Vitamin A-mediated regulation of immune functions and survival of human B cells in
health and disease
Kristine Lillebø Holm
UNIVERSITY OF OSLO Faculty of Medicine
Institute of Basic Medical Sciences Department of Molecular Medicine Thesis for the Degree of Philosophiae Doctor
October 2017
© Kristine Lillebø Holm, 2018
Series of dissertations submitted to the Faculty of Medicine, University of Oslo
ISBN 978-82-8377-238-8
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.
Print production: Reprosentralen, University of Oslo.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ... 1
LIST OF PUBLICATIONS ... 2
ABBREVIATIONS ... 3
1 INTRODUCTION ... 7
1.1 The immune system ... 7
1.1.1 General overview of the immune system ... 7
1.1.2 B cells ... 8
1.1.2.1 B-cell development ... 8
1.1.2.2 B-cell activation... 9
1.1.2.3 Long-lived plasma cells and antibody functions ... 11
1.1.2.4 Memory B cells ... 12
1.1.3 Toll-like receptors ... 13
1.1.3.1 TLR9 ... 14
1.1.3.2 RP105 ... 15
1.1.4 B-cell disorders ... 16
1.1.4.1 Common variable immunodeficiency ... 16
1.1.4.2 B-cell malignancies ... 19
1.2 Cell proliferation and cell death ... 21
1.2.1 Cell proliferation and cell cycle regulation ... 21
1.2.2 Cell death ... 22
1.2.2.1 Apoptosis ... 23
1.2.2.2 Intrinsic apoptosis: The role of BCL2 proteins ... 24
1.2.2.3 Caspase cascade... 26
1.2.3 B-cell homeostasis ... 27
1.2.3.1 BCL2 proteins in regulating survival of B cells ... 28
1.3 DNA damage ... 29
1.3.1 DNA damage response pathway ... 29
1.3.2 p53 ... 31
1.3.2.1 Modes of action of p53 ... 31
1.3.2.2 The regulation of p53 ... 32
1.3.2.3 Biological functions of p53 ... 33
1.4 Vitamin A ... 36
1.4.1 Vitamin A metabolism ... 37
1.4.2 Retinoic acid – mechanism of action ... 39
1.4.3 Vitamin A and the immune system ... 40
1.4.3.1 Vitamin A and development of the immune system ... 41
1.4.3.2 Vitamin A and innate immunity ... 41
1.4.3.3 Vitamin A in the adaptive immune system ... 43
2 AIMS OF THE STUDY ... 47
3 SUMMARY OF THE PAPERS ... 48
4 DISCUSSION... 51
4.1 Methodological considerations ... 51
4.1.1 Primary cell cultures ... 51
4.1.1.1 Peripheral blood B cells ... 51
4.1.1.2 CVID-derived B cells ... 52
4.1.1.3 Malignant B cells ... 52
4.1.1.4 Ethical considerations associated with collection of primary cells ... 53
4.1.2 Stimulation of B cells in vitro ... 53
4.1.3 Retinoic acid ... 54
4.1.4 Proliferation assays ... 55
4.1.5 Secretion of cytokines and immunoglobulins ... 56
4.1.6 Knock down of genes ... 57
4.1.7 DNA damaging agents ... 57
4.1.8 Analysis of DNA damage ... 58
4.1.9 Analysis of cell death and apoptosis ... 60
4.1.9.1 Cell death ... 60
4.1.9.2 Apoptosis ... 60
4.2 General discussion ... 61
4.2.1 The role of vitamin A in regulating survival of normal and malignant B cells ... 61
4.2.1.1 RA protects normal TLR9-stimulated B cells against spontaneous and irradiation- induced apoptosis ... 62
4.2.1.2 CpG-ODN and vitamin A in prevention and treatment of B-cell malignancies ... 66
4.2.2 The role of vitamin A in CVID-derived B cells ... 68
4.2.2.1 The ability of RA to normalize B cell functions in CVID-derived B cells ... 68
4.2.2.2 The clinical potential of RA, CpG-ODN and anti-RP105 in treatment of CVID ... 72
5 CONCLUSION ... 76
6 FUTURE DIRECTIONS ... 77
7 REFERENCE LIST ... 78
8 REPRINT PERMISSIONS ... 116
9 APPENDIX: Paper I, II and III ... 118
ACKNOWLEDGEMENTS
The work presented in this thesis was conducted between August 2013 and October 2017 at the Division of Biochemistry, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo. The primary financial support was a three-year doctor fellowship granted by the University of Oslo.
First and foremost, I would like to express my gratitude to my supervisor Heidi Kiil Blomhoff. Thank you for introducing me to the exciting field of vitamin A and B cells, and for providing an inspiring environment for conducting research. Thank you for your invaluable guidance, your presence, patience and for always having faith in me. Your positive attitude could turn a “negative” result into a step in a new direction. I would also like to thank my co-supervisor Børre Fevang for excellent cooperation regarding the CVID-projects. Thank you for recruiting the CVID-patients, for valuable discussions and for your enthusiasm.
Next, I would like to thank Randi Larsen Indrevær and Agnete Bratsberg Eriksen for our fruitful collaborations, invaluable discussions, and for introducing me to the lab-work. I would also like to thank Karin Margaretha Gilljam and Ida Guttormsen for continuing the Vitamin A-project; I am looking forward to see the results. Further, I would also like to express my gratitude to all former and present members of the Blomhoff Lab, especially Seham Skah, Eva Duthil, Nina Richartz, Camilla Solberg Vethe, Jannicke Holmseth Bukve, Virginie Follin-Arbelet and Elin Hallan Naderi. Thank you for providing a friendly environment and for all scientific and non-scientific conversations. In addition, I would like to thank all collaborators and co-authors for your contribution to this thesis.
Finally, I would like to express my deepest gratitude to my family for your love, encouragement and for believing in me. I am grateful for my children, Eline and Ada, thank you for filling my days with happiness and reminding me of what is important in life.
Especially, I would like to thank my dear husband and best friend, Andreas, for continuous love and support.
Oslo, October 2017
Kristine Lillebø Holm
LIST OF PUBLICATIONS Paper I
Indrevær RL, Holm KL, Aukrust P, Osnes LT, Naderi EH, Fevang B, Blomhoff HK.
Retinoic acid improves defective TLR9/RP105-induced immune responses in Common variable immunodeficiency-derived B cells.
J Immunol 2013 Oct 1;191(7):3624-33.
Paper II
Holm KL, Indrevær RL, Myklebust JH, Kolstad A, Moskaug JØ, Naderi EH, Blomhoff HK.
Myeloid cell leukemia 1 has a vital role in retinoic acid-mediated protection of Toll-like receptor 9-stimulated B cells from spontaneous and DNA damage-induced apoptosis.
Immunology 2016 Sep;149(1):62-73.
Paper III
Holm KL, Syljuåsen RG, Hasvold G, Alsøe L, Nilsen H, Ivanauskiene K, Collas P, Shaposhnikov S, Collins A, Indrevær RL, Aukrust P, Fevang B, Blomhoff HK.
TLR9-stimulation of B cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent on DNA damage responses. Implications for Common variable immunodeficiency.
