The chronic lymphocytic leukemia prognostic marker CD38: Evaluation as a therapeutic
target and its possible regulation of CD45 phosphatase activity
Celina Wiik
Thesis submitted for the degree of Master of Science in Molecular Bioscience and Biochemistry
60 credits
Department of Bioscience
Faculty of Mathematics and Natural Sciences
UNIVERSITY OF OSLO
April 2022TABLE OF CONTENT
ACKNOWLEDGEMENTS 4
ABSTRACT 5
ABBREVIATIONS 7
1 INTRODUCTION 9
1.1 THE VERTEBRATE IMMUNE SYSTEM 9
1.1.1 Innate immunity 9
1.1.2 Adaptive immunity 10
1.2 CYTOKINES 12
1.3 ANTIGEN RECEPTORS 13
1.3.1 The Antigen Receptor of B Cells 13
1.3.1.1 BCR Signaling 14
1.3.2 The Antigen Receptor of T Cells 16
1.3.3 V(D)J Recombination 17
1.4 TLYMPHOCYTE DEVELOPMENT 19
1.4.1 T Cell Maturation 19
1.4.2 T Cell Activation and Differentiation 20
1.5 BLYMPHOCYTE DEVELOPMENT 21
1.5.1 B Cell Maturation 21
1.5.2 B Cell Activation and Differentiation 22
1.5.2.1 The Germinal Center Reaction 24
1.6 CHRONIC LYMPHOCYTIC LEUKEMIA 26
1.6.1 Therapeutic Options 27
1.6.2 The Negative Prognostic Marker CD38 28
1.6.3 The CLL Microenvironment 28
1.6.4 BCR signaling in CLL 31
2 THESIS AIMS 33
3 MATERIALS AND METHODS 34
3.1 PATIENT MATERIAL 34
3.2 PBMC ISOLATION 35
3.3 CELL COUNTING 35
3.4 CD8+ CYTOTOXIC T CELL DEPLETION AND ACTIVATION OF CLL-DERIVED PBMCS 35
3.5 POSITIVE CLL CELL ISOLATION 36
3.6 NEGATIVE CLL CELL ISOLATION 36
3.7 CD38+CLL CELL ISOLATION 37
3.8 PHARMACOLOGY 38
3.8.1 CD38 inhibitor 78c 38
3.8.2 Anti-CD38 mAb AT1 38
3.9 FLOW CYTOMETRY 38
3.9.1 Flow Cytometry antibodies 38
3.9.2 Flow Cytometry-based CD45 phosphatase activity 39
3.9.3 Flow Cytometric Analysis 40
3.10 RNA ISOLATION AND Q-RTPCR 40
3.11 CONFOCAL MICROSCOPY 41
3.12 STATISTICAL ANALYSIS 42
4 RESULTS 43
4.1 CD38 EXPRESSION IS UPREGULATED BY ACTIVATED TH CELLS AND IS ASSOCIATED WITH EXPRESSION OF CD45 ACTIVITY REGULATORS IN CLL CELLS 43 4.2 THE EFFECT OF CD38 INHIBITION IN CLL-TH CO-CULTURES 50 4.3 DIRECT EFFECT OF THE CD38 INHIBITOR ON CLL CELLS 56 4.4 EVALUATING THE RECEPTOR FUNCTION OF CD38 BY MAB AT1 63
4.5 CD38 AND CD43CO-LOCALIZATION ASSAY 71
5 DISCUSSION 73
5.1 CD38 EXPRESSION IS UPREGULATED BY ACTIVATED TH CELLS AND IS ASSOCIATED WITH EXPRESSION OF CD45 ACTIVITY REGULATORS IN CLL CELLS 73 5.2 THE EFFECT OF CD38 INHIBITION IN CLL-TH CO-CULTURES 74 5.3 DIRECT EFFECT OF CD38 INHIBITION ON CLL CELLS 74 5.4 EVALUATING THE RECEPTOR FUNCTION OF CD38 BY MAB AT1 75 5.5 CD38 AS A POTENTIAL CD45 ACTIVITY REGULATOR 78
5.6 CD38 AS A THERAPEUTIC TARGET IN CLL 78
6 FUTURE PERSPECTIVES 80
7 REFERENCES 81
Acknowledgements
The work presented in this master thesis was performed at Department of Immunology, Rikshospitalet, Oslo from January 2021 to April 2022.
