Metabolic and epigenetic instruction of plasma cell differentiation from naïve and memory B cells Restricted; Files Only
Price, Madeline (Spring 2020)
Published
Abstract
Antibody-secreting plasma cells are terminally differentiated through a highly regulated process controlled by transcriptional, epigenetic, and metabolic mechanisms. Naïve B cells can be stimulated to differentiate in either a T cell-independent or T cell-dependent manner. T-independent stimulation produces short lived plasma cells after multiple rounds of cell division. Though cell division and antibody secretion demand significant ATP and metabolites, the metabolic processes used during plasma cell formation are not well described. Following T-independent activation, B cells increased expression of oxidative phosphorylation machinery. Such activated B cells have increased capacity to perform oxidative phosphorylation but showed additional dependency on glycolysis. Promoting oxidative phosphorylation increased plasma cell differentiation. Differentiation orchestrated by BLIMP1 was required for increases in oxidative metabolism as BLIMP1-deficient cells proliferate normally, but neither differentiate to plasma cells nor upregulate oxidative phosphorylation. These findings identify a shift in metabolism as B cells differentiate and identify a requirement for increased metabolic potential to support antibody production. On the other hand, T-dependent stimulation generates long-lived plasma cells and memory B cells. Memory B cells respond more quickly and with higher affinity, more isotype-switched antibodies than their naïve counterparts; however, the molecular mechanisms by which memory cells are primed to respond are unknown. Using a model of influenza infection, nucleoprotein (NP)-specific memory B cells and follicular naïve B cells were purified to identify changes in gene expression by RNA-sequencing and chromatin accessibility by ATAC-sequencing. IgM+ and IgG+ memory B cells showed enrichment for expression of iron-related genes compared to naïve B cells. Iron is the central ion in heme. Treatment with exogenous heme augments plasma cell formation from naïve and memory B cells. Furthermore, memory B cells display open chromatin in gene regulatory regions that map to plasma cell specific transcription factors, including Prdm1 and Irf4, and in response to a heterosubtypic influenza challenge, memory mice form significantly more NP+IgG+ plasma cells. These data describe a molecular basis for enhanced the differentiation capacity of memory B cells.
Table of Contents
Chapter 1. Introduction…………..……………………………………………….……………….1
Part A – Metabolism
B cell formation and differentiation………………………………………...…...…….…..1
B cell activation………………………………………………………………………...…3
B cell differentiation…………………………..…………………………………..………8
Plasma cell function……………………………………………………..…….……...….12
Control of metabolic upregulation through PI3K signaling…………………….….……15
mTOR controls multiple aspects of growth and metabolism………………………….....18
Part B – Epigenetics
Epigenetics and transcriptional regulation.………………………………...………...…..22
Part C – Memory B cells
Differentiating memory B cells from naïve B cells and from each other………………..25
The role for germinal centers in memory B cell formation……………………………...26
The role of IgM+ and class-switched B cell receptors in memory……………..……......30
Memory B cell reactivation……………………………………………………….….….31
Maintenance and self-renewal of the memory B cell population…………..……...…….36
Human memory B cells…………...……………………………………….……...……..37
Significance and outstanding questions……..…………………………...………………38
Chapter 2. Oxidative phosphorylation is progressively programmed during plasmablast differentiation…………41
Summary………………………………………………………………..…………....…..42
Introduction………………………………………………………………………………43
Results
Metabolic changes correspond with differentiation state………………………..44
Ex vivo T-independent B cell activation increases oxidative phosphorylation…..48
Plasmablasts rely on oxidative phosphorylation for antibody secretion…………50
Differentiating B cell subsets increase oxygen consumption in a step-wise manner…………51
Oxidative phosphorylation increases plasmablast frequency…………………....54
Activated B cells and plasmablasts contain similar amounts of ATP…...……....54
Activated B cells use glycolysis to supplement their metabolism…...…………..55
BLIMP1-cKO B cells fail to upregulate oxidative phosphorylation...…..…...….55
Discussion…………...……………………………………………………………...……58
Chapter 3. IgM, IgG, and IgA influenza-specific plasma cells express divergent transcriptomes...……61
Abstract…………...……………………………………………………………………...62
Introduction……………………………………………………………………………....63
Results
Optimization of RNA isolation and sequencing from formaldehyde fixed cells.65
BCR-isotype subsetting of ASC by intracellular staining following influenza infection……….…….67
Antigen-specific ASC show isotype-specific gene expression profiles………....68
Influenza-specific ASC display similar gene expression profiles to bulk ASC....72
Sorted ASC populations are enriched for the target isotype………….……….....75
ASC isotypes have distinct repertoires……………………….…………....…….76
Discussion…………………………………………………………………………...…...79
Chapter 4. Heme directs memory B cell reactivation…………………………………………....82
Abstract………………………………………………………………………...……..….83
Introduction………………………………………………………………………………84
Results
Memory mice showed increased kinetics of differentiation and generate more antigen-specific isotype-switched plasma cells………86
Naïve and memory B cells have distinct gene expression profiles………………89
Memory B cells are epigenetically primed to differentiate to plasma cells…..….95
Plasma cell differentiation is enhanced by heme………………………………...99
Epigenetic signatures in memory B cells are conserved between mice and humans………………………………………………………..………...101
Human memory B cells contain increased HO-1 and differentiate to plasma cells strongly in the presence of hemin…………..…..105
Hemin increases plasma cell differentiation through modulation of mitochondrial metabolism…………………………………………106
Discussion……………………………………………………………………………..107
Chapter 5. Discussion…………………………………………………………………………..111
Chapter 6. Methods……………………………………………………………………………..126
Bibliography……………………………………………………………………………………136
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