Components of the antigen processing and presentation pathway revealed by gene expression microarray analysis following B cell antigen receptor (BCR) stimulation

BACKGROUND: Activation of naïve B lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide (LPS) and CD40 ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. For example, although all three of these ligands induce proliferation, only stimulation through the B cell antigen receptor (BCR) induces apoptosis in resting splenic B cells. In order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary B cells by a panel of ligands using cDNA microarrays and a statistical approach, CLASSIFI (Cluster Assignment for Biological Inference), which identifies significant co-clustering of genes with similar Gene Ontology™ annotation. RESULTS: CLASSIFI analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the BCR-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. Proton pump components that were not included in the initial microarray data set were also upregulated in response to BCR stimulation in follow up experiments. MHC Class II expression was found to be maintained specifically in response to BCR stimulation. Furthermore, ligand-specific internalization of the BCR, a first step in B cell antigen processing and presentation, was demonstrated. CONCLUSION: These observations provide experimental validation of the computational approach implemented in CLASSIFI, demonstrating that CLASSIFI-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. Furthermore, this analysis has identified at least thirty-eight candidate components of the B cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to B cell antigen processing and presentation

Prognostic Markers and Mechanisms of Chemotherapy Resistance in Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous disease. Identification of prognostic and predictive markers is important to design individualised and effective treatment strategies. This thesis describes different approaches to search for molecular prognostic markers and an attempt to overcome chemotherapy resistance in DLBCL. In the first study it was investigated whether immunohistochemical expression of CD40, previously associated with a favourable prognosis in DLBCL, had any prognostic impact after the addition of rituximab to anthracycline-based chemotherapy. Results showed better prognosis in CD40-positive patients receiving this combined treatment. An inflammatory stromal process was observed in CD40-expressing tumours when using gene expression profiling, possibly contributing to a better prognosis in CD40-positive patients. A study about the effect of inhibited protein prenylation on the response to CHOP therapy, using an in vitro cell-line-based model, showed that treatment with geranylgeranylation inhibitors in DLBCL cell lines had a chemo-sensitising effect. This indicates that inhibition of geranylgeranylation may prove a useful strategy for overcoming CHOP resistance in DLBCL. A recombinant antibody microarray was used to search for prognostic protein profiles in plasma from DLBCL patients in an attempt to identify new candidate markers for the prognosis and treatment response in DLBCL and to provide a basis for future investigations and validation. The results of this work indicated that the tumour microenvironment and host immune response contribute to the prognosis in DLBCL, and suggest a potential novel strategy to overcome chemotherapy resistance. Further molecular profiling is important for improved treatment of patients with DLBCL

Molecular signatures of the evolving immune response in mice following a Bordetella pertussis infection

Worldwide resurgence of pertussis necessitates the need for improvement of pertussis vaccines and vaccination strategies. Since natural infections induce a longer-lasting immunity than vaccinations, detailed knowledge of the immune responses following natural infection can provide important clues for such improvement. The purpose was to elucidate the kinetics of the protective immune response evolving after experimental Bordetella pertussis (B. pertussis) infection in mice. Data were collected from (i) individual analyses, i.e. microarray, flow cytometry, multiplex immunoassays, and bacterial clearance; (ii) twelve time points during the infection; and (iii) different tissues involved in the immune responses, i.e. lungs, spleen and blood. Combined data revealed detailed insight in molecular and cellular sequence of events connecting different phases (innate, bridging and adaptive) of the immune response following the infection. We detected a prolonged acute phase response, broad pathogen recognition, and early gene signatures of subsequent T-cell recruitment in the lungs. Activation of particular transcription factors and specific cell markers provided insight into the time course of the transition from innate towards adaptive immune responses, which resulted in a broad spectrum of systemic antibody subclasses and splenic Th1/Th17 memory cells against B. pertussis. In addition, signatures preceding the local generation of Th1 and Th17 cells as well as IgA in the lungs, considered key elements in protection against B. pertussis, were established. In conclusion, molecular and cellular immunological processes in response to live B. pertussis infection were unraveled, which may provide guidance in selecting new vaccine candidates that should evoke local and prolonged protective immune responses.Drug Delivery Technolog

Antibodies against CD20 or B-Cell Receptor Induce Similar Transcription Patterns in Human Lymphoma Cell Lines

BACKGROUND: CD20 is a cell surface protein exclusively expressed on B cells. It is a clinically validated target for Non-Hodgkin's lymphomas (NHL) and autoimmune diseases. The B cell receptor (BCR) plays an important role for development and proliferation of pre-B and B cells. Physical interaction of CD20 with BCR and components of the BCR signaling cascade has been reported but the consequences are not fully understood. METHODOLOGY: In this study we employed antibodies against CD20 and against the BCR to trigger the respective signaling. These antibodies induced very similar expression patterns of up- and down-regulated genes in NHL cell lines indicating that CD20 may play a role in BCR signaling and vice versa. Two of the genes that were rapidly and transiently induced by both stimuli are CCL3 and CCL4. 4 hours after stimulation the concentration of these chemokines in culture medium reaches a maximum. Spleen tyrosine kinase Syk is a cytoplasmic tyrosine kinase and a key component of BCR signaling. Both siRNA mediated silencing of Syk and inhibition by selective small molecule inhibitors impaired CCL3/CCL4 protein induction after treatment with either anti-CD20 or anti-BCR antibodies. CONCLUSION: Our results suggest that treatment with anti-CD20 antibodies triggers at least partially a BCR activation-like response in NHL cell lines