PLOS ONE In press
ABBREVIATIONS
AICD activation-induced cell death
AID activation-induced cytidine deaminase AP-1 activator protein-1
APL acute promyelocytic leukemia ATM ataxia-telangiectasia mutated
ATR ataxia telangiectasia and rad3-related protein BAFF B cell activating factor
BCL2 B cell lymphoma 2 BCL6 B cell lymphoma 6 BCR B cell receptor
BH BCL2 homology
BLIMP1 B-lymphocyte-induced maturation protein 1 BrdU 5-bromo-2'-deoxyuridine
BTK Bruton`s tyrosine kinase CCR9 C-C chemokine receptor type 9 CD cluster of differentiation
CDK cyclin-dependent kinase
CFSE 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester CHK checkpoint kinase
CKI cyclin-dependent kinase inhibitor CLL chronic lymphocytic leukemia CpG cytosine-phosphate-guanine CSR class switch recombination
CVID common variable immunodeficiency DCs dendritic cells
DDR DNA damage response
DISC death inducing signaling complex DNA deoxyribonucleic acid
DNA-PKcs DNA-dependent protein kinase catalytic subunit DR direct repeat
DSB double strand break
ELISA enzyme-linked immunosorbent assay ER endoplasmic reticulum
ERK extracellular signal-regulated kinase
FO follicular
Foxp3 forkhead box p3 GC germinal center
3H tritium
HDM2 human double minute 2 homolog HR homologous recombination HSC hepatic stellate cells
ICOS inducible costimulatory
Ig immunoglobulin
IL interleukin
ILC innate lymphoid cell
IRF4 interferon regulatory factor 4
JC-1 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide LTi lymphoid tissue-inducer
MAPK mitogen-activated protein kinase MCL1 myeloid cell lymphoma 1
MHC major histocompatibility complex
MMP mitochondrial membrane potential
MOMP mitochondrial outer membrane permabilization
MRN MRE11-RAD50-NBS1
mRNA messenger ribonucleic acid
MyD88 myeloid differentiation primary response 88
MZ marginal zone
NF-κB nuclear factor-kappa B NHEJ non-homologous end joining ODN oligodeoxynucleotide
PAMP pathogen-associated molecular pattern PARP poly ADP-ribose polymerase
PAX5 paired box 5
PI3K phosphoinositide 3-kinase PI propidium iodide
PKC protein kinase C
PRR pattern-recognizing receptor
PUMA p53 upregulated mediator of apoptosis RA all-trans retinoic acid
RALDH retinaldehyde dehydrogenases RAR retinoic acid receptor
RARE retinoic acid response element RBP retinol binding protein
RE response element ROS reactive oxygen species RP105 radioprotective 105 kDa RXR retinoic X receptor
SHM somatic hypermutation siRNA small interfering RNA SSB single strand break
TACI transmembrane activator and calcium-modulator and cyclophilin ligand interactor
TD T cell-dependent
TFIIH transcription factor II human
Th T helper
TI T cell-independent TIR toll/IL-1 receptor TLR toll-like receptor TNF tumor necrosis factor
TNFRSF tumor necrosis factor receptor superfamily TRAIL TNF-related apoptosis-inducing ligand Treg regulatory T cell
TTNPB 4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl]benzoic acid
VAD vitamin A deficiency XBP1 X-box binding protein 1
XPC xeroderma pigmentosum group C protein
1 INTRODUCTION
1.1 The immune system
1.1.1 General overview of the immune system
The immune system is aimed to protect the host against pathogenic microbes, toxic or allergenic substances and transformed cells. It consists of an integrated network of lymphoid organs, cells, humoral factors and cytokines (1;2). The immune system includes the primary and secondary lymphoid organs, as well as circulating immune cells. The primary lymphoid organs include the bone marrow and thymus, and these are the primary sites for formation and maturation of lymphocytes. The secondary lymphoid organs are the major sites of immune activation, and consist of lymph nodes, spleen and mucosa associated lymphoid tissues.
Immune cells recirculate between the different secondary lymphoid organs, thereby increasing the chance of contact with the relevant antigen.
The immune system comprises two different compartments, termed the innate and adaptive immune system. There is, however, significant interaction between these two compartments, as recognition of pathogens in the innate immune system instructs the adaptive immune response (3;4). The innate immune response is highly evolutionary conserved and is considered as being non-specific. In addition to involving proteins such as complement factors, it consists of physical barriers (i.e. epithelial surfaces of the skin and gastrointestinal tract, mucus and gastric acid) and immune cells such as phagocytes, innate lymphoid cells (ILCs) and dendritic cells (DCs) (1;5). Through receptors such as pattern recognizing receptors (PRRs), innate immune cells recognize and react to conserved structures on pathogens called pathogen-associated molecular patterns (PAMPs). Innate immune responses are rapid, developing within hours, and are important as a first line defense. DCs serve as a link between innate and adaptive immune cells by secreting cytokines and presenting antigens (6).
The adaptive immune system consists of B- and T cells, providing humoral- and cell mediated immunity, respectively (7;8). Compared with innate immune responses, adaptive immune responses are slower - requiring several days when first encountering a pathogen.
Importantly however, the adaptive immune system is considered to be more advanced due to the fact that adaptive immune cells express antigen-specific receptors. T cells eliminate
pathogens by killing infected cells or by secreting cytokines that “help” other immune cells, whereas B cells secrete antibodies that target and neutralize pathogens. A key feature of the adaptive immune response is the generation of long-lived cells that upon re-exposure to a specific pathogen rapidly can re-express effector functions in a process known as immunological memory (9).
1.1.2 B cells
1.1.2.1 B-cell development
In adults, early B-cell development occurs in the bone marrow by a stepwise differentiation of the common lymphoid progenitor cell via a progenitor B cell into the immature B cell (Figure 1) (10;11). At this stage, the immature B cells leave the bone marrow to finalize the differentiation in secondary lymphoid organs. The goal of the early B-cell development is expression of a functional B cell receptor (BCR). During the transition from pro- to pre-B cell, the VDJ gene segments of the BCR heavy chain are rearranged and combined with a constant gene segment (Cµ). The successful expression of an Ig µ-chain on the surface of the B cell, followed by the assembly with the surrogate light chain, results in the expression of the pre-B cell receptor (pre-BCR). The pre-B cells then undergo further differentiation and replacement of the surrogate light chain with a VJ-rearranged light chain. This results in expression of IgM on the surface of the B cell, defining the cell as an immature B cell. The rearrangement of the Ig-genes culminates in the development of a diverse repertoire of functional BCRs. B cells that recognize self-antigens are eliminated by negative selection, where autoreactive B cells either undergo receptor editing in an attempt to create a non-self-reactive BCR, become anergic or undergo apoptosis (12). Only immature B cells with no- or low affinity to self- antigens will migrate to secondary lymphoid organs. In secondary lymphoid organs, the immature B cells differentiate through several transitional stages to eventually becoming mature B cells (11). Mature B cells that express both surface IgM and IgD are termed naïve B cells. There are two subsets of naïve B cells, follicular (FO) B cells and marginal zone (MZ) B cells (13). Whereas MZ B cells reside in spleen and are important for immunity against blood borne pathogens (14), FO B cells migrate between secondary lymphoid organs in search of cognate antigens. The expression of a functional BCR is essential for survival and activation of the B cell at later stages (15)
Figure 1: B-cell development. Adapted from (16).
1.1.2.2 B-cell activation
B cells are activated by ligation of receptors recognizing their cognate antigen. Activation results in a burst of proliferation, and is followed by differentiation of B cells into effector cells. The response to antigen can be divided into the primary or secondary immune response.
