TACI deficient patients who present with normal levels of these antibodies [166].   1.11.3. Role of BCMA  The function of BCMA is mostly limited to the survival of plasma cells residing in the bone marrow. BCMA deficient mice have impaired survival of long-lived bone marrow plasma cells [167].  In humans, BCMA is selectively induced during plasma cell differentiation. It is expressed at higher levels in malignant plasma cells from patients with multiple myeloma. Constitutive BCMA activation in human multiple myeloma cells promotes myeloma cell growth and survival in the bone marrow microenvironment via upregulated NF- B signaling [168]. In 	   40	  vivo, these effects are likely mediated by APRIL rather then BAFF due to a 100-fold higher selectivity of APRIL binding to BCMA over BAFF [143]. Loss-of-function mutations in human BCMA have not been identified.  1.11.4. The Functional Complexity of BAFF Murine models provide valuable insight into the diverse function of BAFF however none of the transgenic and knockout mouse models recapitulate all phenotypes regarding BAFF in human B cell development. This is further complicated by the pleiotropic effect of BAFF in B cell development. Some of this diversity is attributed to which BAFF receptor is activated, but BAFF itself can be structurally modified in a large number of ways potentially further expanding its functional profile. Furthermore, there are multiple factors involved in the control of BAFF secretion including cell of origin, transcriptional and cytokine regulation.  In this sub-section, I describe all the ways in which murine and human BAFF can be modified and whether this affects function.  1.11.4.1. Structure of Human BAFF Human BAFF is a single pass type II transmembrane protein with a total length of 285 amino acids and an unmodified molecular weight of 31.23 kDa. It is composed of a 46 aa cytoplasmic domain, 21 aa transmembrane signal-anchor sequence (47-67) and 218 aa extracellular domain (68-285) (Figure 1.3).  The dominant model states that full-length BAFF is expressed at the plasma membrane and can be cleaved by a furin-like protein convertase to release soluble BAFF from the surface. It has been demonstrated in neutrophils that this cleavage can occur in intracellular stores followed by the secretion of soluble BAFF. The sequence of the 	   41	  cleavage site is RNKR with the last R at amino acid position 133 for human BAFF [170]. This creates a soluble BAFF consisting of 152 aa from A at amino acid position 134 to L at amino acid position 285 (17.2 kDa). Each monomer folds as a sandwich of two antiparallel β-sheets with Greek-key topology [171]. The name comes from the similarity between this β-strand topology and a decorative pattern used in ancient Greece. Mouse BAFF is a slightly longer protein of 309 aa due to an insertion between the transmembrane region and the first of the β strands. However, the β strand-rich ectodomain is almost identical in mouse BAFF and human BAFF (86% identity, 93% homology), suggesting that the BAFF gene has been highly conserved during evolution [170].   1.11.4.2. Structural and Functional Isoforms of BAFF BAFF is modified in a number of different ways including: a) cleavage of full-length BAFF (membrane-bound versus soluble), b) splice variants, c) post-translational modifications and d) multimerization (Figure 1.3). In many cases, these modifications have also been shown to alter the function of BAFF and may explain the heterogeneity of BAFF functional characteristics.          	   42	   Figure 1.3. Schematic representation of BAFF. BAFF is modified in a number of different ways including cleavage of full-length BAFF (membrane-bound) to soluble BAFF, glycosylation and splice variants. Full-length BAFF is a single-pass type II membrane protein made up of 285 amino acids with a molecular weight of 31.2 kDa.   1. Membrane-Bound Versus Soluble BAFF Previous studies have attempted to discover the functional importance of proteolytic cleavage of membrane-bound BAFF. Bossen et al. generated mice expressing BAFF with a 	   N-terminus C 	   124 242 133-134 	   Immunogen for polyclonal Ab – 254-269 Domains 47 67 Isoforms/Splice Variants cytoplasmic TM extracellular 285 N-linked glycosylation sites 	   Furin cleavage site for soluble BAFF 	  134 285 soluble BAFF 	   	  ΔBAFF 142-160 missing in Δ BAFF Δ4BAFF Exon 4 is excised and a new in-frame stop codon within exon 5 is generated   BAFF(ΔEx4-6) Exons 1-3 with an additional C-terminal stretch of 38 amino acids   1 	   	   	   	   	   	   	   	   1 2 3 4 5 6 	   . . 	   	   ΔBAFF Δ4BAFF BAFF(ΔEx4-6) 	   43	  mutated furin consensus cleavage site [172]. These mice had an identical phenotype to BAFF-deficient mice in terms of their B cell populations and responses to immunizations.  Interestingly, they observed an alternative processing event that released some soluble BAFF. They showed that the furin-processed BAFF is essential for murine B cell homeostasis, as peripheral B cell populations and antibody responses were restored by administration of soluble BAFF trimers.  Manetta et al. transfected HEK293 cells with a construct encoding full length human BAFF in which the furin consensus cleavage site was mutated preventing the release of soluble BAFF [173]. Membrane-bound BAFF in the HEK293 cells was a more potent stimulus of T1165.17 cells, a murine plasmacytoma cell line, compared with soluble BAFF. In contrast, primary human B cells responded slightly better to soluble BAFF. They hypothesized that the differences could be due to species’ differences or in receptor expression. For example, T1165.17 cells strongly express TACI whereas primary human B cells predominantly express BAFF-R. The N-terminal extracellular domain prior to the furin cleavage site also appears to mediate BAFF function. Chang et al. used two different forms of recombinant human soluble BAFF to measure the effect of BAFF on human monocyte survival [174]. One form included amino acids 134 to 285 corresponding to the furin convertase cleaved soluble BAFF and the other form contained most of the extracellular domain as well as the stalk region (amino acids 83-285), analogous to the extracellular portion of membrane bound BAFF. Only the long form of BAFF had an effect on monocyte survival when used at the same concentration even though both were able to enhance anti-Ig-stimulated human B cell proliferation to a similar degree. Furthermore, their results suggested that TACI was the responsible receptor for the BAFF-mediated effects in monocytes.  	   44	  These three experimental results suggest that there is a functional difference between membrane bound and soluble BAFF that could be mediated by binding to different receptors; BAFF-R versus TACI.  2. Splice Variants The BAFF gene maps to chromosome 13q33.3 and contains 6 exons and 5 introns corresponding to a length of 39 kb. Several splice variants of BAFF have been observed in mice and humans. (a). ΔBAFF: this alternative splice isoform of BAFF lacks exon 3 and its mRNA has been detected in human cell lines. To our knowledge, the corresponding protein has not yet been detected in humans and its function is unknown. There is a corresponding ΔBAFF splice isoform in mice that has been more extensively studied. This splice isoform is missing exon 4 in mice. The loss of 57 bp maintains the reading frame but the new junction of exon 3 and 5 in murine ΔBAFF encodes a new N residue at amino acid position 155 resulting in an additional N-linked glycosylation at the newly generated residue. In mice, the ΔBAFF protein physically associates with BAFF in disulfide-bounded heteromultimers and the soluble forms of these mixed molecules bind poorly to receptors relative to homomultimers of BAFF. ΔBAFF suppresses BAFF function by competitive co-association. Furthermore, ΔBAFF co-expression appears to regulate the ability of BAFF to appear on the cell surface and to be subsequently shed into the extracellular space. This appears to be due to the retention of ΔBAFF /BAFF multimers inside the cell [175]. Analysis of ΔBAFF transgenic 	   45	  mice revealed that ΔBAFF and BAFF have opposing effects on B cell survival and marginal zone B cell numbers [176]. (b). Δ4BAFF: Δ4BAFF is a variant in humans in which exon 4 is excised and a new in-frame stop codon within exon 5 is generated [177]. This isoform has been shown to act as a transcription factor for its own parent gene, in association with p50 from the NF-kB pathway. Interestingly, Δ4BAFF is located in the nucleus and, contrary to full-length BAFF, absent from the cytoplasm. This isoform has so far been detected in autoimmune and proliferative B cell diseases. Furthermore, the presence of Δ4BAFF is essential for soluble BAFF release by interferon-γ-stimulated monocytes and for B-CLL cell survival. Finally, this splice variant can directly or indirectly regulate the differential expression of a large number of genes involved in the innate immune response and the regulation of apoptosis. (c). BAFF (ΔEx4-6): An investigation of BAFF transcripts in total PBMCs taken from patients with myasthenia gravis and controls revealed several BAFF splice variants containing different parts from intron 3 [178]. A defined alternative exon in intron 3 was evident resulting in a protein consisting of exons 1-3 and an additional C-terminal stretch of 38 amino acids with unknown function. In silico analysis of the aberrant peptide revealed no homology to known functional protein domains. However, the transmembrane domain and proteolytic cleavage site were intact.  3. Post-Translational Modifications  The only described modification of BAFF is N-glycosylation.  BAFF has two potential sites for N-glycosylation at asparagines (N) at position 124 and 242. According to Schneider et al. the complete form is only glycosylated on N at amino acid position 124, therefore making 	   46	  soluble BAFF non-glycosylated as the cleavage site for sBAFF is at position 133 [170]. However, Diao et al. able to demonstrate the production of a 20.2 kDa glycosylated recombinant human sBAFF from the yeast Pichia pastoris [179]. Le Pottier et al. identified 28 and 21 kDa sBAFF isoforms in the serum of 2 patients with Sjogren syndrome using combinations of 4 different anti-BAFF monoclonal and polyclonal antibodies. Deglycosylation with N-glycosidase F (PNGaseF) caused a shift from 28 to 21 kDa. The authors presumed that the difference between the 21 kDa band and the 17 kDa recombinant BAFF revealed the presence of O-glycosylation sites [180]. The role of glycosylation in modulating the function of sBAFF is poorly understood.   4. Multimerization Processed sBAFF forms a homotrimer but can also further assemble as an ordered, capsid-like structure comprising twenty trimers (60-mer). In this capsid-like structure the receptor-binding sites remain exposed and accessible [181]. BAFF can also form heterotrimers with APRIL (a proliferation-inducing ligand), another member of the TNF ligand family that interacts with the BAFF receptors, BCMA and TACI [182]. As previously described, TACI is solely activated by higher-order BAFF and APRIL oligomers.  5. Other Evidence for Different Structural and Functional BAFF Isoforms Research in mouse models supports the existence of two distinct pools of BAFF. Gorelik et al. established and analyzed reciprocal bone marrow (BM) chimeras with wild type (WT) and BAFF-deficient mice [183]. They showed that BAFF-/- BM cells transferred into lethally irradiated WT mice gave rise to normal numbers of follicular and marginal zone B cell 	   47	  subpopulations due to BAFF production by radiation-resistant stromal cells. In contrast, transfer of WT BM into BAFF-/- lethally irradiated mice resulted in minimal reconstitution of mature follicular B cells and no marginal zone B cells. Therefore, BAFF production by BM-derived cells such as macrophages, dendritic cells and neutrophils was not at all sufficient to support normal B cell homeostasis. However, B cell antibody responses were normal in both types of chimera as demonstrated by high levels of antigen-specific antibody secretion after immunization.  The use of different anti-BAFF antibody clones has also provided results suggesting distinct functions for different BAFF isoforms. Roschke et al. identified two BAFF-binding monoclonal antibodies that detected different levels of BAFF in autoimmune serum samples, but recognized recombinant BAFF in a similar fashion [184]. One of the possibilities suggested by the authors was that one of the mAb was recognizing another form of BAFF. Lahiri et al. identified a 21 and 17 kDa form of BAFF produced by epithelial cells and found that only the 17 kDa form of BAFF participated in epithelial cell survival by binding to BAFF-R [185].  There is ample evidence that there are multiple BAFF isoforms with different functions that may also depend on the cell type.    6. Pitfalls in Interpreting the Function of Recombinant Human BAFF  In addition, several caveats should be kept in mind when interpreting the experimental data regarding the use of recombinant human soluble BAFF (rhsBAFF) due to its source. Recombinant human BAFF produced in E. coli cannot be N-glycosylated. A longer soluble version of BAFF (aa 83-285) generated in mammalian 293 T cells is N-glycosylated at N124 but 	   48	  not N242 [170]. This is in contrast to an rhsBAFF expressed in the yeast Pichia pastoris that appears to be N-glycosylated at N242 [186].  Furthermore, it is important to differentiate whether the functional evaluation of rhsBAFF is performed by stimulating primary mouse B lymphocytes, B cell lines (human malignant or other species) or primary human B lymphocytes. For example, in vitro analysis using recombinant human soluble BAFF generated in mammalian 293 T cells showed that BAFF co-stimulates B cell growth in conjunction with B cell receptor activation, yet by itself it did not stimulate proliferation of resting B cells [170]. In contrast, recombinant human BAFF generated in E. coli promoted the proliferation of purified mouse splenic B cells and Raji cells, a human Burkitt’s lymphoma cell line, without any co-stimulation [187].   1.11.4.3. Cellular Origin of BAFF BAFF is primarily produced by cells of myeloid origin. Several studies have shown that monocytes, macrophages, monocyte-derived dendritic cells as well as leukemia myeloid cell lines (HL-60, U937 and THP-1) express both soluble and membrane-bound forms of BAFF [188,189]. However, BAFF expression has also been demonstrated in activated T cells [190] and B cells [191], synoviocytes [192], astrocytes [193], adipocytes [194] and epithelial cells of the salivary glands [195], airways [196] and gut [197].  1.11.4.4. Transcriptional Regulation of BAFF The promoter region of 1020 bp can be activated by classical transcriptional factors such as NFAT (c1 and c2) and NF-κB members [198]. Chromatin immunoprecipitation assays demonstrated that CD40 binds and stimulates the BLyS/BAFF promoter in activated B cells 	   49	  [199]. BAFF-R itself is also a transcriptional regulator of BAFF exerting its effects via chromatin remodeling and canonical NF-κB activation at the membrane surface [200]. Both CD40 receptor and BAFF-R interact with c-Rel to activate BAFF transcription.   1.11.4.5. Cytokine Regulation of BAFF Production  A wide variety of inflammatory cytokines have been shown to up-regulate the expression of BAFF. In the human monocytic cell line THP-1, all type 1 and 2 interferons upregulated BAFF expression (RNA & protein); however, IFN-β was the most potent (IFN-β > IFN-α > IFN-γ) [201]. Interestingly, TNF-α inhibited BAFF expression by IFN-β-stimulated cells. Similar results were obtained with primary cultures of human peripheral blood monocytes. Dendritic cells up-regulate BAFF upon exposure to IFN-α, IFN-γ and CD40L [202]. In the intestine, microbes trigger multiple Toll-like receptors (TLRs), including TLR4, TLR5, and TLR9 to stimulate follicular dendritic cells, dendritic cells and stromal cell release of BAFF [203]. Granulocyte-colony stimulating factor (G-CSF) and IFN-γ-stimulated neutrophils are a prominent source of BAFF [204]. In myeloid cells, the binding of immune complexes to high-affinity Fc receptors for IgG (FcγRI) increases BAFF processing [205].  	   50	  1.11.5. The Role of Elevated BAFF in Disease In humans, elevated levels of soluble BAFF are present in: 1) primary immunodeficiencies associated with defects in B cell development; 2) autoimmune conditions; 3) inflammatory/infectious conditions associated with B cell abnormalities and 4) B cell malignancies (Table 1.1).  Table 1.1: Diseases associated with elevated soluble BAFF Category Diseases Inherited primary immunodeficiencies involving genes required for B cell development Wiskott-Aldrich syndrome [206] BAFF-R, TACI, BTK, CD40L and ICOS deficiency [207] Autoimmune inflammatory diseases with B cell dysregulation – autoantibodies, immune complexes rheumatoid arthritis [208] systemic lupus erythematosus [209] Sjogren’s syndrome [210] chronic graft-versus-host disease [211] myasthenia gravis [212] ANCA-associated vasculitis [213] systemic sclerosis [214] autoimmune pancreatitis [215] idiopathic thrombocytopenic purpura [216] sarcoidosis [217] common variable immunodeficiency [218] chronic inflammatory demyelinating polyneuropathy [219] giant cell arteritis and polymyalgia rheumatic [220] Infections, inflammatory and allergic conditions associated with alterations in peripheral B cell subsets hepatitis C [221] hepatitis B [222] malaria [223] HIV [224] visceral leishmaniasis [225] allergic rhinitis [226] asthma [227] atopic dermatitis [228] B cell malignancies multiple myeloma [229] pre-B acute lymphoblastic leukemia [230] B cell chronic lymphocytic leukemia [231] B cell non-Hodgkin lymphoma [232]   	   51	  In B cell primary immunodeficiencies, the increase in BAFF is hypothesized to be a compensatory effect to overcome absence or delayed/arrested maturation of B cells. This presupposes that steady-state BAFF concentrations depend on the number of B cells as well as the expression of BAFF receptors. Patients with primary antibody deficiencies, including severe functional B cell defects such as BAFF-R deficiency, were found to have higher BAFF levels than asplenic individuals, patients after anti-CD20 B cell depletion, chronic lymphocytic leukemia patients, or healthy donors [233]. In a comparable manner, mice constitutively expressing human BAFF were found to have higher concentrations of BAFF in the absence than in the presence of B cells.  In autoimmune and inflammatory diseases, there is a strong inverse relationship between sBAFF levels and surface BAFF-R expression on B cells.  This inverse relationship is observed in Sjögren’s syndrome, HIV, hepatitis C, malaria, chronic graft-versus-host disease (GVHD) and common variable immunodeficiency (CVID) [233-238]. In the same diseases, there is also a relative expansion of immature transitional B cells at the expense of mature B cell subsets, similar to human BAFF-R deficiency. It is possible that high levels of sBAFF induce BAFF-R internalization and down-regulation. This is a common mechanism for limiting receptor signaling. However the increase in levels of circulating BAFF is unable to overcome the defect in B cell development and function. This suggests that the primary defect in diseases with elevated sBAFF is due to the dysregulated expression or function of BAFF-R. An alternative possibility is that there is a distinct sBAFF isoform whose function is to block BAFF-R signaling by rendering the receptor unavailable through internalization.  	   52	  The last category represents malignant B cell disorders, which aberrantly express BAFF-R and BAFF as part of autocrine and paracrine signaling pathways that increase survival and chemoresistance of malignant B cells. This phenomenon has been demonstrated in cell lines and primary samples from patients with chronic lymphocytic leukemia (CLL), multiple myeloma (MM), B cell non-Hodgkin lymphoma (NHL) and precursor acute lymphoblastic leukemia (pre-B ALL) [307-312]. The production of autocrine BAFF in these diseases protects malignant B cells against spontaneous and drug-induced apoptosis.  This pathway is currently being evaluated as a target in pre-B ALL mouse models with a humanized anti-BAFF-R monoclonal antibody optimized for FcRyIII-mediated, antibody-dependent cell killing by effector cells [313]. There are also a number of anti-BAFF monoclonal antibodies such as the blocking monoclonal antibody, belimumab and Fc fusion decoy receptors, such as BAFF-R-Fc that are clinical approved or currently in clinical trials for the treatment of autoimmune diseases [314], although they are yet to be evaluated in the treatment of B cell malignancies.   The association of high levels of sBAFF with a diversity of clinical phenotypes from immunodeficiency to malignant lymphoproliferation suggests that BAFF may have biological effects hereto unknown that could be modulated by the variety of modifications BAFF undergoes. To date, little is known about the presence of different BAFF isoforms and their role in human B cell biology.      	   53	  1.12. Primary Hypotheses and Aims Primary Hypotheses 1. Studying primary immunodeficiencies associated with peripheral B cell development and reduced BAFF-R expression will provide novel insights into BCR signaling and reveal mechanisms regulating surface BAFF-R expression in human B cells. 2. The soluble BAFF present in immunodeficiencies and autoimmune inflammatory disorders compared to B cell malignancy are different isoforms with opposing functional and structural properties. Aims Specific Aim #1: We will identify the genetic cause of two previously undefined primary immunodeficiencies with reduced surface BAFF-R expression, elevated soluble BAFF and defects in B cell development and then characterize the functional effects of the mutated genes on the BCR and BAFF-BAFF-R signaling pathways.  