IRF8 Governs Expression of Genes Involved in Innate and Adaptive Immunity in Human and Mouse Germinal Center B Cells

IRF8 (Interferon Regulatory Factor 8) is a transcription factor expressed throughout B cell differentiation except for mature plasma cells. Previous studies showed it is part of the transcriptional network governing B cell specification and commitment in the bone marrow, regulates the distribution of mature B cells into the splenic follicular and marginal zone compartments, and is expressed at highest levels in germinal center (GC) B cells. Here, we investigated the transcriptional programs and signaling pathways affected by IRF8 in human and mouse GC B cells as defined by ChIP-chip analyses and transcriptional profiling. We show that IRF8 binds a large number of genes by targeting two distinct motifs, half of which are also targeted by PU.1. Over 70% of the binding sites localized to proximal and distal promoter regions with ∼25% being intragenic. There was significant enrichment among targeted genes for those involved in innate and adaptive immunity with over 30% previously defined as interferon stimulated genes. We also showed that IRF8 target genes contributes to multiple aspects of the biology of mature B cells including critical components of the molecular crosstalk among GC B cells, T follicular helper cells, and follicular dendritic cells

Regulation of B cell development by transcription factors IRF4 and BACH2

B lymphocytes—major cell types mediating an adaptive immunity—recognize a vast variety of structures by B cell antigen receptors (BCR). Activated B cells can form germinal centers, undergo antigen-induced affinity maturation of their BCRs and differentiate into memory B cells sought upon vaccinations or antibody-secreting plasma cells that eradicate infections. Aberrant BCR signaling or transcriptional regulation of B cell development may cause immunodeficien- cies, autoimmune diseases or lymphoid malignancies. Therefore, deeper knowledge of the transcriptional regulation of B cell development is essential for the understanding of B cell derived diseases. Transcription factors IRF4 and BACH2 are crucial regulators of germinal center formation and high-affinity antibody production. However, the molecular mech- anisms how they do these remain unknown. The aim of this study was to investi- gate the role of IRF4 in B cells and clarify IRF4 function in the regulation of an- tibody secretion as well as to examine the role of BACH2 in immunoglobulin (Ig) diversification processes. To do this IRF4-, BACH2-, and IRF4/BCL6 dou- ble-deficient DT40 B cell lines were generated. In this thesis, I demonstrate a new role for IRF4 as a regulator of BCR signaling, which is crucial for the B cell survival, and activation. IRF4 directly represses IRF8 and upregulates SHIP gene expression that results in stronger BCR-induced calcium flux and changed signaling to ERK and PI3K/AKT signaling pathways. In this study, I show nonredundant functions for IRF4 and BLIMP1 in the induction of antibody secretion, as in IRF4- or IRF4/BCL6 double-deficient cells enforced expression of BLIMP1 fails to induce antibody secretion. As the most important finding, I reveal a new role for BACH2 in Ig gene conversion that diversifies BCR

B cell-mediated immunity and signal regulation. The roles of MIM/MTSS1 and IRF4

B cells constitute an important part of the adaptive immune system and produce antibodies, which protect organism against infectious agents. Recognition of the antigen by the B cell receptor and the following B cell activation are key steps toward production of protective antibodies. In recent years it has been shown that the actin cytoskeleton is actively involved in the regulation of early events of B cell activation. Therefore, understanding the factors that influence actin cytoskeleton remodeling in B cells is essential for the understanding of B cell-mediated immunity. The focus of this work is on the membrane and actin cytoskeleton regulatory protein, MIM/MTSS1, and on the B cell-specific transcription factor, IRF4. In order to reveal unexplored MIM functions, the gene and protein sequences of MIM were analyzed by using computational tools, bioinformatic resources and online databases. The analysis demonstrates a high overall degree of MIM conservation in vertebrates and we also report on the presence of various short functional motifs. We also show that expression of MIM is downregulated in samples from patients with poor prognosis chronic lymphocytic leukemia (CLL). The role of MIM in B cells was studied by analysis of MIM knock-out (KO) mice. MIM-deficiency results in impaired BCR signaling upon stimulation with surface-bound but not soluble antigen, and elevated reprogramming toward higher oxidative metabolic state upon TLR4/9 stimulation. MIM KO animals also exhibit lower antibody responses against T cell-independent antigen immunization. Finally, the impact of IRF4-deficiency on BCR signaling was examined in an IRF4-KO DT40 cell line. We show that IRF4 deficiency results in disturbed BCR signaling, characterized by impaired Syk, BLNK, MAPK1/2 activation and F-actin engagement, low SHIP phosphatase levels and upregulation of PI3K, PLC��� and Ca2+ signaling. This work is the first comprehensive study of the actin-regulatory protein MIM in B cell-mediated immunity and provides an evolutionary standpoint on the functional significance of MIM protein regions. This study also highlights the fact that IRF4 is not only a downstream effector of BCR signaling but by itself can influence B cell activation via developmental stage-specific expression in B cells