The primary immune response occurs the first time the body encounters a specific pathogen, whereas the secondary immune response occurs when the pathogen re-enters the body. The secondary immune response is generally more rapid, and is characterized by elevated secretion of antibodies (see more on the secondary immune response in section 1.1.2.4).
There are two principally different groups of antigens that can activate B cells, T cell- dependent (TD) and T cell-independent (TI) antigens (17;18). The vast majority of antigens, such as proteins and glycoproteins, stimulate the B cells in a TD manner. Generally, TD B cell activation requires two principal types of signals. The first signal is mediated by the binding of antigen to the BCR. The antigen is then internalized, processed and presented on major histocompatibility complex II (MHC-II) molecules on the surface of the B cell. The second signal involves T cells, which in addition to recognizing and binding to the antigen/MHC-II complex, provides co-stimulatory molecules like the cluster of differentiation 40 (CD40)-ligand. Activation of B cells results in the generation of extrafollicular short-lived plasma cells or establishment of germinal centers (GC) (Figure 2). GCs are transient structures formed in peripheral lymphoid organs, and the GC reaction promotes the differentiation of B cells to either long-lived plasma cells (see more section 1.1.2.3) or memory B cells (see more section 1.1.2.4) (19). In the GCs, the B cells will proliferate, undergo class switch recombination (CSR) and somatic hypermutation (SHM). B cells generated during the GC reaction are able to secrete antibodies with higher affinity and of other isotypes than the antibodies secreted by extrafollicular plasma cells (20;21). Following
SHM, B cells expressing high affinity BCRs are selected, whereas B cells that express low affinity BCRs either are eliminated or re-enter the GC for new rounds of SHM (19;22). Both CSR and SHM depend on the function of activation-induced cytidine deaminase (AID), an enzyme that converts cytosine to uracil (20). SHM or CSR is induced depending on whether the conversion occurs in the variable- or switch-regions of the Ig genes, respectively (23).
The cytosine to uracil conversion is followed by error-prone DNA repair, and the AID expression is therefore under strict regulation to maintain genomic integrity (24;25).
Figure 2: B-cell activation. Adapted from (26).
TI B cell activation involves recognition of for instance polymeric antigens or PAMPs (17;27). Polymeric antigens, such as polysaccharides on the surface of pathogens, contain repetitive structures that activate B cells by cross-linking and clustering BCRs on the surface of the B cells. Pathogen-derived PAMPs bind to and activate PRRs on the surface of B cells.
Toll like receptors (TLRs) are one class of PRRs, and these receptors are described in more
detail in section 1.1.3. Generally, TI B cell activation is more rapid than TD activation, but results in production of antibodies with lower affinity for the antigen.
1.1.2.3 Long-lived plasma cells and antibody functions
Plasma cells are non-dividing, terminally differentiated B cells with the ability to secrete large amounts of antibodies (26;28). Plasma cells are characterized by the loss of the surface markers CD19 and CD20, and the concurrent expression of the plasma cell markers CD38 and CD138 (28;29). As mentioned in section 1.1.2.2, activation of B cells can result in the generation of extrafollicular short-lived plasma cells or GC-generated long-lived plasma cells.
Whereas the extrafollicular response is the source of the majority of early protective antibodies, long-lived plasma cells are capable of secreting high affinity antibodies for decades (26;28). Long-lived plasma cells reside in the bone marrow and provide long term protection (30). The transition from an activated B cell into an antibody secreting plasma cell requires fundamental changes in gene expression involving silencing of transcription factors important for B cell identity and expression of plasma cell-specific regulators. Important B cell specific genes such as paired box 5 (PAX5), BTB Domain And CNC Homolog 2 (BACH2) and B cell lymphoma 6 (BCL6) are downregulated, whereas interferon regulatory factor 4 (IRF4), X-box binding protein 1 (XBP1) and B-lymphocyte-induced maturation protein 1 (BLIMP1) are upregulated (26). Recent studies have shown that in particular BLIMP1 is indispensable for the generation of antibody-secreting plasma cells (31;32). Differentiation of B cells into plasma cells also involves XBP1-driven expansion of the endoplasmic reticulum (ER), which enable the plasma cell to secrete large amounts of antibodies (33). Extensive secretion of antibodies causes severe ER-stress (34;35), and a recent study suggested that autophagy has an important role in maintaining ER function and for sustaining antibody secretion by plasma cells (36).
Antibodies are tetrameric molecules composed of two pair of polypeptide chains, each pair consisting of one light chain and one heavy chain. Both the light chain and the heavy chain contains a constant and a variable region that determine isotype and specificity of the antibodies, respectively (37). In humans, there are nine different isotypes: IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgE. Each of these isotypes display specific effector functions, aimed to target the pathogen in the most effective manner (38). The antibodies can be directly protective by neutralizing the pathogen or toxin. However, in most instances antibodies have indirect effects on the pathogen by activating other parts of the immune
system such as the complement system (39). IgM is the antibody secreted by activated B cells independently of the GC-reaction, and polymerization of five IgM molecules enhances their affinity to the antigen. Due to the size, soluble IgM molecules are not able to leave the bloodstream and enter tissues. IgG, on the other hand, have the ability to leave the bloodstream and are important in the immune response at the site of infection. IgA is the predominant isotype of antibodies secreted at mucosal surfaces, whereas IgE antibodies are important in the defense against parasites and in allergic reactions (37;38). IgD antibodies are found in trace amounts in serum, and they are mainly considered to function as surface antigen receptors on mature B cells.
1.1.2.4 Memory B cells
Life-long immunity to specific pathogens relies on the ability of the immune system to develop immunological memory. Memory B cells are long-lived resting pathogen- experienced B cells which can be rapidly reactivated to produce high titers of high affinity antibodies (40;41). Memory B cells can be distinguished from naïve B cells by the expression of the CD27 surface marker (42). Furthermore, memory B cell can be subdivided into IgM memory B cells (CD27+IgM+IgDdull) and switched memory B cells (CD27+IgM-IgD-), based on the isotype of BCR expressed on the surface. Generally, the generation of memory B cells is dependent on both GCs and T cell help. However, recent evidence reveals the existence of both GC-independent memory B cells and TI memory B cells (43). The mechanisms whereby GC B cells are selected to undergo memory B cell differentiation are not clearly understood.
According to one hypothesis, a survival advantage is sufficient for memory B cell differentiation to occur (44). Alternatively, a more recent study suggested that GC B cells with lower affinity for antigens were more prone to differentiate into memory B cells (45).
Reactivation of pathogen-experienced memory B cell characterizes the secondary immune response. The secondary immune response differs from the primary immune response in several ways: the response is more rapid with increased amounts of antibodies, the affinity for the antigen is higher and the antibodies are mainly of other subclasses than IgM.
The elevated secondary immune response can partly be explained by the increased proliferative response in memory B cells compared with naïve B cells (46). Moreover, the number of antigen-specific memory B cells is higher than the number of antigen-specific naïve B cells. Finally, the BCRs on memory B cells have higher affinity for the antigen than the BCRs on naïve B cells (41).