Foremost, I would like to thank my head supervisor and head of the research group, Peter Szodoray. Thank you for letting me take part in your exciting research. I deeply appreciate your enthusiasm and consideration. Sincerely, I thank my co-supervisor, Britt Nakken. Thank you for guiding me through the writing process and for helping me plan experiments. My deepest gratitude also goes to my co-supervisor, John F. Imbery. Thank you for guiding me through laboratory techniques, following me up daily and always being available for me to ask questions. I am grateful for being part of such a supportive and humble group. Thank you all for your guidance throughout the project. I would also like to thank Ludvig Munthe for introducing to the research group and letting me work in his lab.
Additionally, I would like to thank my internal supervisor at UiO, Finn Eirik Johansen. Thank you for following me up throughout the course of my thesis and for your lectures in
immunology.
Finally, I would like to thank my friends and family for their support throughout the master project.
Abstract
Chronic Lymphocytic Leukemia (CLL) is a heterogeneous B cell malignancy that is
characterized by the accumulation and clonal expansion of mature-like B cells in the blood, bone marrow, lymph nodes and spleen. Adverse prognosis and the need for earlier, more frequent treatment is associated with a higher percentage of CD38+ CLL clones [1]. CD38 is a type-II transmembrane glycoprotein possessing both enzymatic and receptor functions [2].
Several studies have implicated CD38 in B cell receptor (BCR) signaling [3, 4] and CD38 stimulation was shown to promote CLL proliferation and survival [1].
CLL pathogenesis and proliferation is reliant on activation by T helper cells [5]. Intriguingly, our group has characterized that T helper cell signals enhance BCR signaling in healthy, mature B cells by increasing CD45 phosphatase activity via increasing surface expression of a CD45 ligand Galectin-1 [6]. Galectin-1 binds to CD45 and CD43 in T cells and co-clusters CD43 and CD45 in dendritic cells, leading to downstream signaling in the latter [7, 8]. Our group has also observed that CD43 is upregulated on CLL cells upon T helper cell stimulation and unpublished work shows these glycoproteins are co-localized upon B cell activation and may contribute to regulation of CD45 activity (Imbery et al., under revision, British J
Haematol 2022). Finally, T helper cell stimulation upregulates the expression of CD38 in CLL [9], which may indicate its involvement in BCR signaling, possibly through CD45 activity regulation.
Connecting the above ideas that (1) T helper cells support CLL cell activation and increase CLL cell CD38, CD43, and Galectin-1 expression and (2) Galectin-1 and/or CD43 regulates CD45 activity and BCR signaling in mature and CLL B cells, we hypothesized that CD38 regulates CD45 phosphatase activity through CD43 and Galectin-1 in CLL. We aimed to investigate the effect of pharmacological CD38 inhibition and anti-CD38 monoclonal antibody treatment on CD38 expression, proliferation, CD45 activity and expression of the CD45 regulators CD43 and Galectin-1.
We confirmed the finding that T helper cell signals upregulates the expression of CD38 in CLL. Additionally, we found that CD38 is co-expressed with CD43 in CLL and defines a CLL population with high CD45 phosphatase activity, Galectin-1 expression, and
proliferating capacity. Treatment with the pharmacological CD38 inhibitor (78c) showed reduction in the CD43/CD38 co-expressing population, as well as a decrease in proliferation, CD45 activity and Galectin-1 expression. We also found that the inhibitor exerts its effect directly on CLL cells, without affecting T helper cells. The anti-CD38 mAb (AT1) was used to evaluate the receptor function of CD38 and revealed both a reduction in the CD43/CD38 co-expressing population and CLL cell proliferative output in a concentration dependent manner.
Altogether, this data suggests a causal connection between CD38 with CD43 expression, Galectin-1 and proliferation in CLL, and thus CD38 may be a positive activator of CD45 activity by modulating its regulators CD43 and Galectin-1.
