Le lupus érythémateux systémique est une maladie autoimmune systémique caractérisée par des lésions multiviscérales et la production d’autoanticorps (ex : anticorps anti-ADN double brin (db)) par les lymphocytes B (LB), qui jouent un rôle central dans la physiopathologie lupique. L’étiologie du lupus est à la fois environnementale et génétique. Dans le but d’identifier des anomalies génétiques intrinsèques aux LB, le laboratoire a réalisé une analyse transcriptomique sur les LB de patients lupiques en phase quiescente et a montré une surexpression des gènes TRIB1 et FKBP11 comparé aux sujets sains.Afin d’étudier les conséquences de la surexpression de ces gènes sur la fonction des LB et le développement d’une autoimmunité, nous avons généré une lignée murine conditionnelle spécifique, surexprimant Trib1 dans les LB à partir du stade précoce pro-préB et une lignée murine transgénique surexprimant Fkbp11 de façon ubiquitaire. La surexpression de Trib1 ne modifie pas l’homéostasie lymphocytaire mais induit une diminution de la production de certaines Ig : 1) les IgG1 dans le sérum à l’état basal et après une stimulation de LB in vitro ; 2) les IgM anti-OVA (Ovalbumine) après immunisation in vivo avec de l’OVA ; 3) les IgM anti-ADNdb dans le cas d’une immunisation par le LPS. Cette anomalie de la production des Ig semble provenir d’un défaut de sécrétion. De plus nous avons généré une lignée cellulaire B surexprimant Trib1 qui nous a permis de confirmer le phénotype et d’identifier des partenaires potentiels de Trib1 par technique de protéomique. La surexpression du gène Fkbp11, est, quant à elle, suffisante à induire des signes de la maladie lupique chez la souris âgée de 8 mois, tels qu’une rupture de tolérance (caractérisée par la production d’autoanticorps) et une initiation de la différenciation plasmocytaire. En conclusion, Trib1 pourrait exercer un rôle d’immunosupresseur et sa surexpression dans les LB lupiques pourrait constituer un nouveau mécanisme de régulation des LB pendant la phase de rémission du lupus, alors que Fkbp11 semble contribuer à la pathologie lupique. La description de ces deux nouvelles voies biologiques pourrait mener à une meilleure compréhension de la maladie et conduire à de potentielles applications thérapeutiques. / Systemic Lupus Erythematosous (SLE) is an autoimmune disease characterized by an inflammation of various tissues and a high production of autoantibodies (autoAb) (for example: anti-double-stranded(ds)DNA) by B cells, central actors in the physiopathology of lupus. The etiology of SLE includes both genetics and environmental factors. Looking for B cell genetic abnormalities during lupus, our B cell microarray analysis in quiescent SLE patients pointed to the overexpression of TRIB1 and FKBP11 compared to B cells from healthy controls.In order to study the consequences of these expression deregulations on B cell function and autoimmunity development, we generated a B-cell specific Trib1-KI mouse line, overexpressing Trib1 in B cells, starting from a very immature stage (pro-pre B) and a transgenic mouse overexpressing Fkbp11 ubiquitously. Trib1 overexpression induces a normal B cell homeostasis but a decrease in the production of some immunoglobulins (Ig): 1) IgG1 subclass in the serum, at a basal level and after an in vitro stimulation of splenic B cells; 2) Anti-OVA (Ovalbumine) IgM after immunization in vivo with OVA; 3) Anti-dsDNA IgM after immunization with LPS. This abnormal production of Ig seems to be linked to a defect in Ig secretion process. In addition, we developed a murine B cell line overexpressing Trib1 that let us to confirm the Ig production deficiency and to identify potential Trib1’s partners in B cells. In contrast, Fkbp11 overexpression, leads to some features of lupus disease in 8-month-aged-mice, including a tolerance breakdown (characterized by autoantibody production) and the initiation of plasma cell differentiation. In conclusion, Trib1 could exert an immunosuppressive role and its overexpression in SLE could constitute a new mechanism of B cell regulation during remission phases, whereas Fkbp11 seems rather to contribute to lupus physiopathology. Thus, the description of these two biological pathways could bring new insights into the comprehension of lupus disease and could also potentially lead to the development of new therapeutic applications.