1.1.3 Toll-like receptors
TLRs are classified as a part of the innate immune system, and are encoded by conserved genes inherited through the germline (47;48). TLRs are one class of PRRs, and as presented in section 1.1.1, these receptors recognize conserved pathogen-derived molecular structures called PAMPs. TLRs were discovered in the 1990s, and Jules Hoffman and Bruce Beutler shared the Nobel Prize in Physiology or Medicine in 2011 for their work on TLRs and innate immunity. TLRs are type 1 transmembrane proteins where the extracellular N-domain contains leucine-rich repeat motifs important for recognition of the ligand. The cytoplasmic C-terminal contain a Toll/IL-1 receptor (TIR) domain required for downstream signaling (49).
The human genome encodes 10 TLRs (TLR1-10), whereas in mice 12 TLRs have been identified (TLR1-9, TLR11-13) (50). TLRs can be subdivided into receptors expressed on the cell surface (TLR1, TLR2, TLR4, TLR5, and TLR6) and TLRs expressed in membranes of intracellular vesicles like endosomes and ER (TLR3, TLR7, TLR8 and TLR9). TLRs on the cells surface typically recognize bacterial membrane compounds, whereas intracellular TLRs recognize nucleic acids from bacteria, vira or dead cells (51). Since nucleic acids are not unique to pathogens, it is important for the innate immune system to distinguish between self- and non-self nucleic acids. This discrimination is based on accessibility of nucleic acids to the TLRs. Hence, cellular nucleic acids from dying/dead cells that are present in the extracellular environment are normally rapidly degraded and not accessible to TLRs in intracellular compartments. Binding of ligands to the TLRs leads to dimerization of the TIR-domains, which in turn act as scaffolds for other downstream signaling proteins. One of these proteins is myeloid differentiation primary response 88 (MyD88). MyD88 act as an adaptor protein, and activates downstream signaling pathways resulting in a variety of antimicrobial and inflammatory responses (49).
TLRs are generally regarded as part of the innate immune system. However, TLRs are also frequently expressed in cells of the adaptive immune system and thereby involved in initiating adaptive immune responses (4;52). Thus, TLRs are considered as bridges between the innate and adaptive immune system. For instance, TLR-mediated activation of B cells leads to polyclonal activation and secretion of Igs (more on this in section 1.1.3.1). The role of TLRs in adaptive immune responses have made them attractive targets as adjuvants in vaccines and other treatments to potentiate the immune system (53).
1.1.3.1 TLR9
TLR9 is localized in endosomes and recognizes deoxyribonucleic acid (DNA) rich in unmethylated cytosine-phosphate-guanine (CpG) dinucleotides (54;55). Compared to human DNA, unmethylated CpG-motifs are abundant in bacterial and viral DNA. In humans, TLR9 is mainly expressed in B cells and plasmacytoid DCs (56), and the receptors are expressed at higher levels in memory B cells than in naïve B cells (57;58). Upon activation of naïve B cells by BCR-stimulation or via TLR9, the expression of TLR9 increases (57). Full length TLR9 is located in the ER, but it is rapidly translocated to the endosomal compartment upon uptake of CpG-rich DNA (59). TLR9 is proteolytically cleaved in the endosomal compartment, resulting in a biological active receptor with increased affinity to CpG-rich DNA (60). CpG- rich DNA translocate to early endosomes, where ligation of TLR9 induces dimerization and enables the C-terminal of TLR9 to activate MyD88 (Figure 3) (61). MyD88 will in turn activate the IRAK1-TRAF6-TAK1 pathway, and this culminates in nuclear translocation and activation of transcription factors such as nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1). The ultimate result of TLR9-mediated activation of B cells is transcription of pro-inflammatory genes, considered to have protective roles against infections (62). In vitro, activation of TLR9 can be mimicked by synthetic CpG oligodeoxynucleotides (CpG- ODN). CpG-ODN are short single stranded sequences (typically 24 nucleotides) containing unmethylated CpG motifs. CpG-ODN differs from bacterial DNA by the exchange of phosphodiester backbone to a phosphorothionate backbone, which makes them more resistant to nuclease degradation. There are several classes of CpG-ODN, named A, B and C. TLR9- expressing immune cells differ in their responses to various CpG-ODN isoforms, where B cells respond most favorably to class B CpG-ODN (63).
Ligation of TLR9 leads to polyclonal activation of B cells. TLR9-mediated activation of naïve B cells results in B-cell proliferation and differentiation into plasma cells secreting low affinity IgM antibodies (64). Whereas TLR9 activation is also able to induce CSR and transcription of genes encoding IgG in naïve B cells, additional signaling from BCR and B cell activating factor (BAFF)-receptor are needed for secretion of IgG (65). TLR9 is constitutively expressed in memory B cells, and activation of TLR9 has been shown to be important for maintenance of long-term B cell memory in the absence of further exposure to antigens (66). Several studies have proposed a bridge between TLR9 and BCR signaling, where co-ligation of these receptors increases the activation of the p38 mitogen-activated
protein kinase (MAPK) and NF-κB (67;68). Moreover, interaction between TLR9 and BCRs promotes antigen specific B cell responses such as antibody secretion (69;70). One hypothesis is that this interaction occurs via the fusion of organelles containing the internalized BCR and TLR9 into an autophoagosome, and that TLR9 is activated by CpG-rich DNA present in the antigen cargo bound to the BCRs (69;71;72). Defective TLR9-signaling has been implicated in immunodeficiencies, whereas inappropriate activation of TLR9 has been linked to development of autoimmune diseases (73;74).
Figure 3: TLR9 signaling pathway. Modified from (75).
1.1.3.2 RP105
Radioprotective 105 kDa (RP105, also known as CD180) was first identified as a surface protein that induces proliferation of murine B cells and rescues the cells from apoptosis after exposure to irradiation (76;77). Later, a human RP105 homolog was identified, sharing >70 %
of the nucleotide sequence with the murine protein (78). RP105 is defined as a TLR-related protein, as it contains the leucine-rich repeats motif but lacks the intracellular TIR-domain required for TLR-specific downstream signaling (79). RP105 is regarded as a TLR4 homolog, and it requires the accessory molecule MD-1 for its surface expression and downstream signaling (80). The downstream RP105 signaling pathway is not fully characterized, but it has been reported that RP105-signaling involves the tyrosine kinase Lyn, protein kinase C (PKC) β I/II and MAPK/ERK kinase (MEK) (81). CD19 expression has also been linked to the signaling pathway of RP105, as CD19-/- B cells have reduced proliferative response to RP105 ligation (82). A recent study also implicated PIM-1L kinase in the signaling pathway downstream of RP105 (83). The ligand of RP105 remains unknown, and ex vivo research has therefore relied on the use of anti-RP105 antibodies to activate the receptor.
RP105 is mainly expressed on mature B cells, but are also to some extent found on macrophages and DCs (78). The effects of RP105-stimulation are cell- and context dependent.
Hence, whereas RP105-stimulation inhibits LPS-mediated TLR4 activation of DCs (84), it enhances the TLR9- and TLR4-mediated activation of B cells (85;86). Although the initial identified role of RP105 was radioprotection and proliferation, later studies have shown that activation of RP105 also increases the Ig secretion from both murine and human B cells (76;78;87;88). Further, in a study conducted by Yamazaki and coworkers, it was reported that activation of RP105 potentiate the effects of TLR9-stimulation both regarding proliferation, survival and Ig-secretion (85). Dysregulation of RP105 has been implicated in inflammatory diseases such as arthritis, atherosclerosis and autoimmunity (89-92)
1.1.4 B-cell disorders
1.1.4.1 Common variable immunodeficiency Prevalence and diagnosis
Common variable immunodeficiency (CVID) is the most common and clinically important primary immunodeficiency, affecting approximately 1/10 000 to 1/50 000 Caucasians (93-95).