Specific Aim #2: We will functionally characterize the role of BAFF in pre-B acute lymphoblastic leukemia, a B cell malignancy with aberrant BAFF/BAFF-R expression.       	   54	  Chapter 2: Materials and Methods 2.1. Patient Samples Work done on blood samples from the affected children with MALT1 and PLCγ2 mutations and their family members were covered by the Child & Family Research Institute Research Ethics Board Certificate number H09-01228. Plasma samples from patients’ post-hematopoietic stem cell transplantation were collected from patients enrolled in the Canadian Institute of Health Research (CIHR) funded biomarker study entitled “Biomarker in Chronic Graft vs. Host Disease”. Institutional Review Boards at each participating center approved the study, and informed consent was obtained in accordance with the Declaration of Helsinki from patients. Our local approval is H09-01141. Plasma was collected at onset of chronic GVHD or at 3, 6 or 12 months after HSCT for patients without chronic GVHD. Peripheral blood was collected in heparinized tubes and shipped at room temperature by overnight courier. The blood was spun at 500 x g for 15 minutes at room temperature in the original blood collection tubes. Under sterile conditions, the upper layer of plasma was transferred to a new tube and centrifuged at maximum speed in a tabletop micro centrifuge for 15 minutes at 4°C to deplete patients. The platelet depleted plasma was frozen at -80°C.  Plasma samples were also collected from patients enrolled on the Children’s Oncology Group ASCT0431 trial. ASCT0431 was a randomized phase III study comparing the post-transplant event-free survival of pediatric patients with relapsed acute lymphoblastic leukemia undergoing allogeneic HSCT treated with GVHD prophylaxis comprising tacrolimus and methotrexate with or without sirolimus. Subjects were between 1 and 29 years old at the time of study entry. Institutional Review Boards at each 	   55	  participating center approved the study, and informed consent was obtained in accordance with the Declaration of Helsinki from parents of patients. Plasma was collected at study entry and at 1, 3 and 6 months after HSCT. Peripheral blood was collected in heparinized tubes and shipped at room temperature by overnight courier. The blood was spun at 500 x g for 15 minutes at room temperature in the original blood collection tubes. Under sterile conditions, the upper layer of plasma was transferred to a new tube and centrifuged at maximum speed in a tabletop micro centrifuge for 15 minutes at 4°C to deplete patients. The platelet depleted plasma was frozen at -80°C. Blood was collected from healthy donors and cord blood was collected from deliveries preformed at BC Women’s Hospital as part of a local research study approved by our Institutional Review Board Certificate number H10-01954: Analyses of normal blood as controls for studies of B-ALL and GVHD.  2.2. Cell Lines and Reagents BAFF was captured or inhibited using the clinical anti-BAFF monoclonal antibody, belimumab (BENLYSTA®, GlaxoSmithKline Inc.). Recombinant human BAFF-R:Fc (Enzo Life Sciences, ALX-522-060) was also used to block soluble BAFF. We used G-CSF (Filgrastim, NEUPOGEN®, Amgen, Thousand Oaks, CA) and recombinant IFN-γ (Biolegend). The recombinant human soluble BAFF was an unmodified 152 amino acid polypeptide (aa 134-285 of full length BAFF) produced in E. coli with molecular weight of 17.0 kDa (PeproTech, catalog # 310-13). Mass spectrometry confirmed the molecular weight.   The human 697, RS4;11, 380 and NALM-6 relapsed pre-B ALL cell lines were purchased from DSMZ (Braunschweig, Germany). The monocytic U937 and T-cell ALL 	   56	  Jurkat cell lines were purchased from the ATCC (Manassas, VA, USA). All cell lines were grown in RPMI medium (Gibco), 10% fetal bovine serum, 1% L-glutamine and 1% penicillin/streptomycin. The wild-type and PLCγ2 knockout DT40 chicken B cell lines were a kind gift from Dr. Michael Gold (University of British Columbia) and grown in RPMI medium (Gibco), 10% fetal bovine serum, 1% chicken serum, 1% L-glutamine and 1% penicillin/streptomycin. Viability of cells collected from the medium was determined by examining and manually counting total and Trypan blue excluding lymphoblasts under a microscope.   2.3. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) Peripheral blood was collected via a peripheral blood draw, usually from a vein in the antecubital fossa into blood collection tubes containing either heparin or ACD (trisodium citrate, citric acid and dextrose) (BD Vacutainer®). Peripheral blood was diluted with an equal volume of ice-cold phosphate-buffered saline (PBS) (Gibco®) + 2% fetal bovine serum (FBS) (Gibco®) and separated by density gradient using Ficoll-Paque PLUS (GE Healthcare®). Tubes were centrifuged during 35 minutes at 400 x g at room temperature and without brake. The buffy coat was transferred into a new tube and washed once with 50 mL ice-cold PBS + 2%FBS. The pellet was re-suspended in ice-cold PBS + 2%FBS and centrifuged at 100 x g for 10 minutes to deplete platelets and then re-suspended in PBS + 2%FBS.     	   57	  2.4. Isolation of CD19+ B Cells  Human B cells were purified after isolation of PBMCs as described above by negative selection using the StemSep® Human B Cell Enrichment kit according to the manufacturer’s instructions (StemCell Technologies). The purity of the CD19+ fraction was typically >95% and was determined by CD19 expression by FACS.  2.5. Isolation of Neutrophils  For peripheral blood neutrophil isolation, whole blood collected in ACD was mixed 2:1 volumetric ratio with sterilized 6% sodium dextran in 0.9% NaCl, inverted 15-20 times, and left at room temperature for 45 min to 1 hour to sediment erythrocytes. The straw colored upper layer was transferred into a new tube and centrifuged at 450 g for 12 minutes at 4°C using a low brake. The supernatant was discarded and the pellet re-suspended in 12 mL ice-cold distilled water to lysis red blood cells. After 20 seconds, 4 mL of 0.6 M KCl was added and mixed several times. The solution wass then diluted to 50 mL with PBS and centrifuged at 500 g for 6 minutes at 4°C using a high brake. This step was repeated if red blood cells still remained. The supernatant was discarded and the pellet re-suspended in 2.5 mL of PBS. This cell suspension was then separated by density gradient using Ficoll-Paque PLUS (GE Healthcare®). The supernatant was removed and the neutrophil pellet was resuspended in PBS. The neutrophils are put on ice and used within 4 hours. The purity of the neutrophil fraction was typically >95% as determined by CD66b expression by flow cytometry.   	   58	  2.6. Cell Sorting of Cord Blood  Isolated cord blood CD19+ B cells were sorted in a BD FACS Aria sorter using a panel consisting of PE-CD27 (M-T271; Biolegend), APC-CD10 (HI10a; Biolegend) and PerCP-CD19 (HIB19; Biolegend). After incubation overnight with or without recombinant sBAFF, immunophenotyping was preformed using PE or FITC-Annexin V (Biolegend), PE-BAFF-R (clone 11C1; Biolegend), FITC-CD62L (DREG-56; Biolegend) and FITC-CXCR3 (G025H7; Biolegend).   2.7. Exome Library Construction and Sequencing of MALT1 Deficient Patient Whole exome sequencing was performed on the trio of the affected patient and both parents. Genomic DNA was isolated, acoustically sheared, size-selected by PAGE, and end-polished. Genomic DNA fragments were ligated with Illumina library adapters and amplified with Illumina sequencing primers (Illumina, San Diego, Calif). The library was then enriched for exon sequences by using the Agilent Sureselect Human All Exon 50-Mb kit (Agilent Technologies, Santa Clara, Calif). The exome fraction was subjected to massively parallel sequencing on the Illumina Hi-Seq 2000. Between 122 million and 151 million 100 bp paired-end reads were obtained from each sample, achieving 97.4% to 97.5% mapping to the reference genome (Hg18). Between 91.7% and 93.1% of all protein coding bases are covered at a depth of 10× or more for each of the subjects, with average coverage of all protein-coding bases between 88× and 115×.    	   59	  2.8. Bioinformatic Analysis and Identification of the MALT1 Variant Illumina sequence reads were aligned to the human genome (Hg18) by using Burrows–Wheeler Aligner [239]. SAMtools [240] was used to identify single nucleotide variants (SNV) and indels. Results were filtered by using Annovar [241] and manually curated by using the Integrative Genomics Viewer. After bioinformatic filtering and analysis, 27 coding variants were identified, which were (a) homozygous in the affected subject, (b) heterozygous in both parents, (c) conserved on the basis of SIFT analysis [242], and (d) not reported in dbSNP132 (http://www.ncbi.nlm.nih.gov/snp), the Exome Variant Server (http://evs.gs.washington.edu/EVS/; accessed September 2012), or the 1000 genome project [243] at a minor allele frequency of more than 0.01. Of these 27 variants, 23 were removed because they were predicted to be either nondamaging or polymorphic on MutationTaster analysis [244]. Of the 4 remaining variants, familial segregation in the 2 healthy siblings by Sanger sequencing revealed 3 variants to be also homozygous in at least 1 unaffected sibling. This left only 1 variant in the MALT1 gene (NM_006785:c.1739G>C) predicted to be the causal novel homozygous mutation in the patient. 2.9. Quantification of MALT1 Gene and Protein Expression Total RNA was isolated from PBMCs (RNeasy Plus Mini Kit; QIAGEN, Valencia, Calif) and transcribed to cDNA (iScript; Bio-Rad, Hercules, Calif). MALT1 gene expression was measured by using SYBR Green quantitative PCR (Universal SYBR Green Super Mix, Bio-Rad) using gene-specific primers for MALT1 (forward: 5ʹ-TCCAGAGAAGTGTTGATGGCGTCT-3ʹ, reverse: 5ʹ-TGAGGAATAGGGCTTCCAACAGCA-3ʹ) and housekeeping gene ACTB (forward: 5ʹ- 	   60	  GTTGCGTTACACCCTTTCTT-3ʹ, reverse: 5ʹ-ACCTTCACCGTTCCAGTTT-3ʹ). Reactions were run by using a 7300 Real-Time PCR System (Applied Biosystems, Carlsbad, Calif) and relative gene expression was analyzed by using the 2−ΔΔCt using ACTB as a reference gene. Immunoblotting was carried out to quantify MALT1 protein expression by using standard techniques. The membranes were blocked and blotted with various primary antibodies: anti-MALT1 against both the N terminus (EP603Y, Abcam, Cambridge, MA) and the C terminus (H-300, Santa Cruz, Dallas, TX) and anti–β-Actin (Cell Signaling Technology, Danvers, MA). They were subsequently blotted with fluorescently labeled secondary antibodies and imaged by using a LI-COR Odyssey infrared imaging system (LI-COR Bioscience, Lincoln, NE). The MALT1 expression was quantified by analyzing the band densitometry (ImageJ freeware; National Institutes of Health, Bethesda, MD) normalized with β-actin expression.  2.10. Analysis of MALT1 Paracaspase Activity and Molecular Scaffold Function To study MALT1 paracaspase activity, EBV-immortalized B cells (2 × 106) from the patient and family were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL) and ionomycin (1 µM) for 0-4 hours, and the cleavage of BCL10 (anti-BCL10 antibody from Cell Signalling Technology) was analyzed by using immunoblotting. A peptide inhibitor (z-VRPR-fmk, 75 µM, Enzo Life Sciences, Farmingdale, NY) was used to block MALT1 proteolytic activity (30 minutes pretreatment) in select experiments before PMA/ionomycin stimulation for 2 hours. The scaffold function of MALT1 was analyzed by the ability to bind to BCL10 as assessed by the co-	   61	  immunoprecipitation. Immortalized B cells (1 × 107) were stimulated with PMA/ionomycin for 30 minutes, and the cell lysates were generated. The lysates were incubated with MALT1 antibody (H-300, Santa Cruz), and the co-immunoprecipitation was performed by using Pierce Magnetic IP/Co-IP kit (Thermo Scientific, Waltham, MA). Immunoblotting was conducted to probe MALT1 (anti-MALT1 antibody, EP603Y, Abcam) and BCL10 (anti-BCL10 antibody, 331.3, Santa Cruz) signals before and after immunoprecipitation. The binding of BCL10 to MALT1 was quantified by densitometry analysis. 2.11. Immunologic Phenotyping and Analysis of NF-κB Activation by Flow Cytometry B cells were characterized by staining with anti-CD19-Pacific Blue (HIB19, Biolegend), anti-IgM-APC (MHM-88, Biolegend, San Diego, CA), anti-IgD-fluorescein isothiocyanate (FITC) (IA6-2, BD Biosciences, San Jose, CA), anti-CD27-PE (0323, Biolegend), anti-CD38- phycoerythrin (HIT2, Biolegend), anti-BAFF-R-phycoerythrin (11C1, Biolegend) and anti-CD21-fluorescein isothiocyanate (LT21, Biolegend). By staining for CD27 and IgD, naive IgD+IgM+CD27− B cells, IgD+IgM+CD27+ marginal zone B cells, and IgD−IgM−CD27+ switched memory B cells can be distinguished. The staining for CD21 and CD38 expression allows the additional distinction of CD38lowCD21low B cells, CD38+IgMhigh translational B cells, and CD38+IgM− plasmablasts. T cells were characterized by staining with anti-CD3-Alexa Fluor647 (UCHT1, Biolegend), anti-CD4-Brillant Violet 711 (OKT4, Biolegend), and anti-CD8-V450 (RPA-T8, BD Biosciences). To identify FOXP3+CD4+CD25+ cells, PBMCs were first surface stained with anti-CD3-Alexa Fluor488 (SK7, Biolegend), anti-CD127-Brillant Violet 421 (A019D5, Biolegend), and anti-CD25-PE (BC96, Biolegend); then 	   62	  fixed and permeabilized (Human FoxP3 Buffer A/B, BD Biosciences); and finally stained with anti-FoxP3-Alexa Fluor647 (259D, Biolegend) and anti-CD4-PerCP-eFluor710 (SK3, eBioscience, San Diego, CA). TH17 cells were identified by means of intracellular staining of CD4+ T cells for IL-17 production, as previously described. Briefly, 1 × 106 PBMCs were stimulated with 50 ng/mL PMA and 1 µg/mL ionomycin (Sigma-Aldrich, St Louis, MO) in the presence of GolgiPlug (BD Biosciences). Cells were fixed and permeabilized (Cytofix/Perm2; BD Biosciences) and stained with a mixture of anti-CD3-Pacific Blue (UCHT1, Biolegend), anti-CD4-PE-CF594 (RPA-T4, BD Biosciences), and anti-IL17A-PE (N49-653, BD Biosciences). To quantify NF-κB p65 phosphorylation and IκBα degradation, cells were stimulated with 50 ng/mL PMA and 1 µM ionomycin (Sigma-Aldrich). Cells were fixed and permeablized with Phosflow Fix Buffer I and Perm II (BD Biosciences). After washing, cells were stained with an anti-IκBα mAb (L35A5, Cell Signaling Technology) and a secondary anti-mouse IgG1-FITC (poly4053, Biolegend). Cells were washed and stained with a mixture of anti-phospho-NF-κB p65-Alexa Fluor647 (Ser536) (93H1, Cell Signaling Technology) and anti-CD3-PerCP-eFluor710 (SK7, eBioscience). T-cell proliferation was quantified by incubating PBMCs with PHA and staining the cells with antibodies against CD3 and the proliferation marker, Proliferating Cell Nuclear Antigen. IL-2 and IL-6 production was measured by using ELISA (eBioscience) following the stimulation of PBMCs with 1% PHA and 10 ng/mL lipopolysaccharide, respectively. All flow cytometry studies were performed on an LSRII instrument (BD Biosciences) and analyzed by using FlowJo software (TreeStar, Ashland, OR). 	   63	  Table 2.1. Flow cytometry immunophenotyping of peripheral blood circulating B cell subsets B Cell Subset Cell Surface Markers Early transitional  CD19+CD10+CD27-CD21loCD24hiCD38hi Late transitional CD19+CD10+CD27-CD21hiCD24hiCD38hi Naïve CD19+CD27-CD10-IgD+IgM+ Memory CD19+CD27+CD10-IgD- Switched memory CD19+CD27+IgD-IgM-IgG+ Marginal zone  CD19+CD27+IgD+IgM+  2.12. Cloning and Transfection Studies For MALT1 experiments, plasmids used for transfection studies contained full-length MALT1 (transcript variant 1) cloned into pCMV6-Entry with a C-terminal Myc-DDK (FLAG) tag (#RC214639, Origene, Rockville, MD) or a control empty vector. For flow cytometric analysis of NF-κB p65 phosphorylation, 1 × 106 purified CD3+ T cells were transfected with 2 µg plasmid DNA in PBS by using the U-014 program in an Amaxa Nucleofector II device (Lonza, Allendale, NJ). Cell viability and transfection efficiency were assessed by immunofluorescence staining with Fixable Viability Dye eFluor 450 (eBioscience) and Alexa Fluor488-conjugated DYKDDDDK tag antibody (Cell Signaling Technology), respectively. For PLCγ2 experiments, plasmids used for transfection studies contained full-length wild-type PLCγ2 cloned into pCMV6-Entry with a C-terminal Myc-DDK (FLAG) tag (#RC200442, Origene, Rockville, MD) or a control empty vector. A mutant PLCγ2 plasmid corresponding to our patient’s genetic variant was constructed by site 	   64	  directed mutagenesis (QuikChange II Site-Directed Mutagenesis Kit, Agilent Technologies) as per manufacturer’s instructions. For transfection studies, 1 × 106 PLCγ2-/- DT40 cells were transfected with 0.5 µg of plasmid DNA in Opti-MEM (Gibco) by using the B-023 program in an Amaxa Nucleofector II device (Lonza, Allendale, NJ). After overnight incubation, cells were stimulated with 10 µg/mL of anti-chicken IgM (#8300-01, Southern Biotech) in HBSS (no Ca2+, no Mg2+, Life Technologies) + 1% FBS (Gibco) and calcium flux analyzed as per section 2.16.  2.13. Whole Exome Sequencing of Patient With PLCγ2 Mutation WES was performed at Canada's Michael Smith Genome Sciences Centre. Exome library was constructed using the Agilent All Exon V4+UTR capture kit, and sequenced on Illumina Hiseq2000. Reads were aligned to the human genome (GRCh37-lite) using Burrows–Wheeler Aligner and duplicate marked with Picard [239,240]. Variants were called using mpileup (SAMtools), subsequently filtered with varFilter, and annotated using an in-house pipeline that combines SnpEff [245], Ensembl variant database (including pathogenicity prediction), dbSNP [246], NHLBI exome sequencing database (https://esp.gs.washington.edu/drupal/), COSMIC [247], and an in-house human variation database [248]. Annotated variants were filtered according to the inheritance mode and previous reported pathogenicity, with a phenotype of B cell defects.   2.14. B Cell Immunophenotyping  Immunophenotyping on fresh or previously frozen isolated PBMCs was preformed using various combinations of Brilliant Violet 605™-CD19 (SJ25C1; 	   65	  Biolegend), PE-Cy7-CD19 (HIB19; Biolegend), Pacific Blue™-CD19 (HIB19; Biolegend), PerCP-CD24 (ML5; Biolegend), Brilliant Violet 421™-CD38 (HIT2; Biolegend), V450-CD38 (HIT2; BD Bioscience), PE-CD38 (HIT2, Biolegend), PE-CD27 (M-T271; Biolegend), FITC-IgD (IA6-2; Biolegend), FITC-CD21 (Bu32; Biolegend), PE-Cy5-CD21 (B-ly4; BD Pharmingen), Alexa Fluor® 700-CD10 (CB-CALLA; eBioscience), APC-CD10 (HI10a; Biolegend), APC-IgM (MHM-88; Biolegend), FITC-BAFF receptor (8A7; eBioscience), PE-BAFF receptor (11C1; Biolegend). Intracellular staining was done with a combination of BD Cytofix™ incubation on ice for 15 minutes and BD FACS Permeabilizing Solution 2 incubation at room temperature for 10 minutes. Cell apoptosis and necrosis was assessed with FITC-Annexin V with 7-AAD (Biolegend). Cells were analyzed using a LSR II flow cytometer (BD Biosciences) and data was analyzed using FlowJo (Tree Star, Ashland, OR). In chapter 5, cell lines were incubated with PE-BAFF-R (11C1; Biolegend), PE-TACI (1A1; Biolegend) and PE-BCMA (19F2; Biolegend) with appropriate isotype matched controls. Surface expression of BAFF-R on human CD19+ B cells was measured with PE-BAFF-R (clone 11C1; Biolegend). Pacific Blue™-CD19 (HIB19; Biolegend) and PE-CD138 (DL-101; Biolegend) were also used. Cells were analyzed using a LSR II flow cytometer (BD Biosciences) and data was analyzed using FlowJo (Tree Star, Ashland, OR).  2.15. Quantification of BAFF and BAFF-R Levels in Plasma and Media  Measurements of soluble BAFF were done in non-previously thawed plasma using a quantitative sandwich enzyme immunoassay according to the manufacturer’s 	   66	  instructions (Quantikine Human BAFF/BLyS kit, R&D Systems Cat. SBLYS0). Soluble BAFF-R in cell culture supernatant was measured using an ELISA kit according to the manufacturer’s instructions (Elabscience).   2.16. Calcium Flux Assay  PBMCs were washed once in HBSS (no Ca2+, no Mg2+, Life Technologies) + 1% FBS (Gibco) and resuspended in dye loading buffer consisting of 4 uM FLUO-4AM (Molecular Probes) and Probenecid (Life Technologies) in HBSS+1% FBS for 45 minutes at 37°C at a concentration of 1 × 106 PBMCs/mL. Cells were washed again with HBSS+1%FBS and then incubated on ice for 20 minutes with 5 uL of Pacific Blue™-CD19 (HIB19; Biolegend) followed by addition of 1 mL HBSS+1%FBS. Sample was warmed again to 37°C and within 10 minutes baseline fluorescence was detected with FITC filter on LSR II flow cytometer in the CD19+ positive fraction. Intracellular calcium flux was induced by BCR stimulation with 10 ug/mL anti-IgM antibody (Jackson Immunoresearch) followed by the addition of extracellular Ca2+ to measure extracellular flux.    2.17. Intracellular ERK Phosphorylation  Freshly isolated PBMCs were stimulated with 10 ug/mL anti-IgM antibody (Jackson Immunoresearch) at 37°C. At indicated time-points, cells were fixed with BD Phosflow™ Fix Buffer I for 10 minutes at 37°C followed by a wash and permeabilization with BD FACS Permeabilizing Solution 2 for 10 minutes at room temperature. After two washes, cells were stained with V450-CD20 (L27, BD Bioscience), Alexa Fluor 488 – 	   67	  phosphor-PLCg2 (pY759) (K86-689.37; BD Biosciences) and PerCP-eFluor® 710 phospho-ERK1/2 (T202/Y204) (MILAN8R, eBioscience) for 20 minutes at room temperature and analyzed on a BD LSR II flow cytometer.   2.18. Collagen Stimulation of Platelets   Whole blood is collected in a BD Vacutainer® Plus plastic citrate tube (0.109 Molar/3.2% sodium citrate) after a 3 mL discard. 