B cell signalling in mechanisms of central and peripheral tolerance

Thalhamer, Theresa (2009) B cell signalling in mechanisms of central and peripheral tolerance. PhD thesis, University of Glasgow.

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Tolerance against self is a necessary feature of the immune system to prevent autoimmunity. Hence during B cell development, a number of central and peripheral developmental checkpoints ensure the deletion of self-reactive B cells and selection of tolerant B cells. For example, antigen-driven ligation of the B cell receptor (BCR) on immature B cells results, by default, in receptor editing, anergy and/or apoptosis, whereas it provides survival, proliferative and effector differentiation signals to mature B cells. Moreover, various factors can influence the functional outcome downstream of BCR ligation. Thus, T helper cell-derived signals, such as those following ligation of the CD40 receptor, can rescue pathogen-specific immature B cells from growth arrest and apoptosis, thereby providing a mechanism in which cells programmed to die because of their immature status can survive after receiving appropriate T cell help. On the other hand, mitogenic BCR-mediated signalling of mature B cells can be suppressed by co-ligation of the inhibitory receptor FcγRIIb. This allows the maintenance of B cell homeostasis in the periphery, as cross-linking of the BCR and FcγRIIb by immune complexes enables the system to terminate ongoing immune responses following clearance of pathogens. The precise signalling mechanisms involved in dictating these differential functional outcomes remain to be elucidated but it is becoming increasingly clear that the developmental stage as well as the integration of various extracellular signals decide the cell’s fate. The core aim of this study has therefore been to characterise the signalling pathways coupling BCR ligation to survival, proliferation and apoptosis during development. In particular, it was planned to focus on the differential signalling mechanisms involved in the negative selection of immature B cells and FcγRIIb-mediated homeostatic regulation of mature B cells.
Negative selection of self-reactive immature B cells constitutes a major mechanism of sustaining central tolerance. The WEHI-231 cell line provides a well-established model system for dissecting the signalling mechanism underlying such clonal deletion of immature B cells as these cells, which have the phenotype of immature B cells, undergo growth arrest and apoptosis following stimulation of their BCR. Previously, this laboratory has identified ERK signalling as a key regulator of immature B cell fate. Thus, spontaneously proliferating WEHI-231 B cells exhibit a sustained yet cyclic pattern of ERK activation that is necessary for their survival and proliferation. By contrast, BCR-ligation induces a strong transient activation of ERK followed by sustained downregulation of the cyclic activation pattern observed in spontaneously proliferating cells. However, the pathways linking BCR ligation with suppression of ERK signalling and consequent growth arrest have not been delineated in full. For example, in B cells, the Ras/Raf-1/MEK cascade is considered to be a major pathway regulating ERK activation and consistent with this, this laboratory has previously shown that overexpression of Ras can rescue WEHI-231 B cells from BCR-mediated growth arrest. However, although MEK activation was found to be compromised under conditions of BCR-driven growth arrest, such BCR-signalling did not suppress Ras activation indicating that other pathways normally contributed to the MEK-dependent activation of ERK in spontaneously proliferating WEHI-231 cells.
The data presented in this thesis now provide evidence for the existence of a second pathway controlling ERK activation in immature B cells, the Rap/B-Raf/MEK cascade, which has now been implicated in the maintenance of cycling ERK activation observed in spontaneously proliferating WEHI-231 B cells. BCR signalling was found to reduce the levels of active Rap, hence providing a mechanism for the BCR-mediated uncoupling of the Ras-independent, MEK-dependent ERK activation contributing to the cycling ERK signalling responsible for survival and proliferation of WEHI-231 cells. Consistent with the role of Rap in promoting such ERK signalling, additionally, it has now been demonstrated that levels of SPA-1, a negative regulator of Rap, increase upon BCR-stimulation indicating that this may be the mechanism by which the BCR signals to uncouple the Rap/B-Raf/MEK pathway.