CVID is a heterogeneous disease, where the main phenotype is loss of B cell functions resulting in antibody deficiency and increased susceptibility to infections. CVID can present itself at any age but usually between the ages of 20 and 40, and it gives a life-long immunodeficiency. However, due to variable presentation of symptoms, diagnosis is often delayed for 4 to 6 years after onset (96;97). There is no universally accepted definition of
CVID. However, the International Union of Immunological Societies’ scientific committee defines CVID according to the following criteria: Hypogammaglobulinemia, with significant decrease in serum levels of IgG, IgA and/or IgM (at least 2 standard deviations below the mean for age), together with exclusion of all other defined causes of hypogammaglobulinemia.
Other diagnosis criteria also include clinical manifestations such as poor response to vaccines, absence of isohemagglutinins and that the onset of symptoms occurs above 2 years of age.
As CVID is a heterogeneous disease, there have been attempts to sub-classify CVID patients based on immunological parameters or on clinical phenotypes (98-101). These have aimed to identify mechanisms underlying development of CVID, and thereby to improve prognostic criteria and provide new targets for therapy.
Pathology and treatment of CVID
There is a great variability in the clinical presentation of CVID, but the main features include respiratory tract infections, non-infectious gastrointestinal and lung disease, autoimmunity (most common is immunologic thrombocytopenic purpura and autoimmune hemolytic anemia), lymphoproliferative disease, and cancers such as gastric cancer and B-cell malignancies (96;97;102). Whereas infections are the main causes of morbidity in these patients, the non-infectious complications are the main reasons for the reduced life expectancy associated with CVID (102). The increased risk of CVID patients to develop gastric cancers might be explained by the inability to secrete antibodies to target Helicobacter pylori (103).
The reason for the increased risk of developing B-cell malignances is still somewhat unclear.
It is believed that a complex interplay between chronic antigen stimulation, genetic abnormalities and immune dysregulation may lead to the increased risk of such cancers (104).
However, it has also been suggested that the increased chromosomal instability documented in CVID-derived lymphocytes may contribute to the increased risk of B-cell malignancies (105-107).
There is today no cure for CVID, but standard treatment is antibody replacement therapy by subcutaneous or intravenous injection of Igs to prevent recurring infections. In addition to the replacement of Igs, intravenously administered Igs have immunomodulatory effects that may contribute to the treatment outcome (108;109). It should however be emphasized that Ig-replacement therapy does not address the many non-infectious complications associated with CVID. It is therefore an urgent need to improve the treatment
of CVID to prevent complications like chronic lung disease, autoimmunity and malignancies (93;110).
Etiology and immunological defects of CVID
Despite many attempts to identify the causes of CVID, the etiology of this disease is still largely unknown. In only approximately 20 % of the cases a disease-causing mutation has been identified (111;112). The most frequent mutation linked to CVID (8-10 % of CVID patients) is localized to the gene tumor necrosis factor receptor superfamily member 13 B (TNFRSF13B). This gene encodes the protein transmembrane activator and calcium- modulating protein and cyclophilin ligand interactor (TACI) (113;114). TACI is expressed on mature B cells, and binding of the ligands BAFF and APRIL activates signaling pathways resulting in CSR and antibody secretion (115). Mutations in the gene encoding the protein inducible costimulatory (ICOS) are also identified in CVID patients (116). ICOS is a surface receptor expressed on T cells, and the interaction between ICOS on T cells and ICOS-ligand on B cells is important for the interleukin (IL)-10-induced generation of memory B cells and plasma cells (117). Other genes known to be mutated in CVID patients include CD19, TNFRSF13C and CD81.
Although CVID primarily is regarded as a B cell disorder leading to reduced Ig production, CVID patients may exhibit functional abnormalities in both the adaptive and innate immune system, including T cells (96;118). Reduced total numbers of B- and T cells are reported in CVID patients, but abnormal distribution of different subsets of B- and T cells are more common. Defective CD4+ and regulatory T cells (Treg) have been shown to contribute to the immunodeficiency by affecting B cell maturation (119;120). Regarding B cells, there have been reports on defects in differentiation of B cells into plasma cells, resulting in hypogammaglobulinemia and defective antibody responses (94;121). In particular, it is believed that defective B cell maturation results in reduced numbers of switched memory B cells in CVID patients. Hence, the secretion of Igs of classes other than IgM is frequently reduced (100;122). Deficient SHM may result in abnormal affinity maturation, and it is evidence for defective repair of AID-induced mutations in some cases of CVID (123).
Furthermore, it has been reported that lymphocytes from CVID patients more readily undergo both spontaneous and induced apoptosis, contributing to the observed lymphopenia and possibly also the hypogammaglobinemia (124). Increased expression of the death receptor FAS and of the pro-apoptotic cytokine TNF-related apoptosis-inducing ligand (TRAIL) could
in part explain the increased sensitivity to apoptosis (125;126). Furthermore, reduced expression of co-stimulatory molecules such as CD38 could contribute to reduced survival of CVID-derived lymphocytes (125).
Abnormalities in the innate immune system have also been linked to CVID, with both DCs and natural killer cells being affected (127;128). Further, several studies have revealed that CVID-derived B cells have impaired ability to respond to activation of the innate receptor TLR9. This has been demonstrated by the reduced proliferation (129), CD86 upregulation (73;130), AID-expression (129;131), IL-10- (73;132) and antibody secretion (131) in TLR9- stimulated CVID B cells. Whereas the mechanisms behind the reduced TLR9 responses are not entirely revealed, reduced expression of TLR9 itself may account for some of the defects (73). Diminished responses to activation of RP105 has also been described in CVID-derived B cells, where the effects of combined stimulation of TLR9 and RP105 is reduced (85).
Furthermore, studies have also reported that CVID patients frequently suffer from vitamin A deficiency (133-135), and that vitamin A supplementation can increase Ig secretion in B cells from CVID patients (133).
1.1.4.2 B-cell malignancies
B-cell malignancies are diseases that occur in B cells at various stages of B-cell development (136-138). Shaffer and coworkers defined B cell differentiation as a “disaster waiting to happen” (137), where B cells put their genomic integrity in danger during several stages of development and in response to activation. Both rearrangements of the genes encoding the BCR during early B-cell development and CSR/SHM in the GCs involve double-strand DNA breaks. These breaks are normally repaired by non-homologous end-joining repair mechanisms. However, occasionally these processes may lead to chromosomal abnormalities resulting in cancer development (139). Hence, chromosomal translocations where a proto- oncogene comes under the control of the Ig-promoter is involved in pathogenesis of several B-cell malignancies (140). Clonal expansion of B cells in response to antigens and other stimuli also provide a threat to genomic integrity.
B-cells malignancies can be divided into leukemias that arise in the bone marrow and lymphomas that originate from lymph nodes. Chromosomal translocations and mutations in genes at the B-precursor/pre-B cell stages give rise to B cell precursor acute lymphoblastic leukemia. Mature B cell subsets such as FO B cells, MZ B cells, GC B cells, plasma cells and
memory B cells can give rise to various types of B-cell malignancies such as chronic lymphocytic leukemia (CLL), FO lymphoma, splenic MZ lymphoma or multiple myeloma (136-138) (Figure 4).
Figure 4: Origin of B-cell malignancies. Adapted from (136).