Abbreviations
ADPR Adenosine diphosphate ribose
APC Antigen presenting cell APRIL A proliferation inducing
ligand
BAFF B cell activating factor BCMA B cell maturation antigen BCR B cell Receptor
BLNK B cell linker BR3 BLyS receptor 3 Btk Bruton tyrosine kinase cADPR Cyclic adenosine
diphosphate ribose CD Cluster of differentiation CDR Complementarity-
determining region CLL Chronic lymphocytic
leukemia
CRP C-reactive protein Csk C-terminal Src kinase CTL Cytotoxic T Lymphocyte DAG Diacyl glycerol
DC Dendritic cell
Fab Fragment, antigen binding FACS Fluorescence-activated cell
sorting
Fc Fragment, crystallizable FCS Fetal calf serum
FDC follicular dendritic cell Gal-1 Galectin-1
GC Germinal center
HEV High endothelial venules M-CLL Mutated CLL
MHC Major Histocompatibility Complex
NAADP Nicotinic acid adenine dinucleotide phosphate NF-kB Nuclear factor kappa B NK cell Natural Killer Cell NLC Nurse-like cell PBMC Peripheral blood
mononuclear cell
PC Plasma cell
pCAP phosphocoumaryl amino propionic acid
PI3K Phosphoinositide 3-kinase PIP2 Phosphatidylinositol 4,5-
bisphosphate
PIP3 Phosphatidylinositol (3,4,5)- trisphosphate
PLC Phospholipase C
IGHV Immunoglobulin heavy chain variable
IP3 Inositol 1,4,5-trisphosphate ITAM Immunoreceptor tyrosine-
based activation motif RAG Recombination activating
gene
RSS Recombination signal sequence
SFK Src-family kinase
SH2 Src Homology 2
SHM Somatic hypermutation (p)Syk (phosphorylated) Spleen
tyrosine kinase
TACI Transmembrane activator and calcium modulator and cyclophilin ligand interactor
TCR T cell receptor TD T cell-dependent
Tfh Follicular helper T cells
Th Helper T cell
TI T cell-independent TNF Tumor Necrosis Factor TRAF TNF receptor-associated
factors
Treg Regulatory T cell UM-CLL Unmutated CLL
IFN Interferon
Ig Immunoglobulin
IL Interleukin
q-RT PCR Quantitative reverse transcription PCR
1 Introduction
1.1 The Vertebrate Immune System
The body defends itself against infectious disease through its immune system, which can be broken down into innate immunity and adaptive immunity. The innate immune system serves as the first line of defense against a pathogen and provides a rapid response against it. The adaptive immune system takes longer to elicit a response and this response is highly specific toward a particular portion of the pathogen, called an antigen [10]. Some infections can be dealt with by the innate immune system alone, although most require the additional defense of the adaptive immune system [11].
The cells constituting the immune system are called leukocytes, commonly known as white blood cells. These either reside in tissue or circulate through the blood stream and lymphatics.
Leukocytic interactions, either with each other or other immune molecules, enables the cells to translocate from the circulation to infected tissue where they carry out their effector functions [11]. The ability of leukocytes to readily migrate is what makes immunity systemic [10]. Leukocytes derive from pluripotent hematopoietic stem cells in the bone marrow, yolk sac, and fetal liver and are divided into two cell lineages: myeloid cells and lymphoid cells [11]. Lymphoid cells include B cells, T cells and Natural Killer (NK) cells. NK cells are involved in innate immunity, while T and B cells are part of adaptive immunity [12]. Myeloid cells primarily include phagocytes and cells of the innate immune system [13].
Epithelial barriers of the cutaneous and mucosal membranes are the initial defense against pathogens. If there is a breach of the epithelia and pathogens enter the body, an innate immune response is initiated as leukocytes are recruited to the infected area where they activate complement, eliminate pathogens, and induce an inflammatory response. The innate immune response usually occurs within minutes after infection and acts temporarily to impair the pathogen until an adaptive immune response occurs, which can take several days [11].
1.1.1 Innate immunity
The innate immune system includes the anatomic barriers of the skin and mucosal surfaces, as well as a variety of leukocytes and immune molecules. When a pathogen evades the defense
of the epithelial barrier and enters the tissue, the complement system becomes activated as plasma proteins bind to the pathogen, coating it in a process called opsonization. Opsonins and other proteins involved in complement are the effector molecules of innate immunity.
Complement activation leads to rapid recruitment of macrophages and neutrophils to the infected area, both of which are phagocytes. Phagocytes are specialized cells that ingest and kill pathogens along with damaged cells. Additionally, phagocytes secrete cytokines to recruit other immune cells to assist in combating pathogens1. This results in inflammation, which is the accumulation of immune cells and proteins, including pro-inflammatory cytokines and C- reactive protein (CRP), which is involved in complement activation [10, 14]. NK cells are among the leukocytes being recruited during the inflammatory process and kill infected cells by secreting granules that contain perforin and granzymes. Perforin mediates the entry of granzymes into the cytosol of the target cell. After entry, granzymes induce apoptosis of the infected cell through their proteolytic activity [15].
There are many types of phagocytes, some of which have central roles in innate immunity, like dendritic cells (DCs). While macrophages and neutrophils phagocytose cells to eliminate them, dendritic cells, and macrophages capture, degrade, and display peptide antigens for presentation and activation of T lymphocytes, which are cells of the adaptive immune system.