90 uL of whole blood is then immediately mixed with 10 uL of PBS containing indicated collagen (Chrono-log Corporation) concentration for 15 minutes. Subsequently, 5 uL of this mixture is incubated with a flow cytometry antibody mix of 3 uL of FITC-CD61 (VI-PL2; Biolegend) and APC-CD62P (P-Selectin) (AK4; Biolegend) each for 15 minute on ice in the dark. At the end of the incubation, 1 mL of 1% paraformaldehyde solution is added and the final mixture is analyzed on a BD LSR II flow cytometer.  	  2.19. RNA Extraction and cDNA Synthesis  Total RNA was prepared from cells using the RNeasy Mini Kit (Qiagen). cDNA were synthesized using the QuantiTect Reverse Transcription Kit (Qiagen).   2.20. Quantitative Real-Time PCR               The quantitative PCR was performed using SSoFast™ EvaGreen® Supermix (Bio-Rad) in a Bio-Rad CFX96™ Real-Time PCR System. Cycling conditions were as follows: enzyme activation at 95°C for 30 s followed by 40 cycles of 5 s at 95°C and 5 s at 60°C. To confirm the purity and specificity of the reaction, a melting curve analysis 	   68	  was preformed at the end of the PCR by slowly increasing the temperature of the reaction from 65 to 95°C. The RT-PCR products were also analyzed by gel electrophoresis.  Primer pairs used to detect BAFF-R mRNA consisted of the forward primer 5’-GTGGGTCTGGTGAGCTG-3’ and the reverse primer 5’-ACAGAATGATGACCTTGTCCAG-3’ (Integrated DNA Technologies). Primer pairs used to detect PIM-2 mRNA consisted of the forward primer 5’-GCCTCACAGATCGACTCCA-3’ and the reverse primer 5’-CACCCACTTTCCATAGCAGT-3’ (Integrated DNA Technologies).  The control β-actin primers were forward 5’-ACAGAGCCTCGCCTTTG-3’ and reverse 5’-CCTTGCACATGCCGGAG-3’ (Integrated DNA Technologies). Relative gene expression was analyzed by using the 2−ΔΔCt using β-actin as a reference gene.  2.21. Cell Lysis, Immunoprecipitation, SDS-PAGE and Western Blotting  Cell-conditioned media was concentrated using Pierce Protein Concentrators with a 9K molecular-weight cutoff. Cell lysates were prepared using either Cell Lysis Buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin; Cell Signaling) or RIPA Buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin; Cell Signaling) in the presence of protease inhibitor cocktails (Roche). Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membrane and blocked with 5% BSA. Proteins were detected by primary antibodies including: rabbit anti-BAFF (C-	   69	  terminus) polyclonal antibody raised against peptide corresponding to amino acids 254 to 269 of human BAFF (Millipore). After washing bound antibody was detected with HRP-conjugated anti-rabbit secondary antibody and Novex ECL chemiluminescent substrate (Invitrogen).  Soluble BAFF was removed from cell-conditioned media by immunoaffinity chromatography using the same mouse anti-human BAFF monoclonal antibody (clone 137314; R&D Systems) used in the R&D ELISA kit or the clinical anti-BAFF monoclonal antibody, belimumab (BENLYSTA®, GlaxoSmithKline Inc.). The mAb was immobilized in a AminoLink column (Thermo Scientific). Soluble BAFF was affinity purified by gravity-flow through the column.  2.22. Mass Spectrometry Intact protein mass determination was performed on a Waters Xevo GS-2 mass spectrometer as previously described [249]. BAFF was digested in solution as previously described [250]. Digest products were then loaded onto a Bruker Impact II Q-ToF mass spectrometer. Peptide separation was carried out on a 50cm in-house packed 75 um C18 column by a Proxeon EasynLC UPLC system. MS/MS data were extracted to mascot generic format (MGF) file format, and submitted to the Global Proteome Machine for database search using the X!Tandem algorithm. Search parameters were as follows: +/- 20 ppm precursor mass accuracy, 0.4 da fragment ion mass accuracy, semi-specific trypsin enzyme specificity, and a refined search for all known post-translational modifications for identified proteins.  	   70	  2.23. Enzymatic Removal of N-Glycans  Concentrated cell-conditioned media or cell lysates were incubated with PNGase F (New England Biolabs) at 25°C for 4-5 days as per the manufacturer’s instructions.   2.24. Statistical Analysis  Descriptive statistics were generated on all data using Prism version 6 for Mac (GraphPad Software, San Diego, CA). Significance of observed changes was determined using paired and unpaired T tests and two-tailed Mann-Whitney test.                	   71	  Chapter 3: Combined Immunodeficiency Due to MALT1 Deficiency  3.1. Overview The first patient with an undiagnosed primary immunodeficiency we chose to study to test the first hypothesis was a 15-year old girl with severe B cell lymphopenia and near absent surface expression of BAFF-R on her peripheral B cells. Her peripheral B cell phenotype was characterized by reduced transitional, but elevated percentage of mature naïve B cells, near absent marginal zone B cells and reduced switched memory B cells. We hypothesized that characterizing the underlying molecular defect in this patient would provide novel insights into BCR signaling and the regulation of BAFF-R expression in human B cells.   We discovered a novel homozygous mutation inherited from consanguineous heterozygous carrier parents in mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), a crucial intermediary in NF-κB signal transduction in lymphocytes. Based on ex vivo and in vivo experiments and successful hematopoietic stem cell transplant of the patient, we demonstrate that MALT1 is necessary for the surface expression of BAFF-R on B cells and the presence of transitional B cells.  3.2. Case History The patient is a 15-year old girl with normal intellectual development who was born after an uncomplicated term pregnancy to parents of Kurdish descent, who are first cousins through their fathers (Fig. 3.1A). Family history was otherwise unremarkable. Although birth weight and length were normal, she has had short stature and low weight 	   72	  since age 2 years (Fig. 3.1B). An eczematous rash developed at 2 weeks of age, and severe, widespread, treatment-resistant dermatitis has persisted. There have been multiple episodes of bacterial super-infection of the skin, primarily with Staphylococcus aureus, and also vesicular dermatitis due to varicella-zoster virus and herpes simplex virus 1. Severe inflammatory gastrointestinal disease has been a challenge since infancy, requiring Nissan fundoplication and esophageal stricture dilatation. She is currently fed a hypoallergenic elemental formula-diet via jejunostomy tube. Multiple episodes of pneumonia, with organisms including Staphylococcus aureus, Streptococcus pneumonia and cytomegalovirus, have contributed to the development of chronic inflammatory lung disease, airways hyper-responsiveness, bronchiectasis, and nail clubbing. She has fractured her femur and both tibiae after low impact injuries. Other notable features include recurrent generation of chronic granulation tissue involving the vocal cords, larynx, and external auditory canal and severe periodontal disease.  Physical examination is notable for multiple non-specific dysmorphic facial features, nail clubbing without cyanosis, and widespread excoriated and lichenified plaques of maculopapular dermatitis with diffuse hypopigmented scarring. Radiologic imaging revealed no evidence of skeletal dysplasia or metabolic bone disease but her bone age was significantly delayed (by 4 years ± 11 months) and bone mineral density was very low (Z-score=-7.5). Histopathology showed an atypical, non-specific chronic CD3+ T cell lymphocytic infiltration in the skin and throughout the gastrointestinal tract interfering with the normal epithelial maturation sequence (Fig. 3.1C).   	   73	     Figure 3.1. Family pedigree, growth chart and histopathology. A. Family pedigree. B. Growth chart demonstrating profound failure of linear growth and weight gain. C. Patient (MALT1mut/mut) skin demonstrating dense chronic lymphocytic infiltrates (arrow) associated with acanthosis, basal cell hyperplasia (star), and parakeratosis. Patient’s antral-type gastric mucosa demonstrates inflammatory infiltrates (star) with 	  	  	   	  	   74	  intraepithelial lymphocytes and neutrophils. Surface epithelium is immature and lacks tall columnar differentiation (arrow) as is seen in normal antral-type epithelium  3.3. Laboratory Investigations   3.3.1. Lymphocyte Cell Counts Lymphocyte numbers were normal for age but there was persistent moderate eosinophilia (maximum = 1.67 × 109 /L; reference range <0.5× 109 /L). The most striking finding was the very low number of B cells in the peripheral blood (CD19+ fraction = 1% (reference range 8-24%); absolute = 0.05 × 109/L (reference range: 0.2-0.6× 109 /L)).  3.3.2. B Cell Function  Humoral immunity was characterized by chronically elevated serum IgE (maximum = 9856 IU/mL; reference range <200 IU/mL) with normal serum IgG, IgA and IgM levels. The patient was able to generate protective antibody titers against a variety of infectious agents and vaccine antigens (i.e. tetanus, diphtheria, measles, mumps, rubella, varicella zoster), as well as isohemagglutinins against blood group antigens.   3.3.3. Flow Cytometric Immunophenotyping of B Cell Subsets  Due to B cell lymphopenia, the B cell compartment was analyzed by flow cytometric immunophenotyping to identify a possible B cell differentiation arrest. Immunophenotyping showed a: 1) near absence of transitional B cells (Figure 3.2A, B, C and D); 2) abundant fraction of naïve B cells (Figure 3.2C, D and E); 3) reduced fraction 	   75	  of memory B cells (Figure 3.2C, D and E) and 4) absent marginal zone B cells (Figure 3.2E). There was also significantly reduced surface expression of BAFF-R (clone 11C1) on the patient’s CD19+ B cells (Figure 3.2F).     MALT mut/mut MALT mut/+ Healthy Control CD21 CD38 CD27 CD10 A. B. C. 	   76	     Figure	  3.2. B cell subset immunophenotyping of CD19+ B cells from patient (MALT1mut/mut), sibling (MALT1mut/+) and healthy donor. A. CD21+/lowCD10+ transitional B cells. B. CD38highCD10+ transitional B cells. C. CD27+CD10- memory B CD24 D. CD38 CD27 E. IgD F. %	  of	  Max F. 	   77	  cells, CD27-CD10- naïve B cells and CD27-CD10+ transitional B cells. D. CD24highCD38high transitional B cells, CD24highCD38low memory B cells and CD24lowCD38low naïve B cells. E. CD27+IgD+IgM+ marginal zone B cells, CD27+IgD-IgM- switched memory B cells and CD27-IgD+IgM+ naïve B cells. F. Surface BAFF-R (clone 11C1) expression on CD19+ B cells from patient (MALT1mut/mut) (light gray), sibling (MALT1mut/+) (dashed black line) and healthy donor (black line) with an isotype control (solid gray). 7AAD was used to exclude dead cells. Each of these plots represents three independent experiments.   3.3.4. Other Immune Cell Subsets Due to the near absence of circulating B cells, the fraction of CD3+ T cells was elevated at 0.97 (reference range 0.52-0.78) with an abnormal skewing towards the CD4+ T helper subset (fraction of 0.75 (reference range 0.25-0.48)) resulting in an elevated absolute count of 2.61 × 109/L (reference range 0.40-2.10× 109/L). CD25+CD127lowFoxP3+ regulatory T cells were present at the same percentage of CD4+ T cells in the patient, heterozygous family members, and healthy controls (Figure 3.3). TH17 cells comprised 0.5% of the patient’s CD4+ T cells, which was comparable to heterozygous family members and healthy controls, and greater than 0.2%, which has been suggested as the normal threshold [251]. Natural killer (NK) cell counts were normal. Lymphocyte stimulation testing revealed the absence of proliferation and blast formation in CD3+ T cells following exposure to phytohemagglutinin (PHA), as well as a failure to secrete IL-2 following TCR stimulation (Figure 3.4 and 3.5). Interestingly, the 	   78	  level of IL-2 secretion in the heterozygous sibling (MALT1mut/+) was approximately one half the level of the normal controls.         Figure 3.3. CD25+CD127lowFoxP3+ regulatory T cells as a percentage of CD4+ T cells in the patient (MALT1mut/mut), sibling (MALT1mut/+), and healthy controls. Representative of two independent blood draws.    	   79	   Figure 3.4. Change in cell size of MALT1-mutated CD3+ T cells (MALT1mut/mut) after stimulation with phytohaemagglutinin (PHA) for 5 days. The forward scatter (FSC) measurement on a flow cytometer was used as a measure of cell size. 	   80	    Figure 3.5. Secretion of IL-2 by primary T cells after PHA-dependent T cell receptor stimulation. IL-2 secretion was measured by ELISA (eBioscience) in patient (MALT1mut/mut), siblings (MALT1mut/+) and heathly controls. Individual points represent data from individual samples with 2 biological repeats while the horizontal line denotes the mean ± SEM. Statistical comparisons were made by using an unpaired t test. * P < .05, ** P < .01   3.4. Bioinformatic Analysis and Identification of the MALT1 Variant Whole-exome sequencing of the trio of affected patient and unaffected parents, combined with targeted Sanger sequencing of the 2 healthy siblings, identified 1 variant in MALT1 predicted to be the casual novel homozygous mutation.     	   81	  Table 3.1: Variant prioritization based on autosomal recessive inheritance. dbSNP – Single Nucleotide Polymorphism Database, TGP – The Gene Partnership, NHLBI EVS – National Heart, Lung, and Blood Institute Exome Variant Server, MAF – minor allelic frequency  Number of Variants Homozygous variants present in patient that are heterozygous in both parents 13 Not present in dbSNP, TGP, or NHLBI EVS (MAF>1%) 9 Predicted to be damaging 4/9 Homozygous in unaffected brother(s) 3/4 Homozygous, damaging, novel variant 1   This variant, a single base pair substitution (NM_006785:c.1739G>C), converts a tryptophan to serine (NP_006776.1:p.Trp580Ser) in the C-terminal domain. Multiple sequence alignment showed that Trp580 is highly conserved (Fig. 3.6A). The crystal structure of MALT1 revealed Trp580 to be part of an alpha-helical linker region, and its aromatic group interacts with hydrophobic residues of the caspase-like domain (Fig. 3.6B).      	   82	     Figure 3.6. Site of mutation in MALT1 protein. A. Domain structure of MALT1 protein and amino acid sequence alignment of the Trp580 mutation site of MALT1 in Homo sapiens with homologs of various metazoan species. Side chains shown to interact with Trp580 in tertiary structure are boxed. *, Identical amino acids; :, conserved substitutions; ., semi-conserved substitutions. B. Crystal structure of MALT1 highlighting the caspase-like domain (green) and the remaining C-terminal domain (gray). Crystal structure was generated using Swiss-PdbViewer.  	   83	  3.5. Functional Evaluation of MALT1 Mutation in Patient  3.5.1. Quantification of MALT1 Gene and Protein Expression  The MALT1 mutation did not affect its own gene expression (Fig. 3.7), but it did result in very low expression of MALT1 protein, suggesting an effect on protein stability, structure, or dynamics (Fig. 3.8A and 3.8B).     Figure 3.7. Quantification of MALT1 gene expression. MALT1 gene expression relative to β-actin gene expression was measured in cDNA isolated from peripheral blood mononuclear cells by quantitative PCR in healthy controls, heterozygous family members (MALT1mut/+), and the homozygous patient (MALT1mut/mut). Individual points represent data from separate blood samples while the horizontal line denotes the mean ± SE 	   84	     A.	   B. 	   85	   Figure 3.8. Quantification of MALT1 protein expression. A. MALT1 protein expression in isolated PBMCs was measured in MALT1mut/mut patient (IV1) compared to MALT1mut/+ siblings (IV2 and IV3), parents (III1 and III2) and healthy controls by immunoblotting using anti-MALT1 antibodies against both the N and C terminus. B. The MALT1 expression was quantified by analyzing the band densitometry normalized with β-actin expression. Individual points represent data from separate blood samples while the horizontal line denotes the mean ± SEM. Statistical comparisons were made by using an unpaired t test. * P < .05    3.5.2. Effect of Mutation on MALT1 Activity Based primarily on murine studies, MALT1 has been shown to promote canonical NF-κB signaling through proteolytic cleavage as a paracaspase and by acting as a scaffold for the CBM complex. We interrogated the proteolytic and scaffold function of MALT1 in our MALT1 deficient patient to confirm the roles of human MALT1. We hypothesized that the patients’ lymphocytes would lack MALT1-mediated BCL10 binding and cleavage.    3.5.2.1. Scaffold Function in CBM Complex Formation  A co-immunoprecipitation assay was used to examine the protein-protein interactions involved in forming the CBM signalosome complex.  The MALT1-mutated cells lacked scaffold function as demonstrated by the inability of MALT1mut/mut protein to stably bind to BCL10 (Figure 3.9A and B).  	   86	   Figure 3.9. Scaffold function of MALT1 as determined by ability to bind BCL10. A. EBV-immortalized B cells from the patient (MALT1mut/mut) and all four heterozygote family members (MALT1mut/+) were stimulated with PMA/I for 30 minutes. Cell lysates were incubated with MALT1 antibody and co-immunoprecipitation was performed. Immunoblotting was conducted to determine MALT1 and BCL10 signals before and after immunoprecipitation. B. The binding of BCL10 to MALT1 was quantified by densitometry analysis. Individual points represent data from individual samples with 2 biological repeats while the horizontal line denotes the mean ± SEM. Statistical comparisons were made by using an unpaired t test. **P < .01. 	   87	   3.5.2.2. Paracaspase Activity MALT1 paracaspase activity was studied by visualizing the cleavage of a known substrate, BCL10. Uncleaved BCL10 contains a full-length carboxy-terminal Glu-C peptide (amino acids 220–233); the cleaved form of BCL10 contains a carboxy-terminal Glu-C peptide (amino acids 220–228) lacking the terminal 5 amino acids. MALT1 activity is triggered via the activation of PKC isoforms by phorbol 12-myristate 13-acetate and ionomycin (PMA/I).  A lower band of cleaved BCL10 lacking 5 terminal amino acids failed to appear in EBV-immortalized B cells from the MALT1-deficient patient after PMA/I stimulation (Figure 3.10A). Pretreatment with a selective MALT1 inhibitor, z-VRPR-fml confirmed BCL10 cleavage was mediated by MALT1. The inhibitor was able to prevent PMA/I-stimulated MALT1 cleavage of BCL10 in all four heterozygote family members  - parents (III1 and III2) and siblings (IV2 and IV3) (Figure 3.10B).   	   88	     Figure 3.10. Paracaspase activity as visualized by cleavage of BCL10. Immunoblotting was used to detect a cleaved form of BCL10 lacking the terminal 5 amino acids (arrows) after A. stimulation with phorbol 12-myristate 13-acetate and ionomycin in EBV-immortalized B cells from the MALT1-deficient patient (MALT1mut/mut) compared to one of her heterozygote parents (III1); B. MALT1 specific BCL10 cleavage was confirmed in MALT1mut/+ family members (parents: III1 and III2; siblings: IV2 and IV3) with pretreatment with a paracaspase inhibitor, z-VRPR-fmk.    3.5.2.3. Activation of the Canonical NF-κB Pathway To quantify the impact of MALT1 deficiency on the activation of the canonical NF-κB pathway, we examined 2 key signaling steps: 1) degradation of the inhibitory protein IκBα and 2) phosphorylation of the p65/relA subunit. Following stimulation with PMA and ionomycin, there was an absence of both IκBα degradation and NF-κB p65 A.	   B.	   	   89	  subunit phosphorylation in the patient’s primary CD3+ T cells (Figure 3.11A and B) and CD19+ B cells (Figure 3.12A and B) as determined by flow cytometry intracellular staining. Interestingly, the peak level of NF-κB p65 subunit phosphorylation in the primary CD3+ T cells of the heterozygous sibling (MALT1mut/+) was approximately one half the level of the normal controls.   	   90	    Figure 3.11. IκBα degradation and NF-κB p65 subunit phosphorylation in primary CD3+ T cells. Primary CD3+ T cells from MALT1mut/mut patient, MALT1mut/+ family members and healthy controls were stimulated with PMA/I for indicated time length and (A) IκBα A. B. 	   91	  degradation and (B) NF-κB p65 subunit phosphorylation were measured by flow cytometry. Each data point is presented as a ratio of MFI to baseline MFI at start of incubation. Values represent means ± SEM compared by using ANOVA with Bonferroni posttest. PMA, Phorbol 12-myristate 13-acetate. ∗P < .05, **P < .01, and ***P < .001.                                            	   92	     Figure 3.12. IκBα degradation and NF-κB p65 subunit phosphorylation in primary CD19+ B cells. Primary CD19+ B cells from MALT1mut/mut patient, MALT1mut/+ family members 5 15 30 600.00.51.01.5PMA/Ionomycin Stimulation  (min)Iκβα Protein ExpressionHealthy ControlsMALT1mut/+MALT1mut/mut*****A.	   5 15 30 60012345PMA/Ionomycin Stimulation  (min)Phospho-NF-κB-p65Protein Expression Healthy ControlsMALT1mut/+MALT1mut/mut**********B.	   M	   93	  and healthy controls were stimulated with PMA/I for indicated time length and (A) IκBα degradation and (B) NF-κB p65 subunit phosphorylation were measured by flow cytometry. Each data point is presented as a ratio of MFI to baseline MFI at start of incubation. Values represent means ± SEM compared by using ANOVA with Bonferroni post-test. PMA, Phorbol 12-myristate 13-acetate. ∗P < .05, **P < .01, and ***P < .001.  3.6. Establishing Causality of MALT1 Mutation  To definitively establish that the MALT1 gene variant is responsible for the patients’ immunological phenotype we: 1) reconstituted expression with a transfected MALT1 plasmid in the patient’s primary lymphocytes and 2) evaluated for changes in the immunological phenotype after correction of MALT1 deficiency in hematopoietic cells through allogeneic bone marrow transplantation.   3.6.1. MALT1 Transfection of  Primary T Cells Restores NF-κB Signaling Transfection of MALT1mut/mut primary CD3+ T cells with a normal MALT1 protein expressing plasmid rescued their ability to activate NF-κB (p65 phosphorylation), while control transfection with an empty vector had no effect (Figure 3.13).        	   