Le syndrome de Gougerot-Sjögren (SGS) est une épithélite auto-immune caractérisée par des lésions des glandes exocrines et manifestations systémiques. Une des complications majeures est la survenue chez 5% des malades, d’un lymphome non-hodgkininen (NHL). La contribution majeure des lymphocytes B (LB) a récemment été démontrée. Dans ce travail, nous avons voulu aborder des sujets cliniques et fondamentaux concernant le rôle des LB dans le SGS. Dans un premier temps, nous avons démontré que des LB mémoires sont visibles dans des infiltrats des échantillons de la peau et sa présence peut aider au diagnostic. Dans un deuxième temps, nous avons démontré que la cytokine FLT3-L augmentée (une cytokine impliquée dans l’ontogenèse des LB et lympho-prolifération) est associée à une distribution anormale des LB dans les malades. En plus, le rôle prolifératif de FLT3-L sur les LB pourrait expliquer l’évolution vers le NHL. Dans un troisième temps, nous avons étudié une autre cytokine dérégulée dans le SGS (la cytokine BAFF) et nous avons confirmé le rôle d’un nouveau variant de BAFF produit par l’épissage alternatif de l’exon 4 (∆4BAFF) comme un facteur de transcription de son propre gène. Ce nouveau variant est beaucoup plus exprimé au cours des maladies autoimmunes, et son expression est contrôlée par l’interferon gamma et la protéine SC35. Tous ces données montrent pour la première fois, un nouveau concept à savoir la possibilité pour une cytokine d’être régulée par un variant provenant de l’épissage alternatif de son propre gène. Ensemble, ces résultats montrent le rôle des cytokines impliquées dans l’ontogenèse et la survie des LB, dans la physiopathologie du SGS. / Primary Sjögren’s Syndrome (pSS) is a systemic autoimmune disease characterized by sicca symptoms and a broad variety of systemic manifestations. The most severe complication of the disease is the development of non-Hodgkin’s lymphoma (NHL) in 5% of patients. Recent evidence indicates a major contribution of B cells. In this work, we developed clinical and basic research subjects, related to the role of B-cell in the pathogenesis of pSS. In the first section, we showed that memory B-cell infiltrates are present in pSS and may be used as an additional diagnostic and follow-up tool. In the second section, we showed that high serum levels of the cytokine called FLT3-L (a cytokine implicated in B-cell ontogenesis and lymphoproliferation) are associated with abnormal B-cell distribution, characteristic of pSS; and disease clinical activity. In addition, this cytokine may explain the development of lymphoma. In the third section, we demonstrated that ∆4BAFF (a new variant of BAFF, due to the alternative splicing of exon 4) is a transcription factor of its own gene. Interestingly, this new variant is mainly detected in autoimmune diseases and its expression is regulated by IFN-y and SC35 protein (one of the proteins implicated in the splicing machinery). This finding provides an expanded conceptual view of BAFF gene regulation in autoimmune diseases, and contributes to a better understanding of the mechanisms involved in BAFF up-regulation in autoimmunity. Collectively, these results are of clinical and fundamental basic interest in pSS, in the diagnostic, physiopathology and therapeutic contexts.