By acting as antigen-presenting cells (APC), DCs serve as a link connecting innate to adaptive immunity [16].
1.1.2 Adaptive immunity
The adaptive immune system is a specialized defense constituted of B and T lymphocytes targeting distinct antigens through antigen-specific receptors expressed on their cell surface [10, 12]. In every healthy person there is a large repertoire of lymphocytes with different antigen specificities, as antigen-specific receptors are generated randomly prior to antigen exposure. Before the lymphocytes have been activated by an antigen, they are referred to as naïve lymphocytes. During clonal selection, a cell expressing an antigen-specific receptor becomes activated upon antigen binding. An antigen activates a lymphocyte upon
presentation via an APC, such as a DC [17]. Upon activation, the cell starts to proliferate, generating many clones with the same specificity. This is called clonal expansion and is followed by differentiation. The lymphocyte differentiates to become either an effector cell
that eliminates the antigen, or a long-lived memory cell that provides immunological memory. Immunological memory enables the immune system to give a faster and stronger response if the body is re-exposed to the same antigen. While most effector cells die after the initial antigen response, memory cells live on to readily respond to reoccurring antigens [10, 11].
There are two arms of adaptive immunity: the humoral response and the cell-mediated response. The humoral arm is mediated by B lymphocytes and the antibody-secreting plasma cells2. Plasma cells are terminally differentiated effector B lymphocytes that secrete
antibodies to coat and neutralize intruding microbes, thereby eliminating the threat before it causes an infection [18]. The antibodies also serve as opsonins, activating the complement system of innate immunity [19]. If the pathogen re-enters the body at a later time, memory B cells are activated and differentiate into antibody secreting plasma cells (PC) [20].
In cell-mediated immunity, effector cells of the T cell linage either kill cells that have been infected by microbes or help other immune cells execute their immunological function. Such cells include CD8+ cytotoxic T lymphocytes (CTL) and CD4+ helper T lymphocytes (Th).
CTLs kill infected cells by the same mechanism as NK cells, secreting granule proteins that induce apoptosis. [21] Th cells recruit phagocytes through cytokine secretion and help activate B cells [22].
B and T lymphocytes differ in the structure of the antigen receptor, protein expression, and immunologic function. Additionally, their development takes place at different locations.
Development of B lymphocytes occurs in the bone marrow and T cell development in the thymus. These areas are known as primary lymphoid organs. Following maturation, naïve lymphocytes migrate to secondary lymphoid tissue to become activated [10, 23, 24] . Secondary lymphoid organs are specialized areas where adaptive immune responses are initiated and include tissues such as the lymph nodes, spleen, and Peyer’s patches [25].
Lymphocytes enter the lymph nodes through high endothelial venules (HEVs), arriving at T cell rich zones. While T lymphocytes stay located in the T cell zone awaiting activation, B cells continue migration to B cell follicles [26, 27]. Upon an immune response, germinal
centers are formed inside the B cell follicles. Germinal centers are sites of massive B cell proliferation that dissolve once the infection is cleared [28].
1.2 Cytokines
Immune cells secrete cytokines and express cytokine receptors on their membrane to
communicate with each other and regulate immune responses [29, 30]. Cytokines are divided into several families, including interleukins, interferons, tumor necrosis factors, and
chemokines. Chemokines are chemoattractant molecules that recruit other immune cells through establishment of a chemotaxis [31, 32]. They are named based on positioning of their N-terminal cysteine residues and are divided into four subgroups; C, CC, CXC, and CX3C [31].
Tumor necrosis factors (TNF) family members are pro-inflammatory and include cytokines necessary for B cell activation and survival, such as CD40L, B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) [33]. Importantly, the TNF family also include TNF-α and TNF-β, both of which are involved in inducing cytotoxic immunity provided by CD8+ T cells and NK cells.
Interleukins (IL) represent a large subset of cytokines, many of which take part in humoral immunity. IL-4 induces isotype switching into the immunoglobulin E isotype (IgE), while IL- 21 can promote B cell proliferation and plasma cell differentiation [31]. IL-7 is particularly important in T lymphocyte development, as it maintains expression of Bcl-2 and mediate progression from the double positive stage (CD8+CD4+) into the single positive stage (CD8+/CD4+) [34]3.
Interferons (IFN) are characterized by their ability to interfere with viral growth. There are only three members of the IFN family: IFN-γ, -β and -α. IFN-γ is secreted by different Th subtypes and is important in cell-mediated immunity. Additionally, IFN-γ is involved in B cell maturation and antibody production [10, 31]. IFN-γ also promotes antibody mediated complement activation by stimulating class switching to IgG subtypes [35].