94	    Figure 3.13. Reconstituted expression of the normal MALT1 protein in patient’s primary CD3+ T cells. The patient’s primary CD3+ T cells were transiently transfected with a vector encoding myc-DDK-tagged MALT1 and control myc-DDK vector and stimulated with PMA/I for indicated times. NF-κB p65 subunit phosphorylation was assessed by flow cytometry. All values represent means ± SEM of three separate experiments compared by using ANOVA with Bonferroni posttest. PMA, Phorbol 12-myristate 13-acetate, I , Ionomycin. ∗P < .05, **P < .01, and ***P < .001.      	   95	  3.6.2. Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) of MALT Deficiency Patient  The patient successfully underwent a reduced intensity conditioning HSCT from an HLA-matched sibling (MALT1 mut/+) donor. At one year post-HSCT, XX/XY fluorescence in-situ hybridization (FISH) chimerism testing on total peripheral blood lymphocytes indicated that 87% of interphase cells observed were donors cells. The transplant served as an in vivo experiment to confirm that the abnormalities in the immune system were intrinsic to the immune cells and not due to environmental factors.    Figure 3.14. MALT1 Protein Expression and NF-κB Signaling in Primary Lymphocytes Post-HSCT. MALT1 protein expression (timepoint 0) in isolated PBMCs rested overnight was measured in MALT1 mut/mut patient post-HSCT compared to MALT1 mut/+ and healthy control by immunoblotting using anti-MALT1 antibody against N terminus. Cells were also stimulated with PMA/I for indicated times and phosphorylation of p65 and Iκβα degradation was measured by immunoblotting; PMA, Phorbol 12-myristate 13-acetate, I , Ionomycin  0 15 30 2h 0 15 30 2h 0 15 30 2h 0’ PMA/I MALT1	  mut/+ Patient	  Post-­‐HSCT Normal 100 75 50 37 IκBα β-­‐Actin p-­‐p65 MALT1-­‐N 	   96	   MALT mut/mut MALT  mut/+ Healthy  Donor MALT mut/mut MALT  mut/+ Healthy  Donor p65 phosphorylation  Iκβα degradation  A. B. 0 min 5 min 15 min 30 min 60 min 	   97	   Figure 3.15. IκBα degradation and NF-κB p65 subunit phosphorylation in primary CD19+ B cells and CD3+ T cells post-HSCT. Primary CD19+ B cells (A and B) and CD3+ T cells (C and D) from MALT1mut/mut patient post-HSCT, MALT1mut/+ family members and MALT mut/mut MALT  mut/+ Healthy  Donor MALT mut/mut MALT  mut/+ Healthy  Donor p65 phosphorylation  Iκβα degradation  0 min 5 min 15 min 30 min 60 min C. D. 	  	  	   98	  healthy controls were stimulated with PMA/I for indicated time length and (A and C) NF-κB p65 subunit phosphorylation and (B and D) IκBα degradation were measured by flow cytometry. Black arrows highlight fraction of CD3+ T cell population in MALT1mut/mut patient post-HSCT that had no evidence of NF-κB signaling likely representing mixed chimerism state in CD3+ T cell population post-HSCT.   CD21 CD38 CD27 A. B. C. CD10 MALT mut/mut MALT mut/+ Healthy Donor 	   99	    Figure 3.16. Primary B cell immunophenotyping Post-HSCT. B cell subset immunophenotyping of CD19+ B cells from patient (MALT1mut/mut), parent (MALT1mut/+) and healthy control. A. CD21+/lowCD10+ transitional B cells. B. CD38highCD10+ transitional B cells. C. CD27+CD10- memory B cells, CD27-CD10- naïve B cells and CD27-CD10+ transitional B cells. D. CD27+IgD+IgM+ marginal zone CD27 D. IgD E. %	  of	  Max 	   100	  B cells, CD27+IgD-IgM- switched memory B cells and CD27-IgD+IgM- naïve B cells. E. Surface BAFF-R (clone 11C1) expression on CD19+ B cells from patient post-HSCT (MALT1mut/mut) (light gray), parent (MALT1mut/+) (dashed black line) and healthy donor (black line) with an isotype control (solid gray). 7AAD was used to exclude dead cells.  MALT1 expression is restored in primary lymphocytes after allogeneic HSCT to levels similar to the sibling (MALT1mut/+) donor (Figure 3.14). Re-establishment of MALT1 expression results in the return of NF- κB signaling in primary lymphocytes (Figure 3.15) and transitional B cells, normal surface BAFF-R expression on CD19+ B cells (Figure 3.16) compared to pre-HSCT (Figure 3.2) suggesting that MALT1 plays a crucial role in these processes. It is too early post-HSCT to properly assess the full reconstitution of more mature B cell subsets, such as memory B cells, as recovery of the adaptive immune system often takes two years.   3.7. Discussion and Future Directions  Three criteria have recently been proposed for deciding if the clinical and experimental data suffice to establish a causal relationship based only on one case [252]. This is applicable to our patient as follows: firstly, the patient’s genotype has not been found to occur in any individuals without the clinical phenotype. Secondly, our experimental studies indicated that the genetic variant impaired the expression of MALT1 leading to absent MALT1 dependent paracaspase activity and canonical NF-κB signaling. Lastly, the causal relationship between the candidate genotype and the clinical phenotype were confirmed by restoring NF-κB signaling in primary T cells by 	   101	  transfection with an un-mutated MALT1 construct and the patient clinically improved after allogeneic hematopoietic stem cell transplantation restored intrinsic MALT1 function. Features that should raise suspicion for loss-of-function mutations affecting the CBM complex include combined immunodeficiency with normal T cell numbers, abnormal T cell proliferation, and failure to activate NF-κB following stimulation with PMA. Given the normal T cell numbers, TREC-based newborn screening may well fail to identify patients with mutations in the CBM complex. Therefore, when there is a clinical suspicion of a CBM mutation and standard testing reveals a failure of TCR stimulation-induced proliferation, more specific tests should be pursued including assessment of PMA-induced NF-κB activation and genetic testing. One of the most striking changes observed in mice and humans with targeted deletions in members of the CARD11/MALT1/Bcl10 (CBM) complex is a developmental block in B cell differentiation at the transitional B cell stage. The exact reason for this developmental block remains unknown. In addition to B cell receptor (BCR) signaling, B cell development at the transitional stage also relies on BAFF-receptor expression and signaling. Mice and human subjects with loss of function mutations in the BAFF receptor also have a similar block at the transitional stage. We and other groups have shown that B cells from MALT1 and CARD11 patients have low surface BAFF-R expression [253]. Interestingly, Bcl10 deficiency has no apparent effect on the expression of BAFF-R suggesting that BAFF-R up-regulation occurs via a CARD11/MALT1-dependent, but Bcl10-independent signaling pathway [254].  	   102	  It has been shown that BAFF-R is a potential NF-kB target gene [255]. BCR signaling induces activation of c-Rel, an active NF-κB transcription factor, in B cells leading to increased expression of BAFF-R. It has been shown that c-Rel has affinity for the putative promoter sites upstream of the BAFF-R gene [256]. These data suggest that c-Rel plays a critical role in the expression of BAFF-R.  Activation of NF-κB dimers is due to IKK-mediated phosphorylation-induced proteasomal degradation of inhibitory IκB molecules (IκBα and IκBβ), enabling the active NF-κB transcription factor subunits (RelB, RelA and c-Rel) to translocate to the nucleus and induce target gene expression. BCR signaling in murine MALT1-/- cells induces the degradation of both IκBα and IκBβ but extent of degradation is much lower compared to WT B cells. Therefore MALT1-/- B cells activated NF-κB but with less intensity then WT cells. Similar degrees of RelA DNA binding activities were detected in BCR-stimulated WT and MALT1-/- cells. However, complexes containing c-Rel were only induced in BCR-stimulated WT cells, not in those lacking MALT1. Efficient BCR-mediated c-Rel translocation was only observed in WT cells [257]. BCR ligation in MALT1-/- cells induced a substantial increase in only nuclear RelA, not c-Rel. This suggested that there was a defect in release of c-Rel from IκB molecules; the extent of degradation of IκBα and IκBβ is lower in MALT1-/- B cells after stimulation. Stimulation in WT and MALT1-/- cells liberated similar amounts of RelA from IκBα. In contrast, whereas c-Rel was efficiently released from both IκB molecules in WT cells, c-Rel remained bound to IκBα and IκBβ in MALT1-/- B cells. MALT1 specifically directs BCR signals to the degradation of IκBα and IκBβ molecules that are in complex with c-Rel. IκB molecules do not possess putative MALT1 cleavage sites (LVSR amino acid 	   103	  sequence). Therefore, the substrate responsible for MALT1-dependent c-Rel activation in B cells remains to be identified.  More importantly, is the role of MALT1 activity in this process dependent on its proteolytic or scaffolding function?  NF-κB activating pathways are triggered by a variety of extracellular stimuli and lead to the phosphorylation and subsequent proteasome-mediated degradation of inhibitory IκB molecules. This is classically mediated by the IκB-kinase (IKK) complex. IKK mediates IκBα phosphorylation on N-terminal serine residues (S32 and S36). Surprisingly however, treatment with a cell-permeable MALT1 protease inhibitor did not affect IκB phosphorylation by the IKK complex in murine B cells [258]. This suggests that MALT1 activity potentiates NF-κB activation in an IKK-independent manner. There is a signaling pathway that is independent of IKK, still requires the proteasome and is triggered by DNA damage such as UV or doxorubicin. UV radiation induces IκBα degradation via the proteasome, but the targeted serine residues (S-283, S-289 and S-293) are located within a C-terminal cluster, which is recognized by the p38-activated casein kinase 2 (CK2) [259]. This pathway may be relevant in B cells as it has been shown that Csnk2β, the regulatory subunit of protein kinase CK2, modulates peripheral B cell development by promoting a proper BCR signal transmission [260]. It is not known whether CK2 interacts with MALT1 in B cells. How MALT1 potentiates the activation of c-Rel in BCR-stimulated B cells in an IKK-independent manner is a research question we are currently pursuing. The novel discovery of MALT1 deficiency also provides unique insight into the potential clinical consequences of MALT1 inhibition to help guide the development of MALT1 inhibitors for the treatment of lymphoma and inflammatory conditions. MALT1 	   104	  was initially described in human diseases as a proto-oncogene that contributes to tumorigenesis in diffuse large B-cell lymphoma (DLBCL) of the activated B cell (ABC) subtype [253].  Recent findings suggest that such lymphomas may be sensitive to treatment with MALT1 inhibitors [262]. Several of our results suggested that there was a gene dosage effect present in the heterozygote sibling, which was identified in primary T cells in regards to IL-2 secretion and p65 phosphorylation. In these two experiments, the maximal output was approximately half that of the control samples. There did not appear to be any dosage effect in B cell testing indicating perhaps that primary T cells would be more sensitive to partial MALT1 inhibition leading to side-effects in patients treated with MALT1 inhibitors resembling T cell immunodeficiency. For example, these patients would be more susceptible to viral infections.  	   The patients’ cells also provide a unique opportunity to identify novel targets of MALT1 and previously unidentified biological pathways mediated by MALT1 activity. For example, 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics was recently used to identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate [263].         	   105	    Chapter 4: Primary Humoral Immunodeficiency Due to Gain-of-Function PLCγ2 Mutation  4.1. Overview The second patient with an undiagnosed primary immunodeficiency we chose to study was a 6-year old girl with hypogammaglobulinemia associated with a block in early peripheral B cell development, low surface BAFF-R expression and increased soluble BAFF levels. We discovered a novel heterozygous mutation in phospholipase C gamma 2 (PLCγ2), a transmembrane signaling enzyme that catalyzes the production of the second messenger molecules diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) and is involved in B cell receptor-induced calcium flux in B cells.  4.2. Case History  A 6 -year old girl of Lebanese descent was admitted to our hospital with an acute febrile respiratory illness with a 6-month history of chronic lung problems. She had a history of repeated viral (parainfluenza virus type 2, respiratory syncytial virus (RSV)) and bacterial (Haemophilus influenza) respiratory infections and had been followed as an outpatient by the Respirology service for her chronic cough, persistent bilateral infiltrates on her chest x-ray and restrictive lung disease associated with a reduced forced vital capacity (FVC). Testing for cystic fibrosis, tuberculosis, alpha-1 antitrypsin deficiency 	   106	  and HIV were negative. A bronchoalveolar lavage was negative for fungal disease and pneumocystis jirovecii.  On admission her physical examination, other then her acute respiratory distress (increased work of breathing, decreased air entry bilaterally and oxygen saturations below 90% on room air) was normal.  She had normal growth parameters and no dysmorphisms. There was no lymphadenopathy or organomegaly. Her skin was unremarkable and her development was normal.  Past medical history except for her lung issues was unremarkable. She was fully immunized as per our provincial vaccination schedule.   Her parents were non-consanguineous. Her father passed away at the age of 51 years due to a clinical diagnosis of emphysema. At the time of his death he was awaiting a lung transplant. She has no siblings. There was no other family history suggestive of a primary immunodeficiency.   4.3. Laboratory Investigations  She had a normal compete blood count: hemoglobin levels, platelet and white blood cell counts were normal. The immunological assessment revealed agammaglobulinemia and normal T- and B-cell counts (Table 4.1).      	   107	     Table 4.1. Immunological parameters T and B Lymphocytes Blood  Absolute lymphocyte count (units) Result (normal range) Fraction CD3+ (T cell) CD3+CD4+ CD3+CD8+ CD19+ (B cell) CD3-CD56+ (NK cell) 0.81 (0.55-0.78) 0.42 (0.27-0.53) 0.38 (0.19-0.34) 0.13 (0.10-0.31) 0.05 Absolute Counts (×109/L) CD3+ (T cell) CD3+CD4+ CD3+CD8+ CD4/CD8 ratio CD19+ (B cell) CD3-CD56+ (NK cell) 1.96 (0.70-4.20) 1.01 (0.30-2.00) 0.90 (0.30-1.80) 1.11 (0.90-2.60) 0.32 (0.20-1.60) 0.13 Serum Ig Levels (g/L) IgA IgG IgM <0.04 (0.3-2.9) <0.27 (5.1-13.6) <0.03 (0.31-2.08)  Lymphocyte proliferation in response to mitogens, phytohemagglutinin (PHA), pokeweed (PWM) and Staphylococcus aureus Cowan I (SAC) as measured by flow cytometric analysis of proliferating cell nuclear antigen (PCNA) was within the normal range for our institution. C3 and C4 complement levels were normal.   4.3.1. B Cell Function Even though the patient’s blood type is Group O, anti-A and anti-B isohemagglutinins were not detectable. Furthermore, despite documented hepatitis B vaccination the anti-HBs titer was non-reactive. We hypothesized that the patient had an 	   108	  absence of mature B cell subsets because of the reduced immunoglobulin levels and lack of a specific antibody response to blood group antigens and vaccinations.   4.3.2. Flow Cytometric Immunophenotyping of B Cell Subsets  Because of the patient’s hypogammaglobulinemia, we analyzed her B cell compartment by flow cytometric immunophenotyping in order to identify a possible B cell differentiation arrest. Immunophenotyping showed a: 1) increased fraction of transitional B cells (Figure 4.1A, B and E); 2) absent memory B cells (Figure 4.1C, D and E) and 3) absent marginal zone B cells (Figure 4.1C).  	   109	   	  CD21 A. CD10 CD38 B. CD27 C. IgD IgM IgG D. CD24 E. CD38 PLCγ2 mut/+ Healthy Donor 	   110	  Figure	  4.1. B cell subset immunophenotyping of CD19+ B cells from patient (PLCγ2mut/+) and healthy donor. A. CD21+/lowCD10+ transitional B cells. B. CD38highCD10+ transitional B cells. C. CD27+IgD+IgM+ marginal zone B cells, CD27+IgD-IgM- switched memory B cells and CD27-IgD+IgM- naïve B cells. D. IgG+IgM+, IgG-IgM- and IgG-IgM+ B cells. E. CD24highCD38high transitional B cells, CD24highCD38low memory B cells and CD24lowCD38low naïve B cells. 7AAD was used to exclude dead cells. Each of these plots represents three independent experiments. 	   The reduced immunoglobulin levels and lack of a specific antibody response to blood group antigens and vaccinations in our patient were consistent with a diagnosis of common variable immunodeficiency (CVID). This clinical diagnosis was sufficient for the initiation of intravenous immunoglobulin therapy to prevent infections, but CVID represents a large heterogeneous group of antibody deficiency syndromes associated with a plethora of clinical features and as yet largely undefined molecular causes [264]. We hypothesized that the patient had a molecular defect in BAFF-R, a monogenic disease associated with CVID, because of the increased proportion of early transitional B cells and reduction in mature B cell subsets seen on flow cytometry.   4.4. Abnormal Surface Expression of BAFF-R Not Associated With a BAFF-R Genetic Mutation The increased proportions of early transitional B cells at the expense of mature B cell subsets resembles B cell developmental abnormalities present in murine models and human disease associated with defects in the BAFF/BAFF-R signaling pathway, such as 	   111	  BAFF-R deficiency [265]. BAFF-R expression is absent from early B cell precursors and is acquired by immature transitional B cells newly expressing the B cell receptor. We further investigated the expression of soluble BAFF and BAFF-R in this case. We measured the serum levels of sBAFF by ELISA and the mean concentration from three time-points including one prior to initiation of intravenous immunoglobulin therapy was 5.84 ± 0.83 ng/ml (SD; n = 3). This is elevated compared to BAFF concentrations in healthy donors (0.61 ± 0.27 ng/ml;SD; n=52), but not as high as concentrations (> 10 ng/ml) routinely seen in numerous primary Ab deficiencies [207].  Immunophenotyping of the total CD19+ B cell fraction in peripheral blood using two different clones (8A7 and 11C1) revealed significantly lower or absent BAFF-R surface expression (Figure 4.2A and B). Flow cytometry intracellular staining also showed reduced total BAFF-R expression (Figure 4.2C). Sanger sequencing of the BAFF and BAFF-R genes revealed no variants. 	   112	   Figure 4.2. BAFF-R expression by flow cytometry. Surface BAFF-R expression on CD19+ B cells from patient (PLCγ2mut/+) (solid black line) and healthy control (dashed black line) with an isotype control (solid gray) with clone 8A7 (A), clone 11C1 (B) and total BAFF-R expression after fixation and permeabilization with clone 11C1 (C). 7AAD was used to exclude dead cells. Each of these plots represents three independent experiments %	  of	  Max A. B. C. %	  of	  Max %	  of	  Max 	   113	  The patient does not have a molecular defect in BAFF or BAFF-R in spite of the reduced BAFF-R surface expression and a CVID-like presentation. The mechanism underlying the low surface expression of BAFF-R will be discussed in the next chapter.  Based on the severe B cell defect and young age of our patient, we hypothesized that the patient had an underlying monogenic disease.     4.5. Whole Exome Sequencing and Identification of PLCγ2 Gene Variant  Whole exome sequencing (WES) and bioinformatics analysis was performed on the patient and her mother, the only first-degree relative alive. After variant filtering was performed as described in Chapter 2, we generated candidate lists of novel nonsynonymous substitutions, insertions and deletions for both patient and her mother. Our first attempt to identify a causative variant involved eliminating all variants that were also present in her mother. This approach was tried first based on the assumptions that her mother was unaffected and the lack of consanguinity argued against an autosomal recessive mode of inheritance. A plausible hypothesis is that her father was also affected as he passed away from undiagnosed lung disease, a common manifestation of humoral immunodeficiency. This would suggest an autosomal dominant mode of inheritance from her father rather then de novo in the patient. After eliminating variants common to both patient and mother, we were left with 184 novel variants (see Appendix 1). We first screened this list for genes already known to be associated with clinical phenotypes using the OMIM (Online Mendelian Inheritance in Man) database of known human genetic disorders. There were 44 previously reported disease causing mutated genes of which 	   114	  only one was associated with antibody deficiency – PLCγ2, as previously described in the introductory chapter.  A single heterozygous point mutation in the gene encoding phospholipase C gamma 2 (PLCγ2) was identified. The mutation is in position 81973605 on chromosome 16 in which a T is replaced with an A in 50 of 96 reads resulting in an amino acid substitution (M1141K – methionine to lysine at position 1141). This mutation is located in the C2 domain (aa 1059-1152) of the protein (Figure 4.3). This was confirmed independently by Sanger sequencing.   Figure 4.3. Domain structure of PLCγ2. PLC isoenzymes contain a conserved domain structure consisting of the catalytic X and Y domains located between EF-hand motifs and a calcium-binding C2 domain. There is a pleckstrin homology (PH) domain at the N-terminus. PLCγ2 contains an additional PH domain split by two random Src homology 2 (SH2) domains and an SH3 domain [266].    