Although ERK had previously been identified as the major regulatory element governing survival and proliferation of immature B cells, the pathways linking ERK to the regulation of survival/apoptosis and cell cycle progression have been only poorly characterised. This study has now highlighted the connection between sustained ERK signalling and the stabilisation of c-Myc protein levels. For example, abrogation of ERK activity by BCR-ligation, or pharmacological inhibition, reduced c-Myc levels in a transcription-independent fashion indicating regulation at the post-translational level. This proposal was corroborated by analysis of the phosphorylation status of c-Myc that indicated that ERK signalling promoted the expression of stabilised forms (S62) of c-Myc and reduced the expression of those (T58) targeting c-Myc for proteosomal degradation. The importance of such c-Myc stabilisation was illustrated by Laser Scanning Cytometric analysis that revealed that the increasing levels of c-Myc expressed by individual WEHI-231 B cells correlated with their cell cycle progression, presumably reflecting widely established findings that c-Myc promotes cell cycle progression by increasing the expression and activation of cyclin/Cdk complexes and reducing the levels of Cdk inhibitors such as p27. Consistent with this, negative signalling via the BCR, or suppression of ERK activation by pharmacological inhibition, also increased p27 levels and resulted in the reduction of the hyperphosphorylation of retinoblastoma (Rb) proteins required for transition through the G1-S-phase checkpoint. As Rb is a target of cyclin/Cdks, collectively these data further confirm the links not only between BCR-signalling and c-Myc stability but also the differential cell fate decisions of apoptosis, survival and/or cell cycle progression.
By contrast, in mature B cells, BCR-signalling is linked to survival and proliferation. However, to prevent autoimmunity resulting from an expanded pool of potentially weakly autoreactive B cells, such survival and proliferation of mature B cells needs to be homeostatically regulated. Hence, the immune system has evolved inhibitory signalling cascades such as that triggered by cognate immune-complexes co-ligating the BCR and FcγRIIb to terminate ongoing antigen-driven responses. This study has emphasised the crucial role of caspase 8 in the apoptosis of mature B cells resulting from such co-ligation of the BCR and FcγRIIb and has indicated that such caspase 8 activation is likely to be downstream of Fas signalling. Consistent with this, blocking the Fas/FasL death receptor cascade was found to reduce the levels of apoptosis detected and B cells from MRL/MpJ-Faslpr mice, a strain harbouring a mutation causing the abrogation of Fas expression, exhibited defective apototic responses upon such BCR/FcγRIIb co-ligation. Thus, Fas/FasL death receptor signalling might be a major mechanism underpinning the FcγRIIb-mediated apoptosis pathway.
Finally, to determine whether dysfunctional regulation of FcγRIIb-mediated signalling plays a role in human autoimmune conditions, B cells from patients with Rheumatoid arthritis and Systemic lupus erythematosus were examined. Overall, the homeostatic regulatory responses between B cells from healthy controls and RA and SLE patients were not found to be significantly different. However, the B cells derived from a small proportion of RA and SLE patients were found to exhibit defective FcγRIIb-mediated inhibitory responses. Moreover, significant differences were found in the ratio of FcγRIIb1/FcγRIIb2 expression between the cohorts of healthy controls and RA and SLE patients. The RA and SLE patients expressed relatively higher levels of the FcγRIIb2 isoform which promotes antigen-processing suggesting that these B cells may play some role in priming autoreactive responses in such individuals. Thus, as these inflammatory disorders constitute spectrum diseases, such defects in the regulation of B cell responses could be one of the contributing factors aggravating autoimmune disease development in some subgroups of patients.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: B cells, signalling, tolerance, ERK MAPK, FcγRIIb
Subjects: Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Supervisor's Name: Harnett, Dr. Margaret M.
Date of Award: 2009
Depositing User: Ms Theresa Thalhamer
Unique ID: glathesis:2009-1375
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 15 Jan 2010
Last Modified: 10 Dec 2012 13:38
URI: http://theses.gla.ac.uk/id/eprint/1375

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