1.3 Antigen Receptors
Both B cells and T cells combat infectious agents by recognizing antigens through antigen receptors. However, B cells recognize an antigen directly through its B cell receptor (BCR), while the T cell need a peptide antigen to be presented on a major histocompatibility complex (MHC) expressed on the cell surface of an APC [10, 11]. The antigen receptor of both B- and T lymphocytes shows great variability due to recombination of their antigen receptor genes [36].
1.3.1 The Antigen Receptor of B Cells
Each B cell clone expresses a unique BCR with specificity for a single antigen. These antigen receptors are also often called immunoglobulins (Ig). Immunoglobulins are either membrane bound to the B cell, mediating signaling following antigen binding, or secreted as antibodies.
The BCR molecule consist of a variable region for antigen binding and a constant region anchoring the molecule into the B cell surface membrane. For antibodies, the constant region functions to recruit cells and molecules that destroy pathogens in response to antigen binding to the variable region [11]. Immunoglobulins are divided into five classes based on the constant region structure and effector mechanism: Immunoglobulin M, D, G, A or E (IgM, IgD, IgG, IgA and IgE). The different classes of immunoglobulins are referred to as isotypes [10]. The basic structure of immunoglobulin makes a Y-shape by pairing two identical heavy polypeptide chains with two identical light polypeptide chains [11]. The light chains are either κ or λ type and the heavy chain can be any of the five types: α, δ, ε, γ or μ. The classification of the heavy chain defines the isotype of the immunoglobulin [37]. The antigen-binding portion of the antibody is called the Fab region (fragment, antigen binding) and consists of a variable portion and a constant portion. The antigen binds to specific sites at the variable domains (VL and VH) of the Fab region. A hinge between the heavy chain domains CH1 and CH2 connects the Fab region to the Fc region (fragment, crystallizable), which consists only of heavy chain constant domains. The isotypes IgA, IgD and IgG have three constant heavy chain domains (CH1-CH3), while IgM and IgE has an additional CH4 domain [10]
The variable (V) domain contains three hypervariable intervals, called complementarity- determining region (CDR) loops (CDR1-CDR3) [38]. The V domain show great variability due to recombination that occurs during lymphocyte development of the germline variable
(V), diversity (D), and joining (J) gene segments [36]. CDRs make up the antigen-binding site and possess even greater variability due to somatic hypermutation taking place in the
germinal centers during affinity maturation [39]. The VH domain is encoded by V, D and J gene segments, while the VL domain lacks a D gene segment (Figure 1) [11, 40].
Figure 1: The basic structure of an antibody molecule. A) A monomeric antibody consists of two heavy chains and two light chains. These domains are either variable (V) or constant (C). Separated by a hinge, the antibody is divided into the Fab region and Fc regions. Depending on the isotype, the Fc region consists of either two or three pairs of constant heavy chain domains (CH). The Fab region contains the antigen-binding site, which is located at the V domains. B) The variable heavy chain domain (VH) domain contains V, D, and J gene segments, while the variable light chain domain (VL) only contains V and J gene segments. Figure inspired from [11] and made using BioRender.com
1.3.1.1 BCR Signaling
When the BCR is used as a signaling molecule, it is expressed as a membrane-bound antibody molecule, that in association with the transmembrane signaling molecules Igα and Igβ, make up the BCR complex. BCR signaling is initiated when the Src-family kinases (SFK) Lyn, Fyn or Blk, in response to BCR stimulation, phosphorylate the tyrosine residues of the CD19 cytoplasmic domain and ITAM motifs located on the cytoplasmic tails of the Igα/Igβ
heterodimer [10, 11]. The activity of SFKs is positively regulated by the tyrosine phosphatase CD45, and negatively regulated by C-terminal Src kinase (Csk) (Figure 2). Csk inactivates SFKs by phosphorylating certain tyrosine residues. CD45 dephosphorylates the same residues, hence counteracting the activity of Csk and activating the SFKs [41, 42]. CD45
activity is positively regulated by binding to its endogenous ligand, Galectin-1, whose
expression can be induced by CD40L-CD40 signaling [6, 43]. Additional stimulation with Th cell signals such as the cytokine IL-4 leads to B cell activation [44].