We hypothesized that an alteration in PLCγ2 function was the cause of our patients’ humoral immunodeficiency due to altered B cell receptor (BCR) signaling.     1 1265 M1141K 	  	  	  	  	  * 	   115	  4.6. Assessment of PLCγ2 Activity in Patient Primary Cells  4.6.1. Calcium Flux in B Cells The key role of PLCγ2 in B cells is to trigger calcium flux after BCR engagement. Because there is a finite store of intracellular Ca2+ available in the ER, we hypothesized that any mutation in PLCγ2 would be reflected in intracellular and/or plasma Ca2+ flux. We show that BCR stimulation induced increased external calcium entry in the patient’s primary B cells (Figure 4.4) suggesting that our mutation may lead to a gain-of-function.   Figure 4.4. Calcium flux in primary CD19+ B cells after BCR stimulation. Fresh PBMCs from patient (PLCγ2mut/+) (black line) and healthy control (gray line) were incubated in Ca2+-free media and stimulated via BCR followed by addition of exogenous Ca2+. Representative of three independent blood collections including one prior to initiation of IVIG therapy with three different sex-matched pediatric controls.   4.6.2. Downstream Signaling of Activated PLCγ2 A gain in function in PLCγ2 could also lead to increased production of one of its key second messengers, DAG, which activates downstream effectors such as ERK. We Intracellular Calcium  Flux (MFI) F(ab’)2 anti-human IgM External Ca2+ added 	   116	  hypothesized that the patient would have increased ERK phosphorylation after BCR stimulation. We stimulated isolated primary CD19+ B cells from the patient via BCR crosslinking and measured ERK phosphorylation by flow cytometry analysis. We found that even though there is equivalent phosphorylation of PLCγ2, there is increased and prolonged ERK phosphorylation (Figure 4.5). Equal amounts of total PLCγ2 were also confirmed by flow cytometry.   	   117	    Figure 4.5. ERK1/2 phosphorylation after BCR stimulation. Primary CD19+ B cells from patient (PLCγ2mut/+) (circle) and healthy control (square) were stimulated for indicated time and the phosphorylation of PLCγ2 (A) and ERK1/2 (B) was measured by intracellular flow cytometry. Each data point is presented as a ratio of MFI to baseline MFI at start of incubation. Values represent means ± SEM compared by using ANOVA with Bonferroni posttest. ∗P < .05, **P < .01, and ***P < .001. 5 15 30 600246BCR Stimulation (minutes)Fold Change in PLCγ2 Phosphorylation (Y759)5 15 30 600481216BCR Stimulation (minutes)Fold Change in ERK1/2 Phosphorylation (T202/Y204)*** ***A.	   B.	   	   118	   4.6.3. Platelet Hyperreactivity  In murine models of PLCγ2 gain-of-function mutations, platelets showed enhanced granule secretion upon stimulation with collagen [267]. To confirm the same phenotype in humans, secretion of alpha-granules was measured by flow cytometry analysis of the surface expression of CD62P (P-selectin) on platelets after collagen stimulation. We show that the patient’s platelets have enhanced alpha granule release after stimulation with collagen in vitro (Figure 4.6).        	   119	    Figure 4.6. Collagen-induced platelet alpha granule release. Fresh blood was collected from the patient (PLCγ2mut/+) and healthy controls using atraumatic technique into sodium citrate tubes with a discard tube first and stimulated with collagen. Alpha granule release from platelets was assessed by measuring the surface expression of CD62P on CD61+ platelets by flow cytometry. Each column represents data from 3 individual samples. Statistical comparisons were made by using an unpaired t test. **P < .01  We demonstrated that primary hematopoietic cells from our patient with a novel heterozygous missense mutation in PLCγ2 have: 1) increased external calcium flux in primary CD19+ B cells after BCR crosslinking, 2) increased phosphorylation of ERK in 0510152025303540455055normalpatientPlatelet Alpha Granule Release (Surface Expression of CD62P)After Collagen Stimulation****	   120	  CD19+ B cells after BCR crosslinking and 3) increased alpha granule release from platelets after stimulation with collagen. These results are consistent with a functional  mutation in PLCγ2.  4.7. Confirmation of Increased External Calcium Flux Due to PLCγ2 Mutation  In order to confirm the causative relationship between our patients PLCγ2 genetic variant and the clinical phenotype of increased external calcium flux in the patients primary B cells, we transiently transfected a PLCγ2 knockout (KO) DT40 cell line with a normal and mutant PLCγ2 construct and assessed calcium flux after BCR stimulation. The chicken DT40 cell line, an avian leukosis virus (ALV) induced bursal lymphoma cell line derived from a Hyline SC chicken, has previously been established as an excellent model system for the analysis of the function of genes related to BCR signaling [334]. BCR-stimulation of PLCγ2 KO DT40 cells transfected with our mutant PLCγ2 construct results in increased external calcium flux compared to a normal PLCγ2 construct (Figure 4.7) supporting the role of the patient PLCγ2 mutation in increased BCR-induced external calcium signaling.         	   121	  A.   B.    0 100 200 300 400 500 600012345Normal PLCγ2Mutant PLCγ2Empty PlasmidMock Transfection    BCRStimulation    ExternalCa2+ AddedTime (seconds)%	  of	  Max PLCG2 Phosphorylation (Y759)	   122	  Figure 4.7. BCR stimulation of PLCγ2 KO DT40 cells transfected with PLCγ2 constructs. (A) 1 million PLCγ2 KO DT40 cells were transiently transfected with 0.5 ug of either empty plasmid, normal and mutant PLCγ2 construct corresponding to our patients’ genetic variant. After overnight incubation, the transfected DT40 cells were incubated in Ca2+-free media and stimulated via BCR followed by addition of exogenous Ca2+. Each data point is representative of three independent experiments with measurements shown in 15-second intervals. Viable cells were analyzed for 60 seconds prior to BCR-stimulation in order to establish an average baseline by which the relative fold increase in fluorescence is measured. (B) Similar transfection efficiencies and PLCγ2 activation, indicated by phosphorylation of Y759, of both PLCγ2 constructs is demonstrated by flow cytometric intracellular staining after 5 minutes of BCR stimulation; control plasmid (solid gray), wild type PLCγ2 construct (light gray) and mutant PLCγ2 construct (black line). This FACS plot is representative of three independent experiments.  4.8. Increased Apoptosis of Immature B Cell Subsets Having identified a potential causative functional mutation in PLCγ2, we needed to reconcile this with the significant reduction in mature B cells. The absence of mature B cells and increased fraction of transitional B cells with a normal total CD19+ B cell count, suggested the possibility of either a block in B cell development or increased apoptosis of immature B cells. As described in the background, a potentially harmful side effect of the process of generating B cell diversity through genetic recombination is the generation of self-reactive B cells. The B cell receptor (BCR) plays an important role in eliminating 	   123	  these clones at immature and transitional stages. It has been shown extensively in human immature B cell lines and mice, that early B cells can be eliminated through BCR-induced cell death and this apoptotic mechanism is dependent on calcium signaling [268]. Our patient has exaggerated calcium mobilization responses to BCR signaling and the block in B cell development may be due to enhanced sensitivity to deletion by self-antigens that correlates with exaggerated BCR-induced calcium mobilization [269]. Normal B cells also rely on “tonic” antigen-independent BCR signaling for development and survival [270]. Therefore, transitional immature B cells are highly susceptible to BCR-mediated antigen induced apoptosis, yet at the same time, tonic BCR signals are required for B cell survival throughout development [271]. It is possible that a gain-of-function mutation in PLCγ2 also causes hyper reactive tonic BCR signaling that mimics strong antigen-dependent BCR signaling making every B cell appear self-reactive. We hypothesized that intrinsic BCR hyper activity due to a gain-of-function mutation in PLCγ2 would lead to a higher level of spontaneous apoptosis and cell death of CD24+CD38+ CD19+ B cells (mature naïve and transitional). To test this hypothesis, we incubated the patients B cells overnight in culture media. After incubation overnight in culture media, there was a significantly higher level of apoptotic/dead CD24+CD38+CD19+ B cells in the patient compared to the healthy donor as shown by 7-AAD flow cytometric staining (Figure 4.8).     	   124	   Figure 4.8.  Cell death in immature primary B cells. Freshly collected PBMCs from patient (PLCγ2mut/+) and healthy control were incubated overnight in complete media and cell death was assessed by 7-AAD flow cytometric staining (B and D) in immature B cell population (A and C). At baseline, the percentage of 7-AAD+ B cells in both patient and healthy control immature B cells was <3%.   Representative of three independent experiments with three different healthy controls.  These results confirmed that the patient’s immature B cells had a higher rate of spontaneous apoptosis.   CD24 	  	  CD38 A. C. B. D. 7AAD Healthy  Control PLCγ2 mut/+ 	   125	  4.9. Discussion and Future Directions Applying the three criteria proposed to establish a causal relationship based on a single case, we describe a novel gain-of-function mutation in PLCγ2 associated with severe defects in peripheral B cell development and hypogammaglobulinemia. Firstly, the patient’s genotype has not been found to occur in any individuals without the clinical phenotype. Secondly, our experimental studies indicated that the genetic variant was associated with increased BCR-triggered external calcium flux in primary B cells, increased and prolonged BCR-triggered ERK phosphorylation in primary B cells, platelet hyper-reactivity and increased apoptosis of transitional B cells. Lastly, the casual relationship between the candidate genotype and the clinical phenotype were confirmed by transfecting a PLC γ2 knockout DT40 B cell line with a mutant copy of PLCγ2 which resulted in increased external calcium flux after BCR stimulation similar to the patients primary B cells.  The C2 domain of PLCγ2, in which our mutation was found, has so far been shown to play two roles in PLCγ2 activity: 1) Initial binding of the PLCγ2 SH2 domain to phosphorylated BLNK is stabilized by the PLCγ2 C2 domain in a Ca2+-regulated manner. As intracellular Ca2+ levels become transiently depleted, the C2-BLNK interaction diminishes terminating PLCγ2 activation [272]; 2) the initial Ca2+ flux via receptor activated calcium channels (RACC) such as IP3R promotes lipase-independent translocation of additional PLCγ2 from the cytosol to the plasma membrane by acting on the C2 domain [273]. Ca2+ influx induces translocation of additional PLCγ2 to the plasma membrane via the C2 domain. Here the PLCγ2 binds to BLNK via SH2 domains and becomes activated leading to the hydrolysis of PIP2 and more production of IP3 and DAG. 	   126	  DAG in turn activates more influx of Ca2+ by directly activating transient receptor potential cation (TRPC) Ca2+ channels in the plasma membrane [274]. PLCγ2 also mediates external Ca2+ entry independent of its catalytic activity, probably through protein-protein interactions [275,276]. Any mutation within the C2 domain has the potential to affect the role of this domain in recruiting PLCγ2 to the plasma membrane where it mediates external Ca2+ entry directly and indirectly through second messengers.  To our knowledge, there has never been a report of a germline mutation in the C2 domain of PLCγ2. The previously reported mutations only involved the SH2 domain.  We searched for other proteins with mutations in their C2 domain to support a mechanism for the hypothesized gain of function. For example, mutants that increase the positive surface charge of the C2 domain in p110α lead to a gain of function [277]. This is analogous to our variant as methionine is substituted with positively charged lysine at position 1141. Our next step is to attach a GFP tag to the mutated version of PLCγ2 and visualize its association with the plasma membrane after BCR stimulation. We would predict that it remains associated with the plasma membrane for a longer time then wild-type protein.         	   127	  Chapter 5: Soluble BAFF Regulates Surface BAFF-R Expression  5.1. Introduction The observation of an inverse relationship between high sBAFF levels and low surface BAFF-R expression on B cells is well described in human diseases such as Sjögren’s syndrome, human immunodeficiency virus (HIV), hepatitis C, malaria, chronic graft-versus-host disease (GVHD) and common variable immunodeficiency (CVID) [233-238]. Several independent groups have previously demonstrated that sBAFF modulates the surface expression of BAFF-R in murine and human disease models [278-280]. The underlying mechanisms of modulation are still a matter of speculation.  There are several hypotheses that may explain the observation of decreased BAFF-R surface expression on B cells in association with high sBAFF levels. The underlying principle supposes that reduced surface BAFF-R expression is a normal regulatory mechanism that prevents any further action of excess BAFF on its targets following BAFF ligation.  1. One plausible hypothesis is that the observed BAFF-R downregulation is an artifact of competitive binding between the BAFF-R antibody and sBAFF. With this in mind, all flow cytometric analysis of BAFF-R expression is performed with the 11C1 monoclonal antibody, which has previously been shown to not compete with endogenous sBAFF for binding to BAFF-R at observed concentrations in patients [278,281]. 2. Down-regulation of surface BAFF-R through a post-transcriptional mechanism such as: 	   128	  a. BAFF-R internalization secondary to ligand binding.  Receptor internalization from the cell surface represents a key first step in the inactivation process, by targeting the internalized receptor for lysosomal degradation. b. Binding-induced receptor cleavage and shedding into the extracellular space. 3. Excessive BAFF regulates BAFF-R expression by down-regulating gene transcription through a negative-feedback loop. Both of our patients described in Chapters 3 and 4 with previously unknown immunodeficiencies had low surface expression of BAFF-R concomitant with high sBAFF levels and defects in peripheral B cell maturation. In neither case were we able to identify any variants in the BAFF and BAFF-R genes by direct sequencing. In the first patient with MALT1 deficiency, the results suggested that the reduced expression of surface BAFF-R was due to an intrinsic defect in MALT1 signaling (i.e. there was no correction in surface BAFF-R expression after overnight incubation in media).    In the second case of the patient with a mutated PLCγ2 gene there appears to be a premature arrest in B cell development caused by increased apoptosis of B cells. Therefore, high sBAFF levels in this patient likely reflect a normal compensatory response to B cell lymphopenia. In this chapter we investigate the cause of low BAFF-R expression in this patient. In the process, we will investigate all hypotheses presented earlier in this introduction.   	   129	  5.2. Transient Suppression of BAFF-R Surface Expression in (PLCγ2mut/+) Patient Initially, we observed that the surface expression of BAFF-R increased in non-apoptotic CD19+ B cells after overnight incubation in complete media. The same observation was made when the patient’s CD19+ B cells were incubated overnight in allogeneic serum versus autologous serum (Figure 5.1). Based on these results, we hypothesized that there was a soluble factor in the patient’s serum that suppressed surface expression of BAFF-R.    Figure 5.1. Surface BAFF-R expression on patients (PLCγ2mut/+) primary CD19+ B cells after overnight incubation with various serums. Peripheral blood mononuclear cells (PBMCs) from the patient were incubated overnight in autologous serum (solid gray), %	  of	  Max 	   130	  allogeneic serum (black line) and complete media (dashed black line) and surface BAFF-R expression was analyzed by flow cytometry. Representative of three independent experiments with three different allogeneic donors.  5.3. Overnight Incubation in Patient (PLCγ2mut/+) Serum Reduces Surface BAFF-R Expression on Healthy Donor B Cells In order to determine whether the suppressive effect was extrinsic to the patient’s B cells, we incubated healthy donor CD19+ B cells in the patient’s serum compared to allogeneic healthy donor serum and their own autologous serum. We found that incubation with the patient’s serum overnight at 37°C caused a significant decrease in surface expression of BAFF-R (0.08 ± 0.02 fold decrease; p < 0.001, Figure 5.2). 	   131	    Figure 5.2. Suppression of surface BAFF-R expression on healthy donor CD19+ B cells incubated in patient serum. Isolated CD19+ B cells from healthy donors (n=10) were incubated for 24 hours at 37°C in autologous serum, allogeneic serum (alternate serum from one of the other healthy controls) and serum from the patient (PLCγ2mut/+) at 1×106/mL and the surface expression of BAFF-R was measured by flow cytometry. Statistical comparisons were made by using a paired t test. ***P < 0.001  The effect was titratable (Figure 5.3) as increasing the percentage of the patient’s serum mixed with complete media incubated overnight with healthy donor CD19+ B 24h Incubation of Isolated B Cells with Serum0.000.250.500.751.00Ratio	  of	  BAFF-­‐R	  MFI	  in	  allogeneic	  or	  patient	  compared	  to	  own	  serum Normal	  B	  Cells	  +	  Autologous	  Serum Normal	  B	  Cells	  +	  Allogeneic	  Serum Normal	  B	  Cells	  +	  PLCγ2mut/+	  	  Serum *** *** 	   132	  cells resulted in increasing reduction of BAFF-R surface expression, resembling a dose response curve.   Figure 5.3. Dose response relationship between patient (PLCγ2mut/+) serum and surface BAFF-R expression. Healthy donor CD19+ B cells were incubated with increasing fractions of patient serum mixed with complete media overnight and surface expression of BAFF-R on CD19+ B cells was measured by flow cytometry. The y-axis shows the fold change in BAFF-R surface expression on B cells measured by flow cytometry (MFI) as a fold increase compared to the MFI in 100% serum. The x-axis shows the log of the percentage of serum mixed with complete media; i.e. 2 is equal to 100% serum.  Data points represent the mean ±SD of three independent experiments.    0.1 1 10 1000204060Log (%serum in mix)Fold Change in BAFF-R MFI	   133	  We also investigated whether the reduced binding of anti-BAFF-R antibody in flow cytometric analysis was due to a blocking effect from soluble BAFF in the serum. BAFF is the only known ligand for BAFF-R and the patient had previously been found to have high sBAFF levels (5.84 ± 0.83 ng/mL). We incubated healthy donor CD19+ B cells for 30 minutes, either at 4°C or 37°C, with patient or autologous serum and measured surface BAFF-R expression by flow cytometry. Receptor occupancy is observed at 4°C, while the observations at 37°C reflect both receptor occupancy and modulation of BAFF-R expression. We observed a significant effect on surface BAFF-R expression as determined by flow cytometry due to receptor occupancy at 4°C (0.78 ± 0.03 fold decrease, p < 0.001) but there was also a significant effect at 37°C compared to 4°C (0.23 ± 0.03 versus 0.78 ± 0.03 fold decrease, p < 0.001, Figure 5.4). These results suggest the decrease in surface BAFF-R expression caused by our patients’ serum is due to a combination of competitive binding and cell surface down-modulation of BAFF-R.            	   134	    Figure 5.4. Hindrance due to receptor occupancy. Healthy donor isolated CD19+ B cells (n=4) were incubated in patient (PLCγ2mut/+) serum (1×106/mL) for 30 minutes at either 4°C or 37°C and surface BAFF-R expression was measured by flow cytometry. The measurement from the sample with complete media at either temperature was set at a value of 1 and the measurement obtained from serum was measured as a fraction of the value obtained with complete media. Statistical comparisons were made by using a paired t test. MFI, mean fluorescence intensity. *** P < .001     4°C 37°C0.00.20.40.60.81.0Fold Change in BAFF-R MFI***	   135	   These results support the existence of a soluble factor in the patients’ serum that modulates surface BAFF-R expression.   5.4. Soluble BAFF in the Patients Serum Down-Modulates Surface Expression of BAFF-R  Based on the existing literature, we hypothesized that the soluble factor in the patients’ serum that down-modulated the surface expression of BAFF-R was sBAFF. We incubated healthy donor CD19+ B cells with our patients’ serum in the presence or absence of belimumab (trade name Benlysta, previously known as LymphoStat-B), a fully humanized IgG1 monoclonal antibody that blocks BAFF [282] and measured the surface expression of BAFF-R by flow cytometry. We observed that incubation with belimumab significantly attenuated the BAFF-R lowering effect by the patients’ serum (Figure 5.5). This result demonstrates that sBAFF in the patients’ serum causes a reduction in surface expression of BAFF-R.          	   136	     Figure 5.5. Effect of blocking sBAFF on ability of patient (PLCγ2mut/+) serum to suppress surface BAFF-R expression. Healthy donor isolated CD19+ B cells (n=5) were incubated overnight with patient (PLCγ2mut/+) serum (1×106/mL) that was pre-incubated for 1 hour ± belimumab (10 ug/mL) or a control IgG1 (10 ug/mL) and surface expression of BAFF-R was measured by flow cytometry. The measurement from the sample with belimumab was set at a value of 1 and the measurement obtained from serum alone was measured as a fraction of the value obtained with belimumab. Statistical comparisons were made by using a paired t test. MFI, mean fluorescence intensity. ****P < .0001  **** PLCγ2mut/+ Serum Alone PLCγ2mut/+ Serum + Control IgG PLCγ2mut/+ Serum + Belimumab Ratio of BAFF-R MFI  	   137	   We then assessed the biological activity of the sBAFF in the patients’ serum in another manner then the ability to down-modulate surface BAFF-R expression. It has previously been shown that sBAFF increases the metabolic state, as measured by cell size, of human B cells [283]. To test whether the sBAFF in the patients’ serum is biologically active, we measured the B cell size by forward scatter using flow cytometry after incubation overnight with and without belimumab. We demonstrated that inhibiting sBAFF in patient serum resulted in a smaller B cell size (Figure 5.6).   