CLL is the most common leukemia diagnosed in the western world, and it mainly affects elderly (141;142). Although CLL is defined as a chronic incurable malignancy, the prognosis and the clinical course vary significantly. Thus, survival of patients ranges from a few months to several years after diagnosis. Most of the patients are asymptomatic at diagnosis, and treatment is not required before debut of symptoms or progression of disease.
The presence of unmutated immunoglobulin heavy chain (IGHV) and mutation in TP53 are prognostic markers associated with poor prognosis (142). Standard treatment is chemotherapy with a combination of fludarabine, cyclophosphamide and rituximab. However, patients with comorbidities and progressive disease might not tolerate such intensive treatment. Novel treatment strategies using kinase inhibitors against Bruton`s tyrosine kinase (BTK), phosphoinositide 3-kinase (PI3K) and B cell lymphoma 2 (BCL2) are in clinical trials and might improve the prognosis of CLL (143). The BTK-inhibitor ibrutinib is approved as first- line treatment in all CLL patients in the U.S and EU, whereas the BCL2 inhibitor venetoclax is approved for a subgroup of patients with 17p deletion (144;145). CLL patients have increased risk of fatal infections both due to the leukemia itself and to treatment related immunosuppression (142).
Targeting TLRs, especially TLR9, has been an emerging field in treatment of B-cell malignancies (146). B-CLL cells show low immunogenicity and are not easily recognized by T cells, partly due to low expression of costimulatory accessory molecules (147-149).
Treatment of B-CLL cells with CpG-ODN results in upregulation of costimulatory molecules such as CD80 and CD86, resulting in increased immunogenicity (150). These findings have encouraged the use of CpG-ODN in tumor cell vaccination protocols and in immunotherapeutic strategies like adoptive T cell transfer and monoclonal antibody-mediated therapies (148). Further, in patients with stable disease, TLR9-stimulation has also been shown to promote activation-induced apoptosis (AICD) (151;152). Hence, the therapeutic benefits of CpG-ODN on B-CLL patients may also include pro-apoptotic effects. A phase I trial concluded that CpG-ODN were well tolerated by B-CLL patients, and that immunogenic changes in the tumor cells were induced (153). TLR agonists have also been tested in clinical trials for other B-cell malignancies, with promising results (146).
1.2 Cell proliferation and cell death
1.2.1 Cell proliferation and cell cycle regulation
Cell proliferation is essential for all living organisms. A cell reproduces itself by performing an orderly sequence of events in which the cell duplicates its content and divide. This cycle of duplication and division is known as the cell cycle, and results in the generation of two essentially equal daughter cells. The cell cycle of a mammalian cell is divided into four distinct and chronological events: the first gap (G1) phase, the DNA synthesis (S) phase, the second gap (G2) phase and the mitotic (M) phase (154). G0 is defined as a resting state where the cells are not proliferating. During the two gap phases, the cells grow in size and protein content to prepare for cell division. The progression through the cell cycle is regulated by the cell cycle machinery, including cyclins, cyclin-dependent kinases (CDKs) and cyclin- dependent kinase inhibitors (CKIs) (155).
To ensure proper progression of the cell cycle, the cell cycle contains several checkpoints. The checkpoints ensure that critical events such as DNA replication and chromosome segregation occur with high fidelity. There is one checkpoint in G1, the restriction-point. In this checkpoint mitotic and anti-proliferating signals are integrated to determine whether or not the cell should start replicating its DNA (156). A second checkpoint is located in the G2-phase to ensure that DNA replication is completed prior to the cell
entering mitosis (157). A third checkpoint prevents execution of the metaphase in cases of improper alignment of the chromosomes to the mitotic spindle (158). These checkpoints are also targeted by the DNA damage response (DDR) pathway (see section 1.3.1) (159;160). In response to DNA damage, the DDR will activate proteins in the PI3K-family, such as ataxia- telangiectasia mutated (ATM), ataxia telangiectasia and rad3-related protein (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). These kinases will in turn phosphorylate and activate downstream kinases to block CDK activity. The cell cycle arrest allows time for DNA repair, alternatively the DDR will induce cell death. Proteins involved in cell cycle regulation and cell cycle checkpoints are frequently mutated in cancer (154;155).
1.2.2 Cell death
Cell death is essential for maintaining homeostasis in multicellular organisms. Historically, modes of cell death were divided into two main categories, necrosis and apoptosis. Necrosis was described as a passive (ATP-independent) and unregulated process, whereas apoptosis was defined as an ATP-dependent, programmed and genetically determined form of cell death.
Today we know that this is an oversimplified view of cell death pathways. There are several different forms of programmed cell death pathways, including different forms of apoptosis and autophagic cell death (161). Furthermore, accumulating evidence indicate that also necrosis under certain circumstances can occur as a highly regulated process called necroptosis (162).
Apoptosis occurs through an active and ordered process resulting in cell-intrinsic programmed suicide. The process is characterized by cellular shrinkage and fragmentation of the apoptotic cell into membrane enclosed “apoptotic bodies”. The apoptotic bodies are phagocytosed and degraded by macrophages or by other neighboring cells (163). Apoptosis occurs without the induction of an inflammatory response, since the cellular content is never released into the cell surroundings. Necrosis is caused by pathological stimuli, such as loss of blood supply, infection, toxins or physical trauma. In contrast to apoptosis, necrosis involves cytoplasmic swelling that leads to cell lysis and the release of cellular contents into the extracellular fluid causing inflammation (164). The programmed type of necrosis, necroptosis, is regarded as a backup mechanism in case the apoptotic pathway is hampered in any way (162). Autophagic cell death is defined as a non-apoptotic form of programmed cell death (165). Macroautophagy (hereafter termed autophagy) is an evolutionary conserved catabolic pathway involving lysosome-dependent degradation of various cargo containing cytosolic
contents. Although autophagy is manly considered as a pro-survival stress response, autophagy may also result in cell death (166).
In the next sections, the focus will be on the apoptotic process, the regulation of apoptosis and on the role of apoptosis in regulation of B cell homeostasis.
1.2.2.1 Apoptosis
Apoptosis is a physiological process required for normal development and homeostasis.
Apoptotic cell death is essential for organ development during embryogenesis, whereas its role in adult organisms is to remove aged, damaged and unwanted cells (167). Defects in the apoptotic pathway are implicated in autoimmunity, tumorigenesis, and in resistance to established cancer treatments (168).
Figure 5: Intrinsic and extrinsic apoptosis. Adapted from (169)
The effector enzymes involved in apoptosis are a family of proteases known as caspases (see section 1.2.2.3). Generally, the caspases can be activated through two distinct pathways, the intrinsic and extrinsic apoptotic pathways (Figure 5) (170). The extrinsic
pathway is activated when extracellular ligands binds to death receptors belonging to tumor necrosis factor (TNF) receptor superfamily (such as FAS and TRAIL-receptors). Binding of ligands to death receptors results in the formation of a death inducing signaling complex (DISC) consisting of the death receptor itself, FAS-associated protein with death domain (FADD) and procaspase 8. The DISC is responsible for activation of caspase 8, which mediates the downstream signaling resulting in activation of the caspase cascade. Activated caspases will in turn degrade vital cellular proteins – and thereby promote cell death.
Alternatively, apoptosis can be initiated by intrinsic stress signals, in a pathway involving the mitochondria (more on this in the next section).