Tyrosine phosphorylation serves as docking sites for SH2 domains. Spleen Tyrosine Kinase (Syk), an SH2-containing tyrosine kinase, is recruited to the BCR complex upon ITAM tyrosine phosphorylation (Figure 2) [10, 45]. Association with a phosphorylated ITAM leads to activation of Syk, which subsequently phosphorylates sites in the ITAM motif, leading to recruitment of supplementary Syk molecules. BCR-associated SFKs phosphorylate Syk, which leads to its complete activation. In its fully phosphorylated state, Syk remains active even when disassociated from the ITAM [46]. Syk then phosphorylates tyrosines of the scaffold protein SLP-65/BLNK [47]. Phosphorylation of CD19 leads to recruitment of PI3K and subsequent generation of PIP3. Together with SLP-65/BLNK, PIP3 recruits several SH2- containing proteins, including Bruton tyrosine kinase (Btk) and PLC-γ. Btk phosphorylates and activates PLC-γ, which hydrolyzes PIP2 into DAG and IP3. IP3 binds to its receptor on the endoplasmic reticulum to release Ca2+, ultimately increasing the cytosolic Ca2+ concentration and driving the activation of transcription factors involved in B cell activation [10, 11, 48].
BLNK also recruits and activates Ras, resulting in activation of the Ras/MAP kinase pathway [10].
Figure 2: BCR Signaling. Antigen binding to the BCR leads to phosphorylation and activation of the Igα/β chains and CD19 by SFKs such as Lyn, which itself is regulated negatively by Csk and positively by CD45.
CD45 activity is upregulated by binding to Galectin-1 (Gal-1), whose transcription is induced by CD40-CD40L signaling. Increased CD45 activity leads to increased activity of SFKs. Furthermore, Syk and PI3K are recruited to phosphorylated ITAM motifs and CD19, respectively. Downstream targets become activated, and together with Th cell signaling (CD40L and IL4), induces B cell activation. Figure inspired from [6, 10] and made in BioRender.com.
1.3.2 The Antigen Receptor of T Cells
The T cell receptor (TCR) resembles the Fab region of the B cell immunoglobulin molecule.
The TCR has a V region with homology to the Ig V domain and is linked to a C region with homology to the Ig C domain [11]. The V region possesses peptide-MHC-complex binding properties and is encoded by V, (D) and J gene segments and contains three CDR loops for variable specificity [11, 38]. A stalk segment connected to the C region anchors the TCR to the cell membrane. Two different polypeptide chains comprise the TCR, making it a
heterodimer. The two polypeptide chains are either α and β chains or γ and δ chains, of which the latter is less common. The α/γ chain corresponds to the Ig light chain and the β/δ chain to the Ig heavy chain, as they are encoded by VJ and VDJ segments, respectively (Figure 3) [11].
Figure 3: The structure of the α:β T cell receptor. A) The TCR heterodimer is comprised of a β chain and a α chain, each consisting of a variable region with an antigen-MHC-complex binding site and a constant region.
Two stalk segments connect each chain of the TCR to the cell membrane. Figure inspired from [11] and made in BioRender.com
1.3.3 V(D)J Recombination
One of the most extensive mechanisms contributing to diversity of the antigen receptor repertoire is V(D)J recombination. V(D)J recombination refers to the site-specific
rearrangement of the VDJ gene segments to generate exons encoding the variable (V) regions of the TCR and Ig receptor [36].
The V region of the Ig light chain (IgL) and the equivalent α chain of the TCR (TCRα) consists only of V and J segments [10]. Post transcription, the V-region is spliced to join the constant region (C-region), making a complete IgL or TCRα. Rearranged V, D, and J gene segments make up the V-region of the Ig heavy chain (IgH) and the TCR β chain (TCRβ). As for the light/α chain, RNA splicing joins the V-region to the C-region of IgH/TCRβ,
generating a complete chain [11, 36].
V(D)J recombination is initiated when VDJ recombinase, a complex of RAG-1 and RAG-2, cleaves a sequence of DNA called recombination signal sequence (RSS). These sequences are located 3’ of V segments, 5’ of J segments, and flank the sides of D segments [10]. RSSs
consist of a conserved heptamer (7bp) and nonamer (9bp) that are separated by a non- conserved spacer sequence of either 12 or 23 base pairs (bp) [11, 49]. V(D)J recombination follows the 12/23 rule, which states a gene flanked by an RSS containing a 12bp long spacer can only be joined to a gene flanked by an RSS with a 23bp long spacer (Figure 4) [40, 50].
