IRF4 controls cell fate outcomes during the initial stages of B cell differentiation Open Access

Abstract

Naive B cell (nB) differentiation to antibody-secreting plasma cells (ASC) requires significant transcriptional and epigenetic reprogramming events that are coupled to cell division. Following antigen stimulation, considerable heterogeneity exists between responding activated B cells (actB), such as their cell division capacity and ability to differentiate. However, a complete understanding of the factors that drive such heterogeneity are unknown. Here, we identified the transcription factor interferon regulatory factor-4 (IRF4) as one molecular determinant controlling cell fate outcomes.  Using an in vivo model system and single cell RNA-sequencing, we identified a bifurcation event that occurred during the earliest stages of B cell differentiation, with only one trajectory leading to ASC formation. This differentiation branch, termed the ASC-destined branch, required IRF4 induction and could be distinguished from non-ASC cells by loss of CD62L expression. Comparing bulk RNA-sequencing data from actB that followed each branch indicated the non-ASC cells contained a pre-memory B cell transcriptional signature, indicating they may be destined to become memory B cells. Additionally, these data indicated the IRF4-dependent ASC-destined branch upregulated gene sets necessary for proliferation. To explore the role of IRF4 on proliferation, we performed an adoptive transfer time course covering three days using IRF4-sufficent and -deficient B cells. We found that IRF4-deficient B cells divided but stalled during the proliferative response, indicating IRF4 also controlled the proliferative capacity of responding B cells. To better understand the cell division-coupled IRF4-dependent reprogramming events that occurred during the initial stages of B cell differentiation, CellTrace Violet (CTV)-labeled IRF4-sufficient and -deficient B cells were sorted in discrete divisions for RNA- and ATAC-sequencing. Transcriptional analyses revealed that IRF4 was critical for inducing MYC target genes and metabolic gene sets during the earliest cell divisions. Complementary chromatin accessibility analyses suggested a hierarchy of IRF4 binding activity and identified broad networks of dysregulated transcription factor families, including E-box binding family members. Indeed, IRF4-deficient B cells failed to induce Myc and displayed altered cell cycle distribution. Furthermore, IRF4-deficient B cells exhibited reduced mTORC1 signaling and were unable to increase in cell size. Myc overexpression in IRF4-deficient cells was able to rescue the cell growth defect, indicating an IRF4-MYC-mTORC1 relationship that controls cell growth and the proliferative capacity of actB in the earliest cell divisions. Taken together, we identify IRF4 as a key factor that instructs cell fate outcomes during the initial stages of B cell differentiation, including differentiation, proliferation, and cell growth.

Table of Contents

Chapter 1. Introduction 1

I.               B CELL DEVELOPMENT AND DIFFERENTIATION 2

a.     Revolutionary discoveries contributing to our understanding of humoral immunity 2

b.     B cell formation 3

c.     B cell activation 5

d.     Adaptations necessary to support ASC physiology and function 9

e.     Initiation of the antibody-secreting cell transcriptional program 11

II.             THE ROLE OF IRF4 ON B CELL DEVELOPMENT AND DISEASE 14

a.     Development 14

b.     Differentiation 15

c.     Disease 16

III.           B CELL DIFFERENTIATION IS A COORDINATED MULTISTEP PROCESS 18                    

a.     Cell division is an essential process during B cell differentiation 18

b.     Cell division-coupled hypomethylation and gene regulation coordinate B cell differentiation 20

c.     Chromatin accessibility changes indicate epigenetic control of B cell differentiation 22

d.     Cell divisions represent distinct stages during in vivo B cell differentiation 24               

IV.          RATIONALE AND OVERVIEW 26

Chapter 2. Antibody-secreting cell destiny emerges during the initial stages of  B-cell activation 29

I.               ABSTRACT 30

II.             INTRODUCTION 31

III.           RESULTS 33

a.     Cell division is a critical component of B cell differentiation in vivo 33

b.     Cell division kinetics of B cell differentiation are similar for T independent Type I and II antigens 36

c.     BLIMP-1-dependent reprogramming is defective at division 8 37

d.     Single cell RNA-sequencing captures a continuum of LPS-responding B cells 39 

e.     Pseudotime analysis defines divergent B cell differentiation trajectories 42

f.      Multiple factors regulate the ASC-destined branch 45

g.     Loss of L-selectin expression marks B cells destined to become ASC 48

IV.          DISCUSSION 51

V.            SUPPORTING DATA 56

a.     METHODS 60

b.     Mice and adoptive transfers 60

c.     Flow cytometry and cell sorting 61

d.     ELISPOT 63

e.     Ex vivo B cell differentiation 64

f.      ELISA 64

g.     Bulk RNA sequencing 64

h.     Bulk RNA sequencing analysis 65

i.      Single cell RNA-sequencing and data processing 65

j.      MAGIC transformation of UMI transcript counts 66

k.     SCENIC transcription factor activity prediction 67

l.      KNN classification of single-cells with bulk RNA-seq data 67

m.   scRNA-seq data display 68

n.     Statistics 68

Chapter 3. An IRF4-MYC-mTORC1 integrated pathway controls cell growth and the proliferative capacity of activated B cells during B cell differentiation in vivo 70

I.               ABSTRACT 71

II.             INTRODUCTION 72

III.           RESULTS 74

a.     IRF4-deficient B cells responding to LPS in vivo stall during the proliferative response 74

b.     IRF4-deficient B cells exhibit a proliferation defect to T-independent and -dependent antigens 77

c.     IRF4-deficient B cells display altered cell cycle distribution 79 

d.     Cell division-coupled IRF4-dependent transcriptional reprogramming 81

e.     ATAC-sequencing reveals a hierarchy of IRF4 activity 84

f.      IRF4-deficient B cells fail to upregulate MYC 87

g.     IRF4-deficient B cells exhibit reduced mTORC1 activity and are unable to initiate the UPR 89

IV.          DISCUSSION 92

V.            SUPPORTING DATA 97

VI.          METHODS 100

a.     Mice and adoptive transfers 100

b.     Flow cytometry and sorting 100

c.     Cell cycle analysis and intracellular staining 102

d.     Ex vivo B cell differentiation 102

e.     Retroviral production and transduction 103

f.      Quantitative RT-PCR 103

g.     RNA-sequencing and data analysis 104

h.     ATAC-sequencing and data analysis 105

i.      Statistics 106

j.      Data availability 106

Chapter 4. Work in progress 107

 

Chapter 5. Discussion 112

References 122

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Health Sciences, Immunology Biology, Cell
Keyword	IRF4 MYC L-selectin CD62L mTORC1 B cell Plasma cell
Committee Chair / Thesis Advisor	Deal, Roger B. , Emory University Jacob, Joshy, Emory University Kelly, William G., Emory University Sanz, Ignacio, Emory University Boss, Jeremy M., Emory University

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