Background & aims. DNA methylation induced tumor suppressor gene silencing plays an important role in carcinogenesis. By using methylation-sensitive representational difference analysis, we identified paired box gene 5 (PAX5) being methylated in human cancer. PAX5 locates at human chromosome 9p13.2 and encodes a 391 amino acids transcription factor. However, the role of PAX5 in gastric cancer is still unclear. Hence, we analyzed its epigenetic inactivation, biological functions, and clinical implications in gastric cancer. / Conclusions. Our results demonstrated that PAX5 promoter methylation directly mediates its transcriptional silence and commonly occurs in gastric cancer. PAX5 gene can act as a functional tumor suppressor in gastric carcinogenesis by playing an important role in suppression of cell proliferation, migration, invasion, and induction of cell apoptosis. Detection of methylated PAX5 may be utilized as a biomarker for the prognosis of gastric cancer patients. / Methods. Methylation status of PAX5 promoter in gastric cancer cell lines and clinical samples was evaluated by methylation specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS). The effects of PAX5 re-expression in cancer cell lines were determined in proliferation, cell cycle, apoptosis, migration and invasion assays. Its in vivo tumorigenicity was investigated by injecting cancer cells with PAX5 expression vector subcutaneously into the dorsal flank of nude mice. Chromosome Immunoprecipitation (ChIP) and cDNA expression array were performed to reveal the molecular mechanism of the biological function of PAX5. / Results. PAX5 was silenced or down-regulated in seven out of eight of gastric cancer cell lines examined. A significant down-regulation was also detected in paired gastric tumors compared with their adjacent non-cancer tissues (n = 18, P = 0.0196). In contrast, PAX5 is broadly expressed in all kinds of normal adult and fetal tissues. The gene expression of PAX5 in the gastric cancer cell line is closely linked to the promoter hypermethylation status. In addition, the expression levels could be restored by exposure to demethylating agents 5-aza-21-deoxycytidine. Re-expression of PAX5 in AGS, BGC823 and HCT116 cancer cells reduced colony formation (P < 0.01) and cell viability (P < 0.05), arrested cell cycle in G0/G1 phase (P = 0.0055), induced cell apoptosis (P < 0.05), repressed cell migration and invasion (P = 0.0218) in vitro. It also inhibited tumor growth in nude mice (P < 0.05). The molecular basis of its function were investigated by cDNA expression array and demonstrated that ectopic expression of PAX5 up-regulated tumor suppressor gene P53, anti-proliferation gene P21, pro-apoptosis gene BAX, anti-invasion gene MTSS1 and TIMP1; and down-regulated anti-apoptosis gene BCL2, cell cycle regulator cyclinD1, migration related gene MET and MMP1. ChIP assay indicated that P53 and MET are direct transcriptional target of PAX5. Moreover, PAX5 hypermethylation was detected in 90% (145 of 161) of primary gastric cancers compared with 16% (3 of 19) of non-cancer tissues (P < 0.0001). After a median follow-up period of 15.4 months, multivariate analysis revealed that gastric cancer patients with PAX5 methylation had a significant poor overall survival compared with the unmethylated cases (P = 0.0201). / Li, Xiaoxing. / Advisers: Hsiang Fu Kung; Jun Yu. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

<p>B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (V_H) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin. </p><p>Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by V_H3-21/V_λ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the V_H3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin.</p><p>In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases. </p>

In chronic lymphocytic leukemia (CLL), the presence of multiple subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has implicated antigen(s) in leukemogenesis. These stereotyped subsets display similar immunoglobulin (IG) gene usage, almost identical complementarity determining region 3’s and may share clinical features. For instance, subsets #1 (IGHV1/5/7/IGKV1-39) and #2 (IGHV3-21/IGLV3-21) have inferior outcome compared to non-subset patients, whereas subset #4 (IGHV4-34/IGKV2-30) display a favourable prognosis. The aim of this thesis was to investigate genomic aberrations, gene expression patterns and methylation profiles in stereotyped subsets and compare epigenetic profiles in CLL and mantle cell lymphoma (MCL). In paper I, we investigated genomic aberrations in subsets #2, #4 and #16 and in non-stereotyped samples (n=101) using high-density 250K SNP arrays. Subset #2 and non-subset #2 IGHV3-21 cases displayed a higher frequency of aberrations than subset #4 cases. The high incidence of del(11q) in both subset #2/non-subset #2 may reflect the adverse survival reported for IGHV3-21 patients. In contrast, the lower frequency of genetic events and lack of poor-prognostic aberrations in subset #4 may partially explain their indolent disease. In paper II, we analysed the global RNA expression in subset #4, #16 and non-subset IGHV4-34 CLL patients (n=25). Subsets #4 and 16 showed distinct gene expression profiles, where genes involved in cell regulatory pathways were significantly lower expressed in subset #4, in line with their low-proliferative disease. In paper III, a genome-wide methylation array was applied to investigate methylation profiles in subsets #1, #2 and #4 (n=39). We identified differential methylation patterns for all subsets and found affected genes to be involved in e.g. apoptosis and therapy resistance. When performing functional annotation, a clear enrichment of genes involved in adaptive immunity was observed. These genes were preferentially methylated in subset #1 when compared to either subset #2 or #4, possibly due to different antigen responses. In paper IV, the genome-wide methylation profiles for 30 CLL and 20 MCL patients were investigated. Distinct methylation profiles were observed, where MCL displayed a more homogeneous profile. Homeobox transcription factor genes showed a higher degree of methylation in MCL, while apoptosis-related genes and proliferation-associated genes were methylated in CLL. In summary, this thesis demonstrates that stereotyped CLL subsets display differences in gene expression profiles, genetic aberrations and methylation patterns, underscoring the functional relevance of subgrouping according to BCR stereotypy. The distinct methylation profiles of CLL and MCL suggests that different epigenetic mechanisms are involved in the pathogenesis of these B-cell malignancies.