VDJ recombinase makes a nick in the coding strand, which releases the 3´OH end and allows it to subsequently attack the other DNA strand. This results in a double stranded DNA break and further hairpin formation. The hairpin is opened, and the ends are joined by non-
homologous end joining [36, 49]. Random N-nucleotides and non-random P nucleotides may be added during the joining of gene segments, giving rise to new sequences. VDJ
recombination causes junctional diversity, resulting in antigen receptors with greater variability in their CDR loops [10].
Figure 4: VDJ recombination. An RSS consists of a conserved heptamer sequence, a spacer of either 12 or 23 bp length, and a conserved nonamer sequence. The 12/23 rule prohibits gene segments with RSSs of equal lengths to be joined. Figure inspired from [11] and made in BioRender.com
1.4 T Lymphocyte Development
T cells consist of two main types: CD4+ Th cells or CD8+ cytotoxic T cells. T cell signaling and activation is facilitated by the TCR complex located at the cell membrane. The TCR recognizes linear peptide antigens presented by the MHC expressed on the surface of other cells [51]. CD3 and ζ chains, are mediators of signal transduction, and along with the TCR, constitute the TCR complex [10]. In addition to the TCR complex, T cell activation depends on MHC binding to either one of the co-receptors CD4 or CD8. MHC class I (MHC I) molecules are expressed on all nucleated cells and presents antigens to CD8+ CTLs. MHC class II (MHC II) molecules are expressed only by APCs and display antigens to CD4+ Th cells [51]. APCs include cells such as DCs, B cells, and macrophages [52].
1.4.1 T Cell Maturation
Hematopoietic stem cells expressing the transcription factor GATA-3 and Notch-1 commit to the T cell linage. Presence of these transcription factors leads to expression of genes
comprising the pre-T cell receptor (pre-TCR), as well as RAG1 and RAG2, which are genes enabling V(D)J recombination. Common lymphoid progenitors that commit to the T cell linage migrate to the thymus as pro-T cells. Here, development proceeds, starting with V(D)J recombination of the TCR β-chain genes. A successful rearrangement leads to a functional β chain. This associates with an invariant α chain called pre-T-α to form a temporary αβ heterodimer that make up the pre-TCR complex. This initiates the dual expression of CD4 and CD8, neither of which has been expressed prior to this stage. The double positive state (CD8+CD4+) of the T cells enables V(D)J recombination of the α-chain genes. The resulting rearranged α chain substitutes the invariant alpha chain [10, 53].
Following this, positive selection of T cells with TCRs possessing low affinity towards MHC antigen complexes occurs, resulting in loss of either CD4 or CD8 expression. Whether the cell becomes singularly CD4 or CD8 positive depends on if the TCR recognizes MHC I or MHC II [24, 53]. An additional step of negative selection eliminates self-reactive T cells by
apoptosis [54]. Such autoreactive T cells recognize self-antigens, i.e. antigens derived from within the individuals own body. Deletion of these cells is necessary to maintain central tolerance, a feature that together with peripheral tolerance prevents autoimmunity, which is when the immune system attacks its own tissue and cells [10, 55]. Central tolerance concerns
only immature cells, while peripheral tolerance is directed towards mature cells [55].
Negative selection of self-reactive T cells occurs both at the double-positive and single- positive stage of maturation [56]. Some self-reactive CD4+ Th cells evade apoptosis and develop into regulatory T cells (Tregs) instead. Tregs negatively control responses of other immune cells, providing peripheral tolerance of autoreactive mature T cells that have evaded the mechanisms of central tolerance [55, 57]. Neither central nor peripheral tolerance is perfect and autoimmune diseases can develop if these tolerance mechanisms are not maintained [58].
1.4.2 T Cell Activation and Differentiation
Ultimately, cells surviving selection are referred to as naïve T cells until they recognize and are activated by an antigen [24]. Naïve T cells leave the thymus to circulate through the periphery and migrate to secondary lymphoid organs where they interact with APCs until they encounter the antigen their TCR has specificity for [59]. The encounter activates, or primes, the T cell, thus initiating differentiation of CD8+ cells into CTLs and CD4+ cells to Th cells [24, 60]. In addition to antigen recognition, cell activation requires costimulatory signals.
