Off the beaten path - An unbiased inventory of signal response elements in B cell receptor signaling

Abstract

Signaling through the B cell antigen receptor (BCR) is indispensable for B lymphocyte maturation, activation and differentiation. Antigenic signal transduction thus constitutes a centerpiece of adaptive immunity and its dysregulation results in immunodeficiency, autoimmunity or tumorigenesis. Yet, despite its pivotal relevance to health and disease, most studies on the BCR signal cascade have been limited to few canonical players, while specific evidence as to what distinguishes the diverse, stage-specific responses conveyed through the BCR remains sparse. In this work, I utilize unbiased proteomic and transcriptomic approaches to explore BCR signal transduction off the beaten path and beyond well-described components. In a first endeavor, I investigated the mystery of IgD’s elusive role among the immunoglobulin classes. To that end, I assembled a comprehensive and unbiased inventory of IgD- and IgM-mediated signal response elements. Here, I show equal induction of BCR-induced actin remodeling as well as an equal glucose metabolic response to stimulation through IgD and IgM. This study moreover demonstrates virtually identical IgD- and IgM-mediated signaling responses on the phosphoproteomic scale. While its specific function is under ongoing debate, my thesis thus challenges the proposedly distinct signaling capabilities of IgD and instead supports the opposing hypothesis of redundant IgD- and IgM-mediated responses on the signal transduction level. BCR stimulation ultimately defines B cell fate through activation of numerous transcription factors, which in concert shape B lymphocyte activation, differentiation and immune responses through transcriptional regulation. Albeit its undisputed critical relevance to B cell physiology, this thesis presents the first unbiased assessment of BCR-mediated nuclear logistics. To that end, I established a novel methodology which combines subcellular fractionation and SILAC mass spectrometry and thus provides the groundwork for investigation of the B lymphoid “Translocatome”. Through application of this method, the presented study uncovers the Transcription Factor EB (TFEB) as a novel effector protein of the BCR. My experiments show that antigenic signaling activates TFEB in primary human B cells as well as in B cell lines of multiple origins and developmental stages, rendering the nuclear translocation of TFEB a novel hallmark of B cell receptor signaling. Furthermore, I could unravel an integrated signaling network initiated through the BCR and its co-receptor CD19 and reveal GSK3β as a constitutive TFEB kinase in B cells and a point of convergence between the two-sided activation cascade. This study further presents strong evidence for the prominent relevance of TFEB to the physiology and immune function of B lymphocytes. In particular, I show central involvement of TFEB in the induction of autophagic flux, as well as in the biogenesis of lysosomes and mitochondria in response to antigenic stimulation. Beyond its canonical role as master regulator of autophagy and lysosomes, this work demonstrates the essential importance of TFEB in the decisive interplay between B cell tolerance and activation: While promoting BCR-induced apoptosis, TFEB simultaneously supports co-stimulation via cytokine receptor expression, lymphocyte homing, as well as antigen presentation pathways. Finally, my results show constant nuclear localization of TFEB in a subset of memory B cells, indicating a role of TFEB in conferring immunological memory. Altogether, the present thesis consigns TFEB to the ranks of established players in antigen receptor signaling and demonstrates its paramount importance to B cell activation and physiology.