Figure 5.6. Soluble BAFF in the patients’ serum increases the cellular size of healthy donor B cells. Healthy donor isolated CD19+ B cells (n=5) were incubated overnight with patient (PLCγ2mut/+) serum (1×106/mL) that was pre-incubated for 1 hour ± belimumab (10 ug/mL) and cell size analyzed by forward scatter (FSC) by flow cytometry. A. Representative histogram of one healthy donor B cells incubated overnight in patient serum without (open area, bold line) and with belimumab (gray shaded area). % of Max A. B. 7500080000850009000095000100000FCSPLCγ2mut/+Serum AlonePLCγ2mut/+Serum + Belimumab****	   138	  B. Median FSC of healthy donor B cells (n=5) after overnight incubation in patient serum ± belimumab. Statistical comparisons were made by using a paired t test. ****P < .0001    5.5. G-CSF and IFNγ Stimulated Neutrophils Secrete sBAFF That Suppresses BAFF-R Expression So far, our analysis of the modulating effect of sBAFF on BAFF-R has focused on a single patient with a very rare primary immunodeficiency. We sought independent confirmation of this phenomenon through incubation of healthy donor B cells with conditioned media from G-CSF and IFNγ-stimulated neutrophils.  Human neutrophils have previously been shown to release high levels of sBAFF when incubated with G-CSF or, to a lesser extent, IFNγ and IFNα [284]. The selective action of G-CSF and IFNγ on neutrophils was highlighted by the fact that a variety of other neutrophil agonists such as LPS, fMLP, TGFβ, IL-4, IL-10, IL-13, GM-CSF and TNFα were unable to upregulate BAFF gene and protein expression. Neutrophil-derived supernatants were able to increase the [3H]thymidine uptake of human B cells in a B cell co-stimulation assay and the effect was completely inhibited by anti-BAFF antibodies.  Furthermore, G-CSF induced a specific increase of intracellular full length 32 kDa BAFF and cleaved 17 kDa BAFF corresponding to unmodified sBAFF. They showed that full-length BAFF is processed intracellularly by a Golgi-associated furin pro-protein convertase prior to secretion as a biologically active sBAFF. The primary reason we choose this model system was that neutrophils have been shown to secrete a biologically active 17 kDa sBAFF similar to the sBAFF present in our patients serum and recombinant sBAFF by Western blotting. Based on this observation, we hypothesized 	   139	  that the sBAFF secreted by G-CSF and IFNγ-stimulated neutrophils would also down-modulate surface BAFF-R expression on healthy donor B cells.  To test this hypothesis, we incubated neutrophils at a density of 5×106/mL in complete media stimulated with G-CSF (1,000 U/mL) or IFNγ (200 U/mL) for 42 hours as previously described [284] and collected cell-conditioned media. We studied the effect of both G-CSF and IFNγ, as G-CSF was shown to have the highest capacity to trigger sBAFF and because of its specificity as a neutrophil agonist and IFNγ because it plays a significant role in inflammation and the development and severity of systemic autoimmunity [285]. The conditioned media was incubated with isolated healthy donor CD19+ B cells overnight with or without the BAFF blocking monoclonal antibody, belimumab. BAFF-R expression was measured on the B cells by flow cytometry. There was a significant reduction in the surface expression of BAFF-R by media from neutrophils incubated with either G-CSF and IFNγ that was inhibited by co-incubation with belimumab (Figure 5.7). These results suggest that human G-CSF and IFNγ–stimulated neutrophils secrete sBAFF capable of down-regulating surface BAFF-R expression.  	   140	    Figure 5.7. Effect of G-CSF and IFNγ stimulated neutrophil conditioned media on surface BAFF-R expression in CD19+ B cells. Conditioned complete media (RPMI 1640 + 10%FCS) from neutrophils (5×106/mL) stimulated with G-CSF (1000 U/mL) or IFNγ (200 U/mL) for 42 hours was collected and pre-incubated with belimumab or control IgG1 at 10 ug/mL for 1 hour and then incubated with isolated CD19+ B cells from healthy adult donor peripheral blood  (n=4). Surface expression of BAFF-R was measured by flow cytometry and fold change was calculated versus the BAFF-R MFI on + -­‐ -­‐ -­‐ -­‐ + -­‐ -­‐ + -­‐ + -­‐ -­‐ -­‐ + -­‐ + -­‐ -­‐ -­‐ -­‐ + -­‐ + -­‐ -­‐ + -­‐ -­‐ + -­‐ -­‐ + -­‐ -­‐ -­‐ -­‐ + + -­‐ -­‐ -­‐ + -­‐ + 	   Neutrophil supernatant G-CSF neutrophil supernatant IFNγ neutrophil supernatant Control IgG1 Anti-BAFF mAb Fold	  Change	  in	  BAFF-­‐R	  MFI 	   141	  CD19+ B cells incubated with neutrophil supernatant alone. Each column represents 4 healthy donors. Controls not included on this chart are CD19+ B cells incubated overnight in complete media with only G-CSF or IFNγ alone, which showed no change in BAFF-R surface expression. Statistical comparisons were made by using a paired t test. MFI –median fluorescence intensity. * P< .05 **P < .01  5.6. The Effect of Recombinant Soluble BAFF on Surface Expression of BAFF-R   Up to this point, we have only demonstrated the modulatory effect of sBAFF by blocking endogenous activity in human serum or neutrophil-conditioned media using a blocking antibody. We next sought to duplicate the effect using recombinant human sBAFF. We used a 17 kDa unmodified (i.e. non-glycosylated) protein corresponding to amino acids 134-285 of full-length BAFF produced in E. coli (Peprotech #310-13). The molecular weight and lack of any post-translational modification was confirmed by mass spectrometry.  5.6.1. Recombinant Soluble BAFF Down-Modulates Surface BAFF-R Expression  We incubated isolated healthy donor CD19+ B cells overnight with increasing concentrations of recombinant sBAFF at 4°C and 37°C. As previously demonstrated with the patient’s serum there appears be a combination of competitive binding and down-modulation of surface BAFF-R at high concentrations of sBAFF (100 and 1000 ng/mL). At a concentration of 10 ng/mL, which is similar to the levels seen in diseases associated with high sBAFF levels, there was no significant down-regulation of surface BAFF-R 	   142	  expression at 4°C suggesting that there was minimal effect from competitive binding between sBAFF and the anti-BAFF-R flow cytometry antibody (Figure 5.8).      Figure 5.8. Effect of recombinant sBAFF (rsBAFF) on surface BAFF-R expression in healthy donor CD19+ B cells. Healthy donor isolated CD19+ B cells (n=4) were incubated overnight in complete media (10% FCS in 1640 RPMI) at 37°C with stated concentrations of rsBAFF and surface expression of BAFF-R was measured by flow cytometry and compared to complete media alone (white bars). To measure the effect of receptor occupancy on flow cytometry antibody binding caused by rsBAFF binding to BAFF-R, the same concentrations of rsBAFF were incubated with the same healthy donor CD19+ B cells at 4°C (gray bars). The bars represent means ± SEM compared using paired T test. MFI, mean fluorescence intensity. *P < .05 ***P < .001  0 10 100 10000.10.20.30.40.50.60.70.80.91.01.1 ******  *rsBAFF (ng/mL)	   143	    5.6.2. Recombinant Soluble BAFF is Biologically Active and Acts on Early Human Transitional B Cells We then sought to confirm the biological activity of recombinant sBAFF as we had done with endogenous sBAFF. This also gave us an opportunity to expand the functional profile of sBAFF in a novel manner. We focused our attention on the ability of sBAFF to modulate the properties of an early transitional human B cell population. This population, characterized by CD19+CD27-CD10hiCD21loBAFF-R+, is the first stage at which BAFF-R becomes expressed.  CD27 is widely considered a reliable marker of memory B cells and CD10 is an immature B cell marker that is most highly expressed on pre-B and early transitional B cells. Differential expression of CD21 identifies developmentally and functionally distinct subsets of human transitional B cells; the development of CD21lo transitional B cells precedes that of CD21hi transitional cells [286]. The CD21lo transitional B cell subset is enriched with polyreactive and autoreactive clones that have yet to be deleted by tolerance mechanisms.  During normal B cell development, the expression of chemokine receptors and their response to ligands increases on B cells expanding their capacity to exit the circulation and enter secondary lymphoid tissue.  Lymphocyte entry into the lymphatic circulation is dependent on CD62L, an adhesion molecule that facilitates migration through the high endothelial venules (HEVs). HEVs constitute a specialized postcapillary network in the lymph node, playing a critical role in lymphocyte recirculation. Expression of CD62L steadily increases as transitional B cells develop into naïve B cells. 	   144	  The migration of lymphocytes from the bloodstream into lymph nodes via HEVs is a prerequisite for the detection of processed antigen on mature antigen-presenting cells such as dendritic cells and the initiation of immune responses [287,288]. In mice treated with anti-CD62L antibody and mice deficient for CD62L, the number of B and T cells is drastically reduced in the lymph nodes but is increased in the spleen [289]. Human cells that lack both CD21 and CD62L expression are excluded from the lymphatic circuit and show an enhanced migration to the spleen [290]. In addition to the inverse relationship between high sBAFF levels and low surface BAFF-R expression on B cells in many human diseases, these same diseases are also associated with an increase of circulating immature/transitional B cells at the expense of mature B cell populations. [291,292]. We hypothesized that increased sBAFF is associated with an increased population of transitional B cells in the peripheral blood of patients because sBAFF decreases the surface expression of CD62L.  To evaluate this hypothesis, we incubated isolated CD10+ transitional B cells from fresh human cord blood with recombinant sBAFF and measured the surface expression of CD21, BAFF-R and CD62L by flow cytometry. Briefly, mononuclear cells were isolated from umbilical cord blood by Ficoll density gradient centrifugation and CD19+ B cells were further isolated by negative immunomagnetic selection. Isolated CD19+ B cells were then separated using flow cytometric cell sorting into two immature populations: 1) CD27-CD10hi and 2) CD27-CD10dim (Figure 5.9) which were cultured overnight in medium alone or with recombinant sBAFF. 	   145	   Figure 5.9. Gating strategy used to sort 1) CD27-CD10hi and 2) CD27-CD10dim CD19+ B cell populations from cord blood. Representative of 4 independent experiments  Firstly, we measured CD21 expression to further characterize the developmental stage of the sorted B cell populations and whether sBAFF affected CD21 expression. The CD27-CD10hi population had uniformly lower expression of CD21 compared to the CD27-CD10dim CD19+ B cell population after overnight incubation in media (Figure 5.10A). 24-hour stimulation with sBAFF did not alter CD21 expression in each population (data not shown). These results indicate that the CD27-CD10hi population is the more immature population and sBAFF does not alter CD21 expression in this B cell population 	   	   	   	   	   	   CD10 CD27 1 2 	   146	   Figure 5.10. CD21 and BAFF-R expression on human transitional B cell subsets. A. Surface expression of CD21 on CD27-CD10hi (black line) and CD27-CD10dim (gray solid) CD19+ B cells after overnight incubation in media. B. Surface expression of BAFF-R on CD27-CD10hi (black line) and CD27-CD10dim (gray solid) CD19+ B cells after overnight incubation in complete media. Representative of 4 independent experiments. MFI – mean fluorescence intensity  We next measured surface BAFF-R expression on these populations. In unstimulated sorted B cells, surface BAFF-R expression was reduced on CD27-CD10hiCD21lo transitional B cells compared to CD27-CD10dim CD21+ transitional B cells (Figure 5.10B) in keeping with previous observations that BAFF-R expression increases steadily through transitional B cell stages and is maintained at high levels up to the final stage of differentiation as plasma cells. Incubation with recombinant sBAFF led to significant down-modulation of surface BAFF-R expression in both subsets (Figure 5.11A and B). CD21CD21 MFI A  B	   BAFF-R MFI 	   147	    Figure 5.11. Effect of sBAFF on surface expression of BAFF-R in human transitional B cell subsets. A. The surface expression of BAFF-R on CD27-CD10dim B cells after overnight incubation in media with sBAFF (100 ng/mL) (gray solid) or without (black line). B. The surface expression of BAFF-R on CD27-CD10hi B cells after overnight incubation in media with sBAFF (100 ng/mL) (gray solid) or without (black line). Representative of 4 independent experiments. MFI – mean fluorescence intensity  We then measured the surface expression of CD62L. Firstly, we demonstrated that CD27-CD10hiCD21lo transitional B cells express less CD62L+ B cells compared to CD27-CD10dim CD21+ transitional B cells after overnight incubation in media (Figure 5.12). Immature B cell development appears to be associated with increasing expression of CD62L. Stimulation with sBAFF led to a significant decrease in the percentage of CD62L+ B cells in each transitional B cell subset (Figure 5.13).  BAFF-R MFI BAFF-R MFI A  B	   	   148	   Figure 5.12. Expression of CD62L in transitional B cell subsets. Distribution of CD62L+ B cells in CD27-CD10hi (black line) and CD27-CD10dim (gray solid) CD19+ transitional B cell populations after overnight incubation in media. Representative of 4 independent experiments. MFI – mean fluorescence intensity  CD62L MFI 	   149	   Figure 5.13. Effect of sBAFF on CD62L expression in transitional B cell subsets. A. Representative histogram. Change in percentage of CD62L positive B cells in CD27-CD10dim (B) and CD27-CD10hi (C) CD19+ transitional B cell populations (n=4) after overnight incubation in media with sBAFF (100 ng/mL) stimulation or without. Statistical comparisons were made by using a paired t test. **P < .01 CD27-CD10dimCD62L60.8CD27-CD10dim + BAFFCD62L38.1CD27-CD10dimCD62L60.8CD27-CD10dim + BAFFCD62L38.1CD62L CD27-CD10dim CD27-CD10dim + sBAFF 	   	   .8 3  CD27-CD10highCD62L11.7CD27-CD10high + BAFFCD62L4.88CD27-CD10highCD62L11.7CD27-CD10high + BAFFCD62L4.88CD62L 	   11.7 	   4.9 CD27-CD10hi CD27-CD10hi + sBAFF A.	   No sBAFF sBAFF20406080% CD62L+ B CellsCD27-CD10dim B Cells**No sBAFF sBAFF05101520% CD62L+ B CellsCD27-CD10hi B Cells**B.	   C.	   	   150	  5.7. Mechanism of Down-Modulation of Surface BAFF-R Expression by sBAFF Having established that sBAFF down-modulates surface expression of BAFF-R, we investigated the possible mechanisms underlying this phenomenon including: 1) reduced gene transcription, 2) release of BAFF-R from surface and 3) internalization of surface BAFF-R. 5.7.1. BAFF-R Gene Transcription We measured the RNA transcript level of BAFF-R in isolated human B cells after incubation in patient (PLCγ2mut/+) serum with or without belimumab. Incubation with patient serum alone was associated with a small (1.52X ± 0.09 fold increase (p = 0.0006); n=4) increase in the RNA transcript level of BAFF-R compared to samples incubated with serum and blocking antibody belimumab. These results indicate that there is no reduction in gene transcript associated with reduced surface expression.  5.7.2. Release of Soluble BAFF-R  We measured the levels of soluble BAFF-R by ELISA in media supernatants from the overnight cultures with recombinant sBAFF and there was no evidence of secreted soluble BAFF-R despite a nearly 30-fold decrease in surface expression of BAFF-R with 1 ug/mL of recombinant sBAFF (n=4). These results suggest that BAFF-R is internalized.  5.7.3. Surface BAFF-R Internalization We preformed intracellular flow cytometry staining for BAFF-R expression. Firstly, previous flow cytometry results from the patient supported internalization as the mechanism of suppression as the total levels of BAFF-R, as determined by intracellular flow cytometry were slightly less then the healthy donor contrasted with near absent 	   151	  surface expression (see Figure 4.2). The reason that the total BAFF-R expression is still lower in the total CD19+ B cell fraction then the healthy donor may be that the high fraction of early immature B cells with differential expression patterns of BAFF-R lowers the median level of total expression [293]. Also need to consider the possibility that there is increased baseline degradation secondary to chronic internalization. To further investigate whether BAFF-R is internalized, we measured total expression of BAFF-R after incubation with recombinant sBAFF using flow cytometry after fixation and permeabilization of the CD19+ B cell population (Figure 5.14). Despite the significant decrease in surface expression of BAFF-R, there was no difference in total expression of BAFF-R.  These results suggest that BAFF-R is internalized and there is no increased production of BAFF-R.  Figure 5.14. Surface and total expression of BAFF-R on normal primary B cells after incubation with recombinant sBAFF. A. Surface staining of BAFF-R by flow cytometry on isolated CD19+ B cells after incubation with 500 ng/mL of recombinant sBAFF (gray line) and without (black line). B. Total staining of BAFF-R by flow cytometry on isolated %	  of	  Max Surface BAFF-R MFI Total BAFF-R MFIA. B. %	  of	  Max 	   152	  CD19+ B cells after incubation with either 500 ng/mL of recombinant sBAFF (gray line) or without (black line) after fixation and permeabilization. Representative of three independent experiments   After testing all three hypotheses, the results support that the mechanism of reduced surface expression of BAFF-R is internalization of the receptor without reducing gene transcription.  5.8. Discussion and Future Directions Our results indicate that exposure to endogenous and recombinant sBAFF induces a decrease in the expression of BAFF-R. Reciprocally, blockage of BAFF with belimumab, a clinically available anti-BAFF monoclonal antibody, prevented this effect. Our results further suggest that this effect is a combination of competitive binding between sBAFF and the anti-BAFF-R antibody and down-modulation of BAFF-R surface expression secondary to sBAFF binding. Contrary to previous reports, we show that the anti-BAFF-R antibody (clone 11C1) does significantly compete with sBAFF for receptor occupancy. There is roughly a 20% decrease in surface BAFF-R expression by flow cytometry at 4°C, meant to prevent receptor signaling, after incubation with sBAFF. There is a significantly larger decrease at 37°C suggesting a combination of competitive binding and receptor internalization. This conclusion assumes that there is a similar binding affinity of sBAFF to BAFF-R at 4°C and 37°C. In order to conclusively determine this biological property of sBAFF, we will need to evaluate binding in a system without any possibility of receptor internalization. This could be achieved by 	   153	  using receptors suspended in artificial membrane-like environments such as lipid vesicles or nanodiscs.   These results suggest that the primary defect in diseases with an inverse relationship between high BAFF levels and reduced surface expression of BAFF-R by flow cytometry is probably due to the increased availability of sBAFF secondary to increased production. In many of the diseases associated with this inverse relationship, it has been shown ex vivo that B cells have preserved responses to BAFF. B cells from patients with chronic GVHD are activated and primed for survival via BAFF-mediated pathways [283]. B lymphocytes from individuals with common variable immunodeficiency have been shown to respond normally to BAFF in vitro [294]. Abnormal B cell activation is associated with BAFF over-expression in chronic hepatitis C virus infection [295].  Our results suggest the amount of sBAFF in the blood controls BAFF-R expression on healthy B cells. The suppression in BAFF-R expression is likely due to a normal feedback loop meant to limit excessive BAFF signaling. The results also suggest that the down-modulation is not due to decreased transcription of the BAFF-R gene or cleavage of BAFF-R from the cell surface, but they are consistent with BAFF-R internalization. The role of receptor-mediated endocytosis is well recognized in the down-regulation of transmembrane signal transduction. This would prevent any further action of BAFF on cells. It is thought that the primary pathogenic consequence of elevated BAFF is to promote the rescue of autoantigen-engaged B cells from rapid competitive elimination due to BCR signaling.  	   154	  Interestingly, this down-modulation is by no means universal in diseases associated with high sBAFF. For example, in systemic lupus erythematosus (SLE), the expression of BAFF-R on B cells from SLE patients was similar to that on the same subsets of B cells from healthy subjects, as determined by analysis with the anti-BAFF-R 11C1 we used in our own flow cytometry analyses. However, they did demonstrate that surface BAFF-R was occupied and that B cells from the blood of SLE patients were less responsive to exogenous BLyS in culture, compared with B cells from healthy donors [281].  