The Aryl Hydrocarbon Receptor Plays a Key Role in the Transcriptional Programme of Regulatory B cells

Regulatory B cells (Bregs) play a critical role in the control of autoimmunity and inflammation. IL-10 production is the hallmark for the identification of Bregs. However, the molecular determinants that regulate the transcription of IL-10 and control the Breg developmental program remain unknown. The aryl hydrocarbon receptor (AHR) is an environmental sensor that binds to a variety of ligands, including physiological compounds derived from the digestion of dietary components by commensal microbiota. Here, we demonstrate that AHR regulates the differentiation and function of IL-10-producing CD19+CD21hiCD24hi Bregs and limits their differentiation into B cells that contribute to inflammation. Chromatin profiling and transcriptome analyses show that loss of AHR in B cells reduces expression of IL-10 by skewing the differentiation of CD19+CD21hiCD24hi B cells into a pro-inflammatory program, under Breg-inducing conditions. B cell AHR-deficient mice develop exacerbated arthritis, show significant reductions in IL-10-producing Bregs and regulatory T cells (Tregs), and show an increase in T helper (Th) 1 and Th17 cells compared with B cell AHR-sufficient mice. The most abundant source of AHR ligands are derived from the diet and the metabolism of dietary tryptophan. We have previously established a link between microbiota-driven signals in the gut and the differentiation of Bregs. Of the gut microbiota-derived metabolites, the short-chain fatty acids (SCFAs) are the most well characterised. More recently, the SCFA butyrate has been demonstrated to act as an AHR ligand in an intestinal epithelial cell line. Given the association of butyrate with AHR activation and the supporting findings showing that butyrate promotes Treg function, these data led us to hypothesise that butyrate acts via AHR to enhance Breg suppression. Here, we demonstrate that mice supplemented with butyrate reduces arthritis severity by inhibiting the differentiation of GC B cells and plasma cells, whilst maintaining Breg numbers and promoting the suppressive function of Bregs. We show that supplementation of mice with butyrate, changes the composition of the microbiota to favour species which metabolise tryptophan; a major source of AHR ligands. Therefore, we hypothesised that butyrate controls the balance between pro-arthritogenic and regulatory B cell differentiation, through the generation of microbiota derived AHR ligands. To date, the AHR ligands which direct Breg function are unknown. We rule out that butyrate acts as a direct ligand of AHR and establish that supplementation with butyrate increases the availability of 5-Hydroxyindole-3-acetic acid (5-HIAA), a downstream metabolite of serotonin, which we identify as a novel ligand of AHR in B cells. Mice supplemented with 5-HIAA promote Breg function and suppress arthritis severity, only in mice with AHR-sufficient B cells. Thus, we identify AHR as a relevant contributor to the transcriptional regulation of Breg differentiation and show that microbiota in the gut influence Breg differentiation by increasing the availability of AHR ligands

Role of TNFSF9 in germinal centre B cells and lymphoma formation

Members of the TNF superfamily play critical regulatory functions in hematopoietic cell lineage and function. Tnfsf9 is expressed in various hematopoietic cells, including B cells. Its receptor CD137, also known as 4-1BB, has been characterised and studied in the context of T cell activation and cancer immunotherapy. Loss of Tnfsf9 is recurrently found in human germinal centre (GC) B cell derived lymphomas and animals lacking Tnfsf9 develop B cell lymphomas. The use of genetically engineered mouse models may help to understand better the biology and function of Tnfsf9 in physiology and cancer. Here, we discover that animals completely deficient for Tnfsf9 display early in life enlarged lymph nodes and spleen, accompanied by spontaneous formation of large GCs. In addition, we investigate in a B cell specific manner the effects of Tnfsf9 loss and report a similar phenotype of spontaneous GCs. Protein microarray analysis of the sera of these mice shows the presence of both IgM and IgG autoantibodies, accompanied by immunoglobulin deposits in kidneys later in life. However, Tnfsf9 deficient animals do not show signs of overt autoimmune mediated pathology; they develop GC B-cell derived lymphomas with 100% penetrance as they age. We show that B cells deficient for Tnfsf9 display increased survival in vitro, and that this phenotype is accompanied by elevated Bcl-2 and Bcl-xL expression. Mechanistically loss of Tnfsf9 increases the activity of the alternative NF-kB signalling pathway upon BCR activation, by allowing increased nuclear translocation of p52. Our findings have implications for the understanding of B cell survival through the modulation of signals emanating from the BCR, and we report here a potential novel role of Tnfsf9 in linking BCR signalling and the alternative NF-kB pathway