1.2.2.2 Intrinsic apoptosis: The role of BCL2 proteins
The intrinsic pathway, also called the mitochondrial pathway, is activated in response to a wide range of intracellular stress factors, such as excessive DNA damage, high intracellular levels of reactive oxygen species (ROS) or calcium, and growth factor deprivation. The regulation of mitochondrial membrane potential (MMP) is fundamental for the regulation of intrinsic apoptosis, as initiation of intrinsic apoptosis involves mitochondrial outer membrane permabilization (MOMP) and subsequently the release of pro-apoptotic factors into the cytoplasm (171).
The BCL2 family of proteins is required for proper regulation of the MMP (172-174).
These proteins are characterized by the presence of one or several BCL2 homology (BH) domains, and these are essential for their function. The BCL2 superfamily consists of three subfamilies; one anti-apoptotic, one pro-apoptotic and one subfamily comprising proteins with indirect pro-apoptotic properties. It is the balance between the pro- and anti-apoptotic proteins that determines the fate of the cell. The pro-apoptotic members BAX and BAK are essential for the execution of the intrinsic apoptotic program, as they are responsible for inducing MOMP. The anti-apoptotic BCL2 members, consisting of proteins such as BCL2, BCL-XL, MCL1, BCL2A1 and BCL2L2, preserve mitochondrial integrity by binding and inhibiting the effects of BAX and BAK. BH3-only proteins such as BAD, BID, BIK, HRK, BIM, NOXA and PUMA act as sensors of cellular apoptotic signals and transduce the signal to the mitochondria (172;174;175). BH3-only proteins cannot kill in the absence of BAX and BAK, but contribute to apoptosis by deactivating anti-apoptotic BCL2 proteins (“sensitizers”
such as BAD/BIK) and/or activating pro-apoptotic proteins (“activators” such as BID/BIM) (176). The structure of the anti-apoptotic and pro-apoptotic BCL2 members are similar, with
the exception of BH3-only proteins that only share homology in the BH3-domain. Pro- and anti-apoptotic members such as BCL2 and BAX are globular multi-domain proteins that contain up to four BH-domains with high sequence homology (177). The BH1, BH2 and BH3 domains form hydrophobic grooves, which can interact with the BH3 domains of the BH3- only proteins and other BCL2 family proteins (178;179). In the pro-apoptotic BAX and BAK proteins, the BH1 and BH2 domains are essential for interaction with the mitochondrial membrane and subsequently for pore formation.
In a normal state, the balance between anti-apoptotic and pro-apoptotic BCL2 proteins is in favor of cell survival. Upon apoptotic signaling, BH3-only proteins are activated and exert their function by binding their BH3-domains to the hydrophobic groove of pro- and anti- apoptotic BCL2 proteins (180). Activation of BH3-only proteins is induced by several mechanisms, including phosphorylation (of BAD), proteolytic cleavage (of BID) or transcriptional induction (of NOXA, PUMA, HRK) (181). Binding of BH3-only proteins to anti-apoptotic BCL2 proteins inhibits their function and results in release of the pro-apoptotic proteins BAX and BAK. Activation of BAX by BH3-only protein leads to exposure of the carboxyl terminal helix and causes integration of BAX into the mitochondria membrane (182).
These combined events, leads to oligomerization of BAX and BAK and the formation of the
“apoptotic pore” and release of pro-apoptotic factors (172). There has long been debated whether BAX and BAK proteins are able to cause pore formation in the mitochondrial membrane, due to the inability to observe such pores in the microscope. However, recent research based on super-resolution atomic force microscopy revealed a ring-like structure of BAX monomers in apoptotic cells, and found that these structures were able to perforate the outer mitochondria membrane (Figure 6) (183;184).
Figure 6: Organization of BAX into ring-like structures resulting in MOMP. Adapted from (184).
MOMP results in the release of several factors from the mitochondria, such as cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low pI (SMAC/DIABLO) and apoptosis inducing factor (AIF).
The interaction between cytochrome c and the apoptotic protease activating factor 1 (APAF1) results in a conformational change that allows APAF1 to bind ATP and form the apoptosome.
The apoptosome interacts with procaspase-9, resulting in the cleavage of this procaspase and thereby generation of the active form of caspase-9 (171).
1.2.2.3 Caspase cascade
Both the extrinsic and intrinsic apoptotic pathways converge in the activation of caspases and the caspase cascade. Caspases are cysteine dependent aspartate-specific proteases, members of the IL-1β-converting enzyme family, and serve as executors of apoptosis (185;186). All caspases are expressed as relatively inactive zymogens known as procaspases, and requires proteolytic cleavage for activation. Procaspases are activated either by auto-cleavage in response to apoptotic signals or by other caspases in a caspase cascade.
The caspase cascade is an orderly series of signals that serves to induce, transduce and amplify intracellular apoptotic signals. Initiator caspases (caspase 2, -8, -9, -10) are auto- activated in response to apoptotic signals. Activation leads to dimerization of the caspases, and this is followed by proteolytic cleavage that contributes to stabilization of the dimer (186;187). Activated initiator caspases will in turn cleave downstream effector caspases (caspase 3, -6, -7) between the large and the small subunit allowing conformational changes that expose the active site of the caspases. In the extrinsic pathway, activation of death receptors and the following DISC formation results in the activation of caspase 8. In the intrinsic pathway, formation of the apoptosome in response cytochrome c release, results in the activation of caspase 9, and the two pathways merge by the activation of the effector caspase 3 (Figure 5) (188). Effector caspases are responsible for the destruction of the cell by cleaving of important cellular proteins. The substrates of caspase 3 include proteins involved in cellular and nuclear structuring, as well as proteins involved in signal transduction and in cell-cell contact (189;190). The caspase 3-mediated cleavage of poly ADP-ribose polymerase (PARP) is frequently used as a hallmark of apoptosis (191). Indirectly, the effector caspases are also responsible for fragmentation of DNA, where caspase-dependent degradation of inhibitor of caspase-activated DNase (ICAD) allows activation of nucleases (192).
The distinction between the intrinsic and extrinsic apoptotic pathways is not rigid, as the extrinsic pathway is able to activate the intrinsic pathway. The outcome of the caspase 8 activation is cell type dependent; in type I cells caspase 8 will activate the caspase cascade directly, whereas in type II cells caspase 8 mediates apoptosis by activating the intrinsic pathway (193). Caspase-8 initiates intrinsic apoptosis by inducing activation of tBID. This is a pro-apoptotic BH3-only protein that contributes to the release of chromosome c by promoting oligomerization and insertion of BAX into the outer mitochondrial membrane (194).
1.2.3 B-cell homeostasis
Apoptosis has a critical role in development, regulation and function of the immune system (Figure 7) (175). During development of lymphocytes, apoptosis has an essential role in removing immune cells with dysfunctional and self-reactive antigen receptors (195). Only proper signaling downstream of functional antigen receptors provides survival signals that allow the cells to further differentiate into mature lymphocytes. Consequently, the majority (>90 %) of lymphocytes die during development due to lack of such survival signals. The BCL2 family members have a central role in the regulation of apoptosis both during B-cell development and in regulation of the immune response. The various members of the anti- apoptotic BCL2 family have both specific and overlapping functions, displaying a redundancy in this group of proteins during B-cell development (196). Defects in the regulation of B cell apoptosis may result in diseases such as autoimmunity, immunodeficiency, and various forms of B-cell malignancies (124;197).