This could be seen as evidence for altered forms of BAFF, for example an isoform that prevents internalization of the receptor despite receptor occupancy. In turn, this could potentially lead to sustained activation due to defective internalization.   In addition to high levels of sBAFF being able to down-modulate surface BAFF-R expression, we show that recombinant sBAFF is also able to down-regulate the surface expression of CD62L on early transitional human B cells. This experiment was performed to verify the biological activity of the recombinant BAFF and to partially explain why diseases with an inverse relation between sBAFF and BAFF-R expression were also associated with increased numbers of circulating transitional B cells. CD62L expression is necessary for the proper migration of immature B cells into lymph nodes. It is possible that the reduced expression of homing molecules may contribute to the increased frequencies of transitional B cells in peripheral circulation. There is a precedent for this observation as it has previously been shown that stimulation of B cells through Toll-like receptor (TLR)-2, -3, and -9 induces shedding of CD62L, which impacts on their migration patterns and results in their exclusion from lymph nodes and Peyer’s patches and they traffic only to the spleen [296]. The authors of that study propose that 	   155	  this may be a mechanism to nonspecifically attract a polyclonal population of B cells to the spleen whose primary immune purpose is to present blood-borne antigens and pathogens. At the transitional B cell stage, this represents an early B cell population still rich in autoreactive and polyreactive clones that can be activated by TLR agonists. Toll like receptors are part of the innate immune system and recognize specific microbial components derived from pathogens including bacteria, fungi, protozoa and viruses [297]. For example, transitional B cells express higher levels of TLR9 compared with IgM memory and mature B cells. TLR-9 recognizes unmethylated CpG (cytosine and guanine separated by one phosphate) sequences in bacterial DNA molecules. It has previously been shown that upon TLR-9 stimulation, transitional B cells can develop into a variety of mature B cell subsets. Furthermore, TLR-9 agonists have previously been shown to protect immature B cells from negative selection imposed by apoptosis [298-300]. This could serve as an additional role of sBAFF in the emergence and stimulation of autoreactive B cells in disease in addition to directly promoting survival of autoreactive B cells. An interesting hypothesis that needs further exploration is whether the combination of high sBAFF and inflammatory stimuli such as TLR agonists acting on human transitional B cells might lead to increased frequencies of unique B cell populations.   It has previously been shown that the same autoimmune/inflammatory diseases we have described in association with high sBAFF, low BAFF-R expression and increased circulating transitional B cells are also frequently associated with elevated levels of a pathogenic CD21 low B cell population [301-306]. This population is characterized by higher levels of CD19, CD22 and IgM expression; lower levels of 	   156	  CD21, CD24, CD38 and BAFF-R; no CD23 and CD27; and high levels of CD69, CD86 and CD95 indicating recent activation. They have higher basal calcium levels and basal phosphorylation of BCR-associated signaling molecules such as SYK and ERK. They are polyclonal, have not undergone somatic hypermutation, but have undergone replication as determined by analysis of kappa recombination excision circles (KREC). The population has a high frequency of autoreactive clones and is prone to apoptosis. They are exclusively responsive to TLR9 stimulation. They preferentially home to inflamed peripheral tissues like the bronchoalveolar space in CVID and cGVHD patients or the synovium of RA patients due to increased surface expression of inflammatory type chemokine receptors such as CXCR3 necessary for migration to inflammatory sites. In contrast, they down-regulate CD62L expression, the chemokine receptor we discussed earlier that regulates B cell trafficking to secondary lymphoid organs and germinal centers. The origin of this pathogenic CD21 low B cell population is unknown. It is plausible that high levels of sBAFF may alter the developmental trajectory of early transitional B cells towards pathogenic endpoints such as CD21 low B cells.           	   157	  Chapter 6: Alternative Isoforms of BAFF: Relevance to Pre-B Acute Lymphoblastic Leukemia (pre-B ALL)  6.1. Introduction In the previous chapter we described an activity in patient serum consistent with a soluble form of BAFF (sBAFF) that down-modulates surface expression of its primary receptor, BAFF-R, likely through a mechanism of internalization. This is consistent with a normal negative feedback mechanism to prevent excessive BAFF signaling. It would explain the inverse relationship between high sBAFF levels and reduced surface BAFF-R expression present in many autoimmune and inflammatory diseases. This would suggest that part of the underlying pathophysiology in these diseases appears to involve an abnormal excess production of BAFF. It is thought that excess BAFF promotes the escape of autoreactive B cells from negative selection. As part of normal B cell homeostasis, high BAFF levels should trigger increased ligand-induced internalization of the BAFF-R preventing sustained activation. In contrast to normal B cells, malignant B cells show uncontrolled cell proliferation. There is convincing evidence that autocrine/paracrine BAFF constitutively activates cell survival pathways in several B cell malignancies. Many key growth factors that are produced aberrantly in cancer exist as several isoforms with distinct properties [315]. The biophysical properties of BAFF in B cell malignancies remains poorly understood. The purpose of this section is to investigate the properties of BAFF produced by malignant cells.   	   158	  6.2. Pre-B Acute Lymphoblastic Leukemia as a Model System for Aberrant BAFF-BAFF-R Signaling We choose to study pre-B ALL as a model because of several observations we and others have made: 1) pre-B ALL cells aberrantly express functional BAFF receptors; 2) pre-B ALL cells express and secrete a N-glycosylated sBAFF isoform 3) BAFF is biologically active in pre-B ALL; 4) soluble BAFF is significantly higher in relapsed pre-B ALL patients. Our results suggest that pre-B ALL cells have a functional BAFF signaling pathway that involves a N-glycosylated BAFF that is associated with increased survival.  6.2.1. Pre-B ALL Cells Express BAFF Receptors  Since BAFF may function as an autocrine signal to promote growth and proliferation in leukemic cells, we first analyzed the expression of BAFF receptors in four human relapse pre-B ALL cell lines (697, RS4, 380 and Nalm-6). BAFF receptors are not expressed on normal pre-B cells [310]. All cell lines were positive for surface expression of BAFF-R and TACI. They showed no expression of BCMA  (Figure 6.1). The intensity of expression of TACI was much lower compared to the BAFF-R expression on these cells.  	   159	    Figure 6.1. Surface expression of BAFF receptors (BAFF-R, TACI and BCMA) on relapse pre-B ALL cell lines.  Flow cytometric analysis of surface expression of BAFF-R, BCMA and TACI (black line) on relapse pre-B ALL cell lines (697, RS4, 380 and NALM6) with isotype control (light gray line). Surface expression is represented as MFI. The last column includes positive controls: CD19+ B cells for BAFF-R, CD19+ B cells for TACI and CD138+ plasma cells for BCMA; MFI – median fluorescence intensity  697BAFF-RRS4 380 Nalm6BCMATACIFigure 4: Flow cytometry analysis of surface expression of BAFF-R, BCMA and TACI (black line) on relapse pre-B ALL cell lines with isotype control (light gray line).697BAFF-RRS4 380 Nalm6BCMATACIFigure 4: Flow cytometry analysis of surface expression of BAFF-R, BCMA and TACI (black line) on relapse pre-B ALL cell lines with isotype control (light gray line).697BAFF-RRS4 380 Nalm6BCMATACIFigure 4: Flow cytometry analysis of surface expression of BAFF-R, BCMA and TACI (black line) on relapse pre-B ALL cell lines with isotype control (light gray line).BAFF-­‐R	  MFI TACI	  MFI BCMA	  MFI 697 RS4 380 NALM6 CD19+	  B	  Cells CD19+	  B	  Cells CD138+	  Plasma	  Cells 	   160	  We also evaluated the surface expression of BAFF-R in primary pre-B ALL patient samples that were previously expanded in immune-deficient mice. We measured the surface BAFF-R expression on leukemic blasts in diagnostic samples. We found that primary pre-B ALL cells express varying degrees of surface BAFF-R (Figure 6.2) with several diagnostic samples exhibiting high surface BAFF-R levels (3 of 5).    Figure 6.2. BAFF-R surface expression on primary pre-B ALL cells. Flow cytometry of surface BAFF-R on pre-B ALL patient samples taken at diagnosis (n=5).   6.2.2. Pre-B ALL Cells Express and Release Glycosylated BAFF  To demonstrate BAFF autocrine signaling in pre-B ALL, we determined BAFF expression in whole cell lysates and in concentrated (100-fold) cell conditioned serum-Diagnostic Samples0200400600800100020003000BAFF-R MFI	   161	  free media (soluble BAFF) from the four pre-B ALL cell lines (697, RS4, 380 and NALM6). In these experiments, our primary positive controls for malignant cell lines that produce and release BAFF are the myeloid cell line, U937, and T-cell leukemia Jurkat cell line [188]. We found that all pre-B ALL cell lines express full-length BAFF and release sBAFF (Figure 6.3A, D, E and F). By subjecting lysates to PNGase F treatment we show in 697 and U937 cell lines that both full-length and sBAFF are N-glycosylated. This appears to be the only modification that sBAFF undergoes as PNGase F mediated deglycosylation resulted in a band equivalent to unmodified recombinant sBAFF (Figure 6.3B and C). By increasing the protein load on the Western blot we observed that 697 cells also express a small amount of unmodified sBAFF intracellularly in addition to glycosylated full-length BAFF (Figure 6.3D).    	   162	   28 kDa 35 kDa Jurkat  lysate 697 lysate 697 media A. 697 media U937 lysate U937 media U937 media rsBAFF 21 kDa 28 kDa B. 697 media 697 media PNGase F U937 media U937 media PNGase F C. 35 kDa 21 kDa rsBAFF 697 lysate PNGase F U937 lysate PNGase F 697 lysate U937 lysate 35 kDa 21 kDa rsBAFF U937 lysate 40 U937 lysate 10 U937 lysate 5 U937 lysate 1 697 lysate 40 697 lysate 10 697 lysate 5 697 lysate 1 35 kDa U937 lysate 697 lysate Nalm-6 lysate RS4 lysate 380 lysate E. 28 kDa F. U937 media 697 media Nalm-6 media RS4 media 380 media D. 	   163	  Figure 6.3. Expression of glycosylated full-length and soluble BAFF in pre-B ALL cell lines. Positive controls for soluble BAFF and full-length BAFF used on the Western blots include: 1) recombinant unmodified soluble human BAFF (aa 134-285, Peprotech #310-13) with a molecular weight of 17 kDa (confirmed by mass spectrometry) that runs at 21 kDa on a 15% SDS-PAGE gel, 2) soluble N-glycosylated BAFF (28 kDa on 15% SDS-PAGE gel) from U937 human myeloid cell line-conditioned serum-free media [Need reference 333], 3) full-length N-glycosylated BAFF (35 kDa on 15% SDS-PAGE gel) from U937 and Jurkat T cell leukemia cell line lysates. A rabbit anti-human BAFF (C-terminus) polyclonal antibody raised against a peptide corresponding to amino acids 254 to 269 of human full-length BAFF was used. A. Western blot of cell lysates (10 ug) and concentrated (100X) cell line (1×106 U937 cells/mL or 2×106 697 cells/mL for 24 hours) conditioned serum-free media. B. Western blot of PNGase F-treated concentrated  (100X) cell line (1×106 U937 cells/mL or 2×106 697 cells/mL for 24 hours) conditioned serum-free media. C. Western blot of cell lysates from U937 and 697 cells treated with PNGase F. D. Western blot of cell lysates (1-40 ug total protein loaded) from U937 and 697 cells. E. Western blot of pre-B ALL cell lysates. F. Western blot of concentrated (100X) cell line (1×106 U937 cells/mL or 2×106 cells/mL for pre-B ALL cell lines for 24 hours) conditioned serum-free media  We then tested three primary pediatric pre-B ALL samples acquired from the Child & Family Research Institute Biobank at BC Children’s Hospital and identified one patient sample that released sBAFF (Figure 6.4A and B).  	   164	   Figure 6.4. Primary pre-B ALL cells release sBAFF. A. Thawed diagnostic primary pre-B ALL cells from three different patients were kept overnight in serum-free media and supernatant was concentrated and sBAFF detected by Western blot. A band at 28 kDa is visible in patient #1. B. Patient #1 cell lysate and supernatant was run with positive controls   6.2.3. BAFF is Functional Active in Pre-B ALL  It has previously been shown that BAFF induces the expression of Pim-2, an anti-apoptotic proto-oncogene that is elevated in a number of lymphoid malignancies including ALL [310]. We hypothesized that blocking autocrine/paracrine sBAFF in 697 cell cultures with soluble BAFF-R:Fc would down-regulate Pim-2 gene expression. Patient #1 28 kDa Patient #2 Patient #3 A. 35 kDa 28 kDa B. Patient #1 lysate Patient #1 media U937 media 697 media 	   165	  Incubation of 697 cell cultures with soluble BAFF-R:Fc resulted in reduced gene expression of Pim-2 (Figure 6.5).   Figure 6.5. Gene expression of Pim-2 after blocking sBAFF in 697 cells.  697 cells (2×106 cells/mL) were incubated for 24 hours in serum-free media with or without soluble decoy BAFF-R:Fc (5 ug/mL). Levels of mRNA transcripts for Pim-2 upon culture in the presence or absence of soluble BAFF-R:Fc were quantified by real-time PCR and results expressed relative to β-actin transcripts. There was no significant difference in the number of β-actin transcripts. Representative of two independent experiments of triplicate cultures    Blocking sBAFF in 697 Cell Culture0.00.10.20.30.40.50.60.70.80.91.01.1	   166	  6.3. Potential Differences Between sBAFF Isoforms  6.3.1. Belimumab Does Not Bind Leukemic sBAFF Belimumab (BENLYSTA™), previously known as LymphoStat-B is a fully humanized monoclonal antibody that inhibits sBAFF [318]. To characterize the binding properties of leukemic sBAFF versus recombinant sBAFF, we utilized belimumab to capture sBAFF from serum or cell-conditioned media using AminoLink coupling resin consisting of 4% beaded agarose that has been activated with aldehyde groups to enable covalent immobilization of the antibody. We ran 697 cell line conditioned serum free media along with plasma from our PLCγ2 mutation patient as a positive control and then performed a Western blot with a polyclonal anti-BAFF antibody (Figure 6.6). The belimumab immobilized in the column was unable to bind the sBAFF present in 697 conditioned media (Lane C Figure 6.6).     28	  kDa 21	  kDa A B C D 	   167	  Figure 6.6. Western blot of belimumab immobilized column eluate for sBAFF using polyclonal anti-BAFF antibody. Concentrated 697 cell line conditioned serum free media and plasma from our patient (PLCγ2mut/+) was run through a binding column containing immobilized belimumab to assess binding. Immunoblotting was performed to assess for presence of sBAFF in column flow through and eluate. A. Concentrated 697 cell line conditioned serum free media prior to column flow through. B. Concentrated 697 cell line conditioned serum free media flow through (non-binding component) C. Concentrated 697 cell line conditioned serum free media eluate D. Patient (PLCγ2mut/+) plasma eluate. 28 kDa – leukemia sBAFF, 21 kDa –  patient (PLCγ2mut/+) sBAFF   6.3.2. Leukemic sBAFF Inhibits the Ability of Normal sBAFF to Down-Modulate Surface Expression of BAFF-R 697 pre-B ALL cells were seeded at 500,000 cells/mL in complete media and cell-conditioned supernatant was collected 96 hours later when cell concentration was 4×106 /mL and viability was 98% as determined by trypan blue staining. The cell-conditioned supernatant was first concentrated ~10-fold and then run through either an immunoaffinity column containing the anti-BAFF monoclonal antibody from R&D used in the ELISA kit to detect leukemic sBAFF or a mock column under sterile conditions. The flow through was then pre-incubated with healthy adult donor peripheral blood mononuclear cells for one hour followed by stimulation with recombinant sBAFF for 6 hours. The eluate fraction post-flow through demonstrated the presence of leukemic 28 kDa sBAFF isoform by Western blotting that was extracted from the cell culture media 	   168	  prior to incubation. Using the R&D ELISA kit, even at 100-fold concentration (used in Western blotting to detect 28 kDa soluble malignant BAFF), the maximum concentration of sBAFF obtained was only 165 pg/mL. Despite this, supernatant not depleted of soluble BAFF (mock column flow through) was still able to significantly inhibit the suppressive effect of 1 ng/mL (9.37 % vs. 12.84 % inhibition; p = 0.02) and 10 ng/mL (59.87 % vs. 67.84 %; p = 0.04) rBAFF on surface BAFF-R expression on healthy donor CD19+ B cells compared to supernatant depleted of soluble BAFF. The blocking effect was moderate, likely due to the low concentration of soluble malignant BAFF present.   6.3.3. The Effect of Normal sBAFF on Pre-B ALL is Anti-Proliferative  Having demonstrated that high levels of normal sBAFF cause BAFF-R down-modulation and that leukemic sBAFF disrupts this process, we hypothesized that normal sBAFF would interrupt the autocrine/paracrine BAFF/BAFF-R pathway in pre-B ALL cells. Incubating pre-B ALL cells with normal sBAFF would cause down-modulation of surface BAFF-R expression causing an anti-proliferative effect. Interestingly, it has previously been shown that exogenous recombinant sBAFF significantly increased the dexamethasone-induced apoptosis of 697 cells [319].  We incubated pre-B ALL 697 cells with unmodified recombinant sBAFF and found that surface expression of BAFF-R was decreased (Figure 6.7A), with cells exhibiting decreased proliferation (Figure 6.7B) and increased cell death (Figure 6.7C).  	   169	   Figure 6.7. Effect of normal sBAFF on 697 pre-B ALL cells. A. 697 cells were incubated overnight in complete media at a concentration of 1×106 cells/mL with or without unmodified recombinant sBAFF (100 ng/mL) and the surface expression of BAFF-R was measured by flow cytometer. B. 697 cells were incubated in complete media for 6 days at a starting concentration of 1×106 cells/mL with or without unmodified recombinant sBAFF (100 ng/mL). At the end of incubation total cells were counted with a hemocytometer and concentration determined. Data points represent three independent experiments of four replicates (n=12). C. 697 cells were incubated in complete media for 6 days at a starting concentration of 1×106 cells/mL with or without unmodified recombinant sBAFF (100 ng/mL). At the end of incubation alive and dead cell counts were performed by trypan blue cell counting with a hemocytometer and ratio of % No sBAFF 100 ng/mL sBAFF050010001500BAFF-R MFI****No sBAFF 100 ng/mL sBAFF3×1064×1065×1066×106Total (Alive+Dead) Concentration (cells/mL)**No sBAFF 100 ng/mL sBAFF0.01.02.03.04.05.0Ratio % living/ % dead cells**A.	   B.	   C.	   	   170	  living/% dead was calculated. Data points represent three independent experiments of four replicates (n=12). ** p < 0.01, **** p < 0.0001, two-tailed Mann-Whitney  This set of results provides evidence that normal sBAFF could potentially mediate an anti-leukemic effect versus malignancies that rely on autocrine/paracrine BAFF signaling.  We then sought evidence of this potential mechanism in a human disease model. We thought that the treatment of high risk or relapsed pre-B ALL with hematopoietic stem cell transplantation (HSCT) would be an ideal model to study because we have already demonstrated the anti-proliferative effect of normal sBAFF on pre-B ALL cells and have previously identified that the period post-HSCT is associated with high levels of sBAFF.   6.4. The Presence of Soluble BAFF Post-HSCT Having identified the possibility of different functional sBAFF isoforms, we next sought to identify the presence of these isoforms in human disease. We choose to study the role of sBAFF in the treatment of pre-B ALL with hematopoietic stem cell transplantation. This is the ideal model in which to simultaneously study the presence and effect of both normal and leukemic BAFF. The peritransplant period is one of the most inflammatory settings, potentially leading to increased expression of normal sBAFF. This is countered by the possible expression of sBAFF by leukemic cells trying to counteract the effect of normal sBAFF, further promoting the malignant growth of leukemic cells. We first measured the effect of normal sBAFF in plasma from patient’s post- 	   171	  HSCT by inhibiting surface BAFF-R down-modulation with belimumab. In the second section, we sought to determine whether there was a correlation between sBAFF levels and relapse of pre-B ALL post-HSCT, suggesting the presence of endogenous leukemia-promoting sBAFF.  6.4.1. The Role of Soluble BAFF Post-HSCT on the Surface Expression of BAFF-R in Pre-B ALL Cells  We hypothesized that plasma taken from patients post-HSCT would be able to down-modulate the surface expression of BAFF-R on 697 pre-B ALL cells. We measured the effect of plasma taken from patients 3 and 6 months post-HSCT. The effect of sBAFF was measured indirectly by inhibiting its ability to down-modulate surface BAFF-R expression on 697 cells with belimumab. We predicted that the effect of sBAFF would be stronger at 3 months versus 6 months consistent with sBAFF levels post-HSCT.  