3BP2 is a pleckstrin homology (PH) domain- and Src homology 2 (SH2) domain-containing adapter protein that has been linked through genetic evidence to a rare human disease called cherubism 146. 3BP2 was originally cloned in a screen to identify c-Abl SH3 binding proteins 23,24. In overexpression studies, 3BP2 has been implicated as a positive regulatory adapter molecule coupled to immunoreceptor on T cells 67,69,70, B cells 68, NK cells 71-73 and mast cells 74,75. It was also evident that 3BP2 forms complexes with a number of signaling molecules, such as Zap-70, LAT, phospholipase C-γ1 (PLC-γ1), Grb2, Cbl, and Fyn in Jurkat cells 67 and Vav1, Vav2, PLC-γ, and Syk in Daudi B cells 68. Despite the growing body of biochemical data to support the importance of 3BP2 in cells of the hematopoietic lineage, a clear picture of the biological function of 3BP2 has yet to emerge. To elucidate the in vivo function of 3BP2, our laboratory has generated 3BP2 gene-deficient mice through homologous recombination 452. The 3BP2-deficient (3BP2-/-) mice were born at the expected Mendelian frequency and were fertile and viable. 3BP2-/- mice accumulate splenic marginal-zone (MZ) B cells, possess a reduced frequency of peritoneal B-1 B cells, and have a diminished thymus-independent type 2 (TI-2) antigen response. 3BP2-/- B cells demonstrate diminished proliferation and cell survival following cross-linking of the B-cell receptor (BCR). Following BCR ligation, 3BP2 might be recruited to BCR complex through its inducible interaction with BCR costimulatory molecule CD19. In the absence of 3BP2, the activation of BCR downstream effectors such as MAPK Erk1/2, JNK, and c-Abl is normal; however, 3BP2 deficiency leads to defects in Syk phosphorylation and calcium flux. In addition to defects in peripheral B cell activities, 3BP2 deficiency contributes to defects in neutrophil activities. In response to the chemotactic peptide, fMLF, 3BP2-/- neutrophils fail to establish directional migration in vitro. There is a defect in the accumulation of filamentous actin at the leading edge of migrating 3BP2-/- neutrophils which might be responsible for the random movement of these cells under shallow gradient of fMLF. In vivo, there is a delay in the recruitment of circulating neutrophils to the site of chemically induced inflammation in 3BP2-/- mice. Compared to wildtype neutrophils, 3BP2-/- neutrophils fail to properly produce superoxide anion (O2-) following fMLF stimulation. Defects in both directional migration and superoxide production of 3BP2-/- neutrophils might contribute to the reduction in bacteria clearance and the increased mortality in 3BP2-/- mice post Listeria Monocytogenes infection. In Chapter 1 of this thesis, I have reviewed basic structures and functions of the domain modules found in adapter proteins. In addition, I have reviewed the findings from numerous reports on the function of 3BP2 in different cell types. A discussion of the physical appearance and some of the initial characterization of 3BP2-deficient mice (3BP2-/-) we have generated in our laboratory are included in Chapter 1. The second part of Chapter 1 consists of an introduction on B cell receptor signaling pathway and B-cell development and activation. A discussion of G protein-coupled receptor-mediated neutrophil functions can also be found in Chapter 1. Chapter 2 contains all the methods and materials used in my study. Chapter 3 includes the characterization of peripheral B cell compartment of 3BP2-/- mice as well as the role of 3BP2 downstream of B-cell antigen receptor and in T-independent immune response. In chapter 4, I present data from experiments designed to examine the role of 3BP2 downstream of a G protein-coupled receptor, fMLF receptor, of neutrophils. I also show the requirement of 3BP2 in the clearance of Listeria Monocytogenes. In chapter 5, I propose two models for 3BP2 action based on the findings in B cells and neutrophils and discuss future areas for investigation.