Figure 7: Role of apoptosis in B-cell differentiation. Adapted from (198).
1.2.3.1 BCL2 proteins in regulating survival of B cells
Various members of the BCL2 family of proteins are involved in preventing or initiating apoptosis at distinct stages of B-cell development. Myeloid cell lymphoma 1 (MCL1) is a highly regulated anti-apoptotic BCL2 protein with a prominent role in regulating survival of B cells during development and differentiation (199). First, survival of hematopoietic stem cells requires high expression of this anti-apoptotic protein (200). Before the expression of a functional BCR, MCL1 is induced by IL-7 and is responsible for survival of precursor B cells (201). At later stages of B-cell development, the expression of a functional BCR is essential for survival of the B cell, where induction of anti-apoptotic BCL2 proteins in response to appropriate BCR stimulation contributes to the survival of these cells (175;202). On the other hand, activation of the BH3-only protein BIM is responsible for depletion of both autoreactive and non-reactive B cells (203;204).
The survival of peripheral B cells also depends on the expression of anti-apoptotic BCL2 proteins (201). Tonic BCR stimulation provides an antigen-independent survival signal necessary for long-term survival of mature B cells. This effect is partly enforced by expression of MCL1 (205). There is differential outcome of BCR-signaling in B cell precursors versus mature B cells. Hence, whereas strong BCR stimulation of B cell precursors results in apoptosis, BCR activation of mature B cells promotes survival. One possible reason for this discrepancy is that B cell precursors and mature B cells differ in their expression and activation of downstream mediators of BCRs, which in turn will affect the outcome of BCR activation (206). Moreover, mature B cells receive distinct survival signals provided by receptors such as BAFF-receptor and TLRs, and activation of these results in the induction of anti-apoptotic BCL2 genes (202;207-210). The survival of activated B cells is highly dependent on the expression of MCL1 (196;201;211), and this is particularly relevant long- lived plasma cells responsible for long-term immunity.
Apoptosis has also a regulatory role in determining the duration of an immune response. After resolution of the infection, the surplus of immune cells is removed by AICD.
The balance between BIM and BCL2 is important for regulating the pool of immune cells at the end of the immune response (198). Ideally, this balance should result in the survival of a suitable pool of immune cells that provides long-lasing immunity against the pathogen, without causing chronic immune activation.
1.3 DNA damage
Maintenance of genomic integrity is essential for proper function and survival of cells and organisms. However, damage to our genetic material is an ongoing threat, and to cope with these challenges, eukaryotes have developed a DNA damage response pathway (DDR). DNA damage arise from both intra- and extracellular sources such as ROS, replication-induced damage or from radiation and numerous chemicals (212). These insults results in alterations of the chemical structure of DNA, such as strand breaks and base damage (Figure 7). The various DNA damage lesions are recognized and repaired by distinct DDR mechanisms (213).
Dysregulation of the DDR could lead to mutations and chromosomal aberrations, and potentially result in diseases such as cancer, immune deficiencies and neurodegenerative disorders (214).
Figure 8: Types of DNA damage. Adapted from (215).
Ionizing radiation (hereafter referred to as irradiation) is a highly effective DNA damaging agent, and it is currently widely used in treatment of cancer. Irradiation induces a variety of DNA lesions, where single strand breaks (SSBs) and base damage accounts for 98 % of the overall damages (216). However, the double strand breaks (DSBs) are considered as the most detrimental type of DNA damage. The sensitivity to DNA damage and the outcome of such events are highly cell specific. B cells are particularly sensitive to DNA damage, and readily undergo apoptosis in response to radiation-induced DNA damage (217).
1.3.1 DNA damage response pathway
In response to DNA damage, the DDR ultimately regulate processes such as cell cycle arrest, DNA repair and apoptosis. The DDR consist of a hierarchy of proteins that through the action of sensors, transducers and effectors determine the fate of cells harboring DNA damage (Figure 9) (218;219).
Figure 9: The DNA damage response. Adapted from (220).
Central in the DDR are proteins belonging to the PI3K–superfamily: ATM, DNA- PKcs and ATR (219). Whereas ATR is primarily involved in sensing of SSBs generated at stalled replication forks (221), ATM and DNA-PKcs are essential for the response to DSBs (219;222). The MRE11-RAD50-NBS1 (MRN) complex senses DSBs, and is responsible for recruitment of ATM to the site of damage (223). ATM is activated by autophosphorylation at S1981, probably initiated by changes in higher order of chromatin structure in response to DSBs (224). Phosphorylation of ATM triggers a switch from inactive dimers to active monomers, and activated ATM is able to phosphorylate downstream substrates (224).
Following recognition of DNA lesions, ATM will phosphorylate proteins such as checkpoint kinase 2 (CHK2), a protein important for amplification and transduction of the damage signal (218). Further, ATM will phosphorylate the H2A histone variant H2AX at S139 to form γH2AX. γH2Ax plays a role in maintaining genome integrity, and is required for the concentration and stabilization of DNA repair proteins at the site of damage (225). Other signaling factors like p38 MAPK are also thought to be an integrated part of the DDR (226).
Eventually, DDR activation leads to accumulation and activation of the tumor suppressor p53.
P53 is a central effector protein in the DDR, involved in processes including DNA repair, cell cycle arrest, senescence and apoptosis (for more detailed information, see section 1.3.2).
Whereas p53 has a central role in the DDR, also p53-independent effector functions of the DDR exist. Cell cycle arrest in G1 and G2 can be induced by both p53-dependent and - independent mechanisms. Activation of ATM/CHK2 results in phosphorylation of members of the CDC25 family of phosphatases. This phosphorylation prevents the phosphatases from activating CDK1-cyclin B, thereby initiating cell cycle arrest in G2 (226). Further, ATM is required for efficient repair of DSBs, especially in the G2 phase of the cell cycle (218). Hence, ATM is involved in recruitment and activation of several proteins involved in DNA repair, including BRCA1, BLM1, CtIP as well as the MRN complex itself (227). Furthermore, DNA- PKcs play a role in repair of DSBs (228).
1.3.2 p53
P53 is a crucial tumor suppressor known as the “guardian of the genome”. Previously it was believed that the tumor suppressor function of p53 solely relied on canonical p53 functions such as induction of cell cycle arrest, senescence and apoptosis (229). However, recent research has revealed that the p53 tumor suppressor functions also include non-canonical effects such as regulation of metabolism, autophagy, necrosis and oxidative status of the cell (230;231). Studies have also revealed that p53 has a role in maintaining genomic stability by preventing conflicts between transcription and replication, thereby preventing the induction of DNA damage (232). P53 plays a critical role in the DDR, and numerous signaling pathways that are triggered by cellular stresses merge at the activation of p53. In general, p53 exerts its effects as a transcriptional factor regulating the level of several hundred target genes (231).
However, also transcription-independent functions of p53 exist (233;234). The importance of p53 as a tumor suppressor is highlighted by the fact that more than half of all human cancers harbor a TP53 mutation (235). The majority of p53 mutations are missense mutations leading to loss of vital tumor suppressive functions, or gain of function mutations that via other mechanisms promote tumor development (236;237).
1.3.2.1 Modes of action of p53
In the classical view, p53 acts as a transcription factor binding in a sequence-dependent manner to p53 response elements (REs) in the promoter of target genes (238). Canonical REs consists of two repeats of a decamer motif (RRRCWWGYYY) separated by a spacer. P53