We demonstrated that there was a significantly higher effect on BAFF-R expression in plasma taken from patients 3 months post-HSCT (Figure 6.8A) and this correlated with the level of plasma sBAFF (Figure 6.8B).         	   172	  A.  B.  3 mo post-HSCT 6 mo post-HSCT0246Fold Change in BAFF-R MFI****0 2 4 60200040006000697 pre-B ALL cellsFold Increase in Surface BAFF-R Expression with BelimumabInhibitionsBAFF pg/mLr2 = 0.72p < 0.0001	   173	   Figure 6.8. Plasma from patients post-HSCT down-modulates surface expression of BAFF-R on pre-B ALL 697 cells. A. 697 cells were incubated with a 1:1 mix of complete media and patient plasma with or without belimumab (10 ug/mL) overnight and surface expression of BAFF-R was measured by flow cytometry. The MFI was compared between both conditions for each sample and expressed as fold increase in BAFF-R due to sBAFF inhibition by belimumab. Effect was compared between 3 and 6-month post-HSCT time-points. Statistical comparisons were made by using two-tailed Mann-Whitney. **** p < 0.0001. MFI –mean fluorescence intensity. B. Correlation between plasma sBAFF levels and fold increase in surface BAFF-R expression   6.4.2. High Levels of Soluble BAFF are Associated with Relapse of pre-B ALL and Increased Disease-Related Mortality Post-Hematopoietic Stem Cell Transplantation (HSCT) Plasma levels of sBAFF were measured in patients’ post-HSCT with relapse of pre-B ALL in the absence of graft-versus-host disease (GvHD). GvHD is a devastating disease that occurs post-HSCT in which donor immune cells attack healthy host tissues. It is an unfortunate consequence of transplanting mature allogeneic immune effector cells and the long-term reconstitution of an allogeneic immune system whose primary therapeutic purpose is to mediate an immunological anti-tumor effect referred to as the graft-versus-leukemia (GvL) effect. We specifically excluded samples from patients who developed any form of GvHD as our laboratory and others have previously shown along that chronic GvHD (cGvHD) is associated with high levels of sBAFF [316,317]. 	   174	  Sarantopoulos et al. measured serial BAFF levels 1, 3, 6, 9 and 12 months after HSCT in patients who never developed cGVHD compared with BAFF levels in patients who developed cGVHD between 7 and 12 months after transplantation. In both groups, sBAFF levels were elevated in most patients in the first 3 months post-HSCT. They found that sBAFF concentrations generally decreased by 6 months after transplant in patients who did not develop cGvHD, while BAFF levels remained high for at least 10 to 12 months in patients who subsequently developed cGvHD. Because of this we used control samples in this experiment (patients with no relapse post-HSCT) taken at 6 months post-HSCT. Furthermore, the implications of high levels of sBAFF early post-HSCT (i.e. 3 months post) will be discussed in the last section of this chapter. This experiment utilized specimens obtained from patients enrolled on the Children’s Oncology Group ASCT0431 trial, a randomized phase III study comparing the post-transplant event-free survival of pediatric patients with relapsed acute lymphoblastic leukemia undergoing allogeneic HSCT. Soluble BAFF levels were measured by ELISA in plasma samples taken at 6 months post-HSCT in patients with pre-B ALL who relapsed (n=11; 174 ± 15 days post-HSCT) versus those who did not (n=13; 187 ± 12 days post-HSCT) in the absence of any acute or chronic GvHD. The R&D QuantikineTM sBAFF ELISA kit used in this analysis pairs the 137134 anti-BAFF mAb with a goat HRP-conjugated anti-BAFF polyclonal antibody. We found that pre-B ALL relapse post-HSCT was significantly associated with a higher level of plasma sBAFF (Figure 6.9) and high sBAFF at 6 months in absence of GvHD post-HSCT was significantly associated with increased mortality (Figure 6.10).  	   175	    Figure 6.9. Soluble BAFF levels at 6 months post-HSCT for relapsed pre-B ALL. Plasma levels of sBAFF (pg/mL) were evaluated by ELISA (R&D) in patients 6 months post-HSCT without any GVHD. Comparison is made between patients with relapsed and those that did not. Statistical comparisons were made by using two-tailed Mann-Whitney. * p < 0.05  Soluble	  BAFF	  at	  6	  Months 	  Post-­‐HSCT	  (pg/mL) Patients	  With	  No	  Relapse Patients	  With	  Relapse * 	   176	    Figure 6.10. Event free survival in GVHD negative patients based on sBAFF concentrations in blood at 6 months from patients on the COG ASCT0431 study. Using the sBAFF optimal cutpoint at 80th percentile of distribution (5412 pg/ml) was the best predictor of relapse with relative risk (RR) of 5.3 (p=0.006). The cutpoint was used to classify 6 months marker value as “high” or “low”. The results are the 1 year EFS with a 95% CI    6.5. Discussion and Future Directions  	   In this section, we have shown that pre-B ALL cells, including cell lines and primary samples, aberrantly express BAFF-R and BAFF ligand simultaneously setting up the necessary parts of an autocrine/paracrine signaling pathway.  The sBAFF isoform expressed by pre-B ALL cells was biologically active as demonstrated by inhibition of Pim-2 gene expression, a proto-oncogene that acts as a 	   177	  serine/threonine protein kinase, after blocking BAFF signaling with a soluble BAFF antagonist, BAFF-R:Fc. The results also suggest that it is biological different then the exogenous recombinant, endogenous neutrophil-derived and PLCγ2mut/+ patient sBAFF characterized in the previous chapter. This was demonstrated by its ability to inhibit the modulatory effect of recombinant sBAFF on surface BAFF-R expression in healthy donor B cells and lack of binding to the clinical monoclonal antibody, belimumab. Potential differences were further emphasized by the ability of non-leukemic sBAFF to decrease surface BAFF-R expression and proliferation and increase cell death of a pre-B ALL cell line. This describes a potential anti-tumor effect that could be mediated by increased levels of non-leukemic sBAFF, whose production is driven by pro-inflammatory cytokines such as IFN-γ. We show preliminary evidence of this mechanism post-HSCT. It is possible that this effect may be more exaggerated in the setting of chronic graft-versus-host disease (cGvHD), a serious complication of HSCT in which donor immune cells attack healthy recipient tissue. Our analysis only evaluated samples up to 6 months post-HSCT. It has previously been shown that a persistent elevation of sBAFF beyond 6 months post-HSCT is associated with cGvHD and there is an inverse correlation between high sBAFF levels and low surface BAFF-R expression. This may explain why cGvHD is associated with a lower incidence of relapse after HSCT in patients with ALL [320]. While the soluble form of cleaved BAFF is thought to be the primary biologically active form, our results suggest that in the case of pre-B ALL a membrane bound full-length that binds BAFF-R in cis or trans may be the primary active form. Despite a 100-fold concentration of cell-conditioned media from pre-B ALL cell lines, the measured 	   178	  amount of sBAFF remained quite low. This is consistent with previous findings as Parameswaran et al. were able to show expression of BAFF mRNA and protein in ALL cells but could not find any evidence of BAFF being shed into the medium. The low amount of leukemic sBAFF also proved to be a technical challenge as we were unable to acquire enough leukemic sBAFF to perform in depth biological studies comparing leukemic and non-leukemic sBAFF directly. Another limitation is that we were unable to identify the type of sBAFF present in the serum of patients who experienced relapse. This was due to a limited amount of sample available for analysis. In the future, we are planning targeted larger serum collections from relapsed patients to identify the properties of the sBAFF. We can only hypothesize that the BAFF in these patients’ serum is N-glycosylated similar to that identified in pre-B ALL cell lines and primary diagnostic samples.             	   179	  Chapter 7: Discussion The original problem statements in this thesis first directed us to identify the underlying molecular defects in two pediatric patients with unknown B cell defects. Our studies were able to provide insights into the diagnosis and treatment of two new primary immunodeficiencies while simultaneously offering novel insights into the critical role of PLCγ2 and MALT1 in peripheral B cell development. Both patients also provided us with a starting point to investigate the regulation of surface BAFF-R expression on B cells. Both of these patients had significantly decreased surface expression of BAFF-R on total B cells, yet this effect appears to be mediated through different mechanisms.  In the case of MALT1 deficiency, the defect was intrinsic to the B cell. This suggests that the proper function of MALT1 is essential for the developmental expression of BAFF-R. BAFF-R is a NF-κB target gene and the NF-κB transcription factor c-Rel has been shown to bind putative promoter sites upstream of the BAFF-R gene [248]. In murine MALT1 knockout models, only the specific nuclear translocation of c-Rel was lacking after BCR ligation suggesting that MALT1 plays a direct role in the cytoplasmic release of c-Rel from inhibitory Iκβ molecules [257]. C-Rel remains bound to IκBα and IκBβ in MALT1-/- B cells. Interestingly, similar degrees of p65/RelA DNA binding activities were detected in BCR-stimulated wild type and MALT1-/- B cells. This demonstrates that murine MALT1 is largely dispensable for canonical NF-κB signaling downstream of the BCR. This is in contrast to the findings in our patient. We demonstrate that there is absent phosphorylation of p65 and degradation of Iκβα in primary B cells after PMA/Ionomycin stimulation, which mimics the actions of diacylglycerol (DAG), one of the second messengers produced by BCR-triggered PLCg2 activation. 	   180	  Furthermore, MALT1-/- mice have also been shown to have normal surface expression of BAFF-R by flow cytometry in contrast to our patient [321].  It has previously been demonstrated that a P21R variant of human BAFF-R does not bind to certain antibody clones by flow cytometry (personal communication from Dr. Warnatz). We did not identify any BAFF-R variants in the whole exome sequencing results in our patient. Also, reduced surface BAFF-R expression in this patient was not due to sBAFF-mediated internalization, as demonstrated in the subsequent chapter, because overnight culture did not restore surface expression. It is possible that the role of the CBM complex in regards to the expression of BAFF-R differs between mice and humans. There are numerous differences in B cell biology between mice and humans, including the impact of deficiencies of several proteins upstream of the CBM complex in BCR signaling such as BLNK and Btk [322]. The drawback is that it may limit the use of mouse models to further study how MALT1 impacts the expression of BAFF-R. It is imperative that we show a similar phenomenon from other patients with MALT1 deficiency linked to B cell defects before we can conclude that murine models are not suitable. Characterization of our patient’s clinical phenotype will help identify patients in the future. Furthermore, studying this pathway in human cell line models will also depend on the developmental stage of the B cell as this pathway may only be relevant at the early transitional stage at which BAFF-R is first expressed.  We were able to generate EBV-lymphoblastoid cell lines that allowed further characterization of MALT1 function. Our next step using these EBV cell lines will be to determine any new binding partners of MALT1 that may shed light on new signaling 	   181	  pathways, such as a hypothesized IKK-independent degradation of IkB inhibitory proteins by CK2 in B cells described in that chapters discussion.  As previously proposed with BAFF we will introduce endogenous protein tags attached to MALT1 in a wild-type EBV LCL using CRISPR/Cas9 genome editing techniques to facilitate co-immunoprecipitation experiments to determine novel MALT1 binding partners in B cells. After stimulation, cell lysates will be run through tag-specific affinity columns and MALT1 scaffolding partners and substrates will be assessed by mass spectrometry. To facilitate this, in addition to adding protein tags we will also directly alter the MALT1 gene in order to have endogenously tagged: 1) wild-type MALT1; 2) catalytically inactive MALT1mutant to help differentiate substrates and 3) a copy of MALT1 with our patients’ mutation to help better understand our patients clinical phenotype.  In chapter 4, we report a novel genetic defect in PLCγ2 in a 6-year old girl presenting with a clinical phenotype of hypogammaglobulinemia, absent mature B cell subsets and reduced surface expression of BAFF-R. A gain-of-function mutation in PLCγ2 was associated with: 1) increased BCR-triggered external calcium entry; 2) increased BCR-triggered ERK phosphorylation and 3) increased α granule release from platelets after stimulation with collagen and 4) increased apoptosis of immature B cells.  One of the earliest mechanisms of negative B cell selection is the elimination of autoreactive B cells between the early transitional (T1) and more mature transtional stages (T2) and between the T2 and mature follicular B cell states, mediated by increased BCR signaling. The BCR hyper-reactivity caused by a gain-of-function mutation in PLCγ2 could be an intrinsic B cell defect that leads to an increased apoptosis of 	   182	  transitional B cells. The increased apoptosis may also be due to a direct toxic effect of increased intracellular calcium concentration. B cells are exposed to a prolonged elevation of [Ca2+] that in turn activates necrotic and apoptotic processes catalyzed by calcium-activated proteases, phospholipases and endonucleases [323].  The prominent arrest in B cell development present in our patient is similar to that observed in PLCγ2-deficient mice despite completely opposite effects on BCR-stimulated ERK phosphorylation and calcium flux [324]. This suggests that PLCγ2 activity is restricted to a limited spectrum in normal B cell development; a decrease in PLCγ2 activity results in diminished tonic BCR signaling that strongly impairs development of transitional B cells [325], while constitutive BCR hyper-reactivity causes every B cell to appear as being self-reactive at the early transitional B cell stage leading to eventual clonal deletion. During the work-up of this patient we also observed a phenomenon by which sBAFF was able to down-modulate the surface expression of its primary receptor, BAFF-R. This effect was observed with sBAFF in the serum of the PLCγ2mut/+ patient, sBAFF released from G-CSF and IFNγ-stimulated human neutrophils and unmodified recombinant sBAFF. A small proportion of this effect was due to receptor occupancy preventing the binding of antibody; a technical problem that needs to be considered but appears not to be significant at sBAFF concentrations seen in human diseases. This does however highlight the need for rigorous antibody testing and a standardized diagnostic approach in order to be able to properly differentiate BAFF-R expression, regardless of occupancy, and differentiate between membrane-bound BAFF and sBAFF bound to its surface receptors. However, we demonstrate that the overall effect of BAFF-R down-	   183	  modulation secondary to normal sBAFF is primarily due to a post-transcriptional mechanism that leads to receptor internalization. It is possible that sBAFF-induced receptor internalization is a concentration dependent regulatory mechanism that prevents any further action of BAFF on its target. This mechanism would serve as a buffer for excess BAFF as part of a negative feedback loop; a natural feedback mechanism in inflammatory conditions meant to turn off excessive B cell activation. Consider a theoretical model in which infection triggers polyclonal B cell activation. After this response has served its purpose, it must be terminated. The immune system produces excess sBAFF isoform activated by inflammation-related IFNγ production and there is down-regulation of excessive B cell proliferation by blocking signaling through the BAFF-R. Viral infections have been shown to induce the production of sBAFF and blocking type 1 interferon (IFN) receptor strongly inhibited BAFF expression [326-328] . This may explain the finding of increased sBAFF and reduced surface expression of BAFF-R in infections such as hepatitis C, HIV/AIDS, Pseudomonas aeruginosa, malaria [329] or tuberculosis [330].  Furthermore, we also demonstrated that sBAFF down-regulated the expression of CD62L, a homing receptor for lymphocytes to enter secondary lymphoid tissues, on early transitional B cells potentially trapping increased numbers of early B cells in the peripheral circulation and preferentially trafficking them to the spleen. In any disease associated with high sBAFF levels, this would expose an increased number of BAFF-primed non-tolerized polyreactive and autoreactive immature B cells to the stimulatory effects of various inflammatory agonists such as TLR9 agonists.   	   184	  We were also able to show that the sBAFF associated with pre-B ALL was able to significantly inhibit the down-modulation of BAFF-R, albeit to a moderate extent likely due to low concentrations of cleaved sBAFF in leukemia. Our observations suggest that the predominantly active form of BAFF in leukemia may be membrane-bound. This model presumes that the down-modulation of surface BAFF-R expression is a normal response to elevated levels of normal sBAFF and the pathogenesis of diseases is due to high levels of normal sBAFF driven by an inflammatory process.  One structural difference that we were able to establish was that full-length and soluble leukemic BAFF were both N-glycosylated. There are only two putative glycosylation sites in full-length BAFF and both of them are extracellular, one being just proximal to the furin cleavage site between aa 133 and 134 at N124 and another distal at N242. There are a number of possible ways that glycosylation could affect the biophysical properties of BAFF. It could be that glycosylation affects the trafficking of BAFF intracellularly and subsequent expression at the cell surface. It is also possible that N-glycosylation prevents furin-mediated cleavage of full length BAFF by blocking access to the cleavage site. However, the fact that we also observe an N-glycosylated soluble BAFF presumes that both putative sites are glycosylated and that blockage is not complete. This would explain how we observed glycosylated sBAFF in concentrated media and may explain the elevated sBAFF levels seen in relapse pre-B ALL patients post-HSCT.  A conceptual model based on these hypotheses could be that in malignant cells, glycosylated full length BAFF is shuttled to the cell surface where it remains and exerts biological activity without undergoing extensive cleavage. In contrast, non-glycosylated 	   185	  full length BAFF is cleaved intracellularly into its truncated, soluble form and stored in Golgi-related compartments that are secreted in response to proinflammatory mediators. Our results and those of others indicate that this may be the mechanism in neutrophils [331]. To definitively address the impact of glycosylation on BAFF we are designing a series of experiments in which we will introduce a 3X FLAG tag and GFP tag to endogenous BAFF in pre-B ALL cell lines using CRISPR/Cas9 mediated targeted genome editing. This will allow the visualization of the intracellular trafficking of BAFF. Furthermore, we will knockout the glycosylation sites simultaneously and assess biophysical properties of BAFF in regards to glycosylation such as trafficking.  It is possible that the clinical heterogeneity of diseases associated with elevated sBAFF may be due to different ratios of each isoform. For example, SLE, in which multiple previous studies have demonstrated no inverse relationship between sBAFF and BAFF-R surface expression, the glycosylated isoform may predominate. In contrast, chronic infections such as HIV and hepatitis C may have a predominance of the unmodified sBAFF. Furthermore, we demonstrate that belimumab, a clinically available humanized monoclonal antibody against sBAFF only binds the unmodified isoform and not the glycosylated isoform. This may explain why belimumab has had mixed success in the treatment of a variety of autoimmune diseases. 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ANKRD63 ZFHX4 FAM179B OAZ3 HAGH CAB39 CAPSL INADL ALDH7A1 SH2D4A ZSWIM5 BCL2A1 ALDOA EHF EP400 ABCA13 SYMPK ACACA C20orf194 KIAA1468 MAP1B ZAN IGFBP4 CCDC88B WAPAL FAP PLEKHM1 CAB39 LTBP4 FREM2 ART1 HSPG2 ZFHX4 ANKRD36C RLF LETM2 C5orf42 GEMIN8 ATHL1 SEMA3B AK7 THAP5 SIPA1 ZSWIM5 RHPN2 CES1 TRANK1 ARL1 RERE FNBP1L ORM1 MYO1A DOCK8 POU4F1 OVOL3 SCYL2 HCK TAL1 AC005841.1 CLCNKA GLTP ATP11A FGF5 CDC14B MYLK3 SPEF2 IMP4 RNF19B OR10G4 HEATR6 GRM3 ZNF232 ZNF628 XRN1 SPTBN2 ZKSCAN2 PLCG2 PHKG2 SEPT7 SRD5A1 SLC16A7 ZNF433 CRYBG3 MYB NAV2 ALDH7A1 	   230	  SDR16C5 ARL1 USP31 BEND2 COL6A3 FAM184A AC007405.2.1 SORT1 RNF151 MUC17 NHSL2 TRIM71 DNAH11 SIPA1 DYM KRT10 PC OSBPL7 KIF18A UTP20 NFATC3 NUBP2 AKAP11 YTHDC2 KARS ZNF439 CAD DVL2 BCAM OR10A3 SEC31B CGNL1 INSC ELMO3 DNAH11 SHROOM4 ABCC5 LTK PRDM4 SH2D4B TLE2 IRF6 IFT122 ATP8A1 ADAMTS13 CRB2 GAL3ST2 MDM1 TMEM133 HACL1 DHX57 MEGF8 ARHGAP18 ZNF578 KNG1 LPHN2 SCN1A C12orf32 AADACL3 PDLIM5 TLE6 AC008686.1 FOXE1 POU6F2 C10orf140 AQP3 AL354984.2 DTYMK GAPDHP69 RP51025A1.2.1 AC010731.4.1 MDGA1 RP1273P12.1.1 TRMU LILRA2 RP11356C4.4.1 AL359392.1 REXO1 CTD2666L21.1.1 ST6GALNAC3 NAP1L3 AL162502.1 NUP214 RAD52 AC002472.14.1 RP11497G19.1.1 PAICS RP11711K1.7.1 FAM154B DPYSL5 LILRB2 ANKRD57 F12 TRPV6 HIVEP3 TCEAL2 SDAD1 CLDN11 	   231	  LINC00442 LGALS3 SLC22A14 RPL19P20 IQCE ZNF268  

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