3BP2 is a pleckstrin homology (PH) domain- and Src homology 2 (SH2) domain-containing adapter protein that has been linked through genetic evidence to a rare human disease called cherubism 146. 3BP2 was originally cloned in a screen to identify c-Abl SH3 binding proteins 23,24. In overexpression studies, 3BP2 has been implicated as a positive regulatory adapter molecule coupled to immunoreceptor on T cells 67,69,70, B cells 68, NK cells 71-73 and mast cells 74,75. It was also evident that 3BP2 forms complexes with a number of signaling molecules, such as Zap-70, LAT, phospholipase C-γ1 (PLC-γ1), Grb2, Cbl, and Fyn in Jurkat cells 67 and Vav1, Vav2, PLC-γ, and Syk in Daudi B cells 68. Despite the growing body of biochemical data to support the importance of 3BP2 in cells of the hematopoietic lineage, a clear picture of the biological function of 3BP2 has yet to emerge. To elucidate the in vivo function of 3BP2, our laboratory has generated 3BP2 gene-deficient mice through homologous recombination 452. The 3BP2-deficient (3BP2-/-) mice were born at the expected Mendelian frequency and were fertile and viable. 3BP2-/- mice accumulate splenic marginal-zone (MZ) B cells, possess a reduced frequency of peritoneal B-1 B cells, and have a diminished thymus-independent type 2 (TI-2) antigen response. 3BP2-/- B cells demonstrate diminished proliferation and cell survival following cross-linking of the B-cell receptor (BCR). Following BCR ligation, 3BP2 might be recruited to BCR complex through its inducible interaction with BCR costimulatory molecule CD19. In the absence of 3BP2, the activation of BCR downstream effectors such as MAPK Erk1/2, JNK, and c-Abl is normal; however, 3BP2 deficiency leads to defects in Syk phosphorylation and calcium flux. In addition to defects in peripheral B cell activities, 3BP2 deficiency contributes to defects in neutrophil activities. In response to the chemotactic peptide, fMLF, 3BP2-/- neutrophils fail to establish directional migration in vitro. There is a defect in the accumulation of filamentous actin at the leading edge of migrating 3BP2-/- neutrophils which might be responsible for the random movement of these cells under shallow gradient of fMLF. In vivo, there is a delay in the recruitment of circulating neutrophils to the site of chemically induced inflammation in 3BP2-/- mice. Compared to wildtype neutrophils, 3BP2-/- neutrophils fail to properly produce superoxide anion (O2-) following fMLF stimulation. Defects in both directional migration and superoxide production of 3BP2-/- neutrophils might contribute to the reduction in bacteria clearance and the increased mortality in 3BP2-/- mice post Listeria Monocytogenes infection. In Chapter 1 of this thesis, I have reviewed basic structures and functions of the domain modules found in adapter proteins. In addition, I have reviewed the findings from numerous reports on the function of 3BP2 in different cell types. A discussion of the physical appearance and some of the initial characterization of 3BP2-deficient mice (3BP2-/-) we have generated in our laboratory are included in Chapter 1. The second part of Chapter 1 consists of an introduction on B cell receptor signaling pathway and B-cell development and activation. A discussion of G protein-coupled receptor-mediated neutrophil functions can also be found in Chapter 1. Chapter 2 contains all the methods and materials used in my study. Chapter 3 includes the characterization of peripheral B cell compartment of 3BP2-/- mice as well as the role of 3BP2 downstream of B-cell antigen receptor and in T-independent immune response. In chapter 4, I present data from experiments designed to examine the role of 3BP2 downstream of a G protein-coupled receptor, fMLF receptor, of neutrophils. I also show the requirement of 3BP2 in the clearance of Listeria Monocytogenes. In chapter 5, I propose two models for 3BP2 action based on the findings in B cells and neutrophils and discuss future areas for investigation.

B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (VH) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin. Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by VH3-21/Vλ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the VH3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin. In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2010. / Härtill 4 uppsatser.

The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.