CD28 on the T cell associates with its ligands, CD80 (B7-1) and CD86 (B7-2), and
glycoproteins expressed on the surface of the APC. Ligand binding leads to activation of the PI3K signaling pathway and consequently expression of the antiapoptotic proteins Bcl-2 and Bcl-X, along with other molecules promoting cell survival, proliferation, and differentiation [10, 61]. A positive feedback mechanism is generated when an antigen is recognized by the T cell, as it induces expression of CD40 ligand (CD40L) on the T cell surface. CD40L binds to CD40 on APCs, causing expression of CD80/CD86, which bind to CD28 on T cells. This costimulatory signal is needed to license APCs, meaning they become more potent activators of T cells and immune responses. Thus, CD28 binding leads to enhanced T cell activation [10, 62]. When they become activated, T cells produce IL-2, a cytokine and autocrine growth factor. Secreted IL-2 bind to its receptor, IL-2R, on the T cell surface, stimulating
differentiation and clonal expansion of itself [59, 63].
CD8+ and CD4+ T cells can become either effector or memory cells, depending on what transcription factors are expressed. Memory cell differentiation is promoted by the
transcription factor Blimp-1, whereas effector cell differentiation is driven by T-bet [64, 65].
Effector CD4+ Th cells can differentiate into one of several subsets, of which the best
characterized are Th1, Th2, and Th17 cells [66]. By secreting IFN-γ, CD4+ Th1 cells activate macrophages to kill microbes phagocytosed by other cells. Th2 cells protect against
extracellular parasites by secreting IL-4 and IL-5 that induce B cell isotype switching into IgE. Secreted IgE recruits and stimulates eosinophils and mast cells to eliminate parasites [66]. CD4+ Th17 cells initiate inflammatory responses through IL-17 secretion, a cytokine that recruits neutrophils to infected areas to kill bacteria and fungi [35]. An additional subtype of CD4+ cells, T follicular helper (Tfh) cells, are involved in B cell differentiation as they help mediate the germinal center reaction. Tfh cells secrete IL-21, which can act as both a
proliferative and apoptotic signal [66, 67].
CD8+ CTLs attach to target cells when they recognize the antigenic peptide presented on the targets MHC I. Antigen recognition leads to synapse formation. Granule content is released into the synapse and is endocytosed into the cytoplasm of the target cell [68, 69]. Once inside, granzymes activate caspases that initiate apoptosis [10]. The CTL then releases itself from the target. Additionally, CTLs secrete IFN-γ to participate in microbe defense [69].
1.5 B Lymphocyte Development
1.5.1 B Cell Maturation
Most B lymphocyte development occur in the bone marrow but can also take place in the fetal liver [70]. Expression of the transcription factors EBF, E2A, and Pax-5 induce expression of BCR genes, and thus commit lymphocyte precursors to the B cell linage. Like T cells, linage- specific transcription factors also induce expression of RAG-1 and RAG-2. At the first stage of development pro-B cells undergo somatic rearrangements of their immunoglobulin genes at the heavy chain locus, bringing the D and J gene segments together (Figure 4). A
productive μ heavy chain rearrangement allows for further differentiation into the pre-B cell stage[71].The cell expresses the resulting μ heavy chain on its surface accompanied by invariant surrogate λ5 light chains and the signaling molecules Igα and Igβ, making the pre-B cell receptor (pre-BCR) [71, 72]. Signaling through the pre-BCR leads to proliferation and enables rearrangement of the light chain as well as restricting further rearrangement of the heavy chain. Allelic exclusion is the process that silences the heavy chain allele that has not been productively rearranged, ensuring only one variation of μ heavy chain is expressed [73].
The light chain recombination involves joining of the V and J segment, as there is no D segment in the light chain locus [10]. Once an in-frame rearrangement has occurred at the light chain locus, the resulting κ light chain protein can associate with the heavy chain and generate a complete IgM protein [11, 72]. Expression of IgM mark the transition into the immature B cell stage. At this point, RAG genes are shut off by PI3K signaling through the BCR, preventing further Ig rearrangement [74, 75]. As for T cells, B cells with autoreactive BCRs should be eliminated to maintain central tolerance [55]. Immature B cells that are autoreactive will undergo receptor editing by reactivating RAG genes, negatively selected and undergo apoptosis or can be tolerated by anergy. Cells surviving the selection leave the bone marrow to complete their maturation in the B cell follicles of the spleen [11, 76]. Mature B cells from the bone marrow, called B-2 cells, develop to become either marginal zone B cells or follicular B cells. Cells that develop from the fetal liver are called B-1 cells. Most B cells are follicular B cells and is this type of B cell that show greatest diversity [10, 77]. Mature B cells produce IgD that is co-expressed with IgM on the membrane [78]. At this point, the survival of the cell depends on stimulation with the cytokines BAFF and APRIL, secreted from cells in the microenvironment. These survival signals lead to differentiation into the naïve B cell stage [79, 80] .
1.5.2 B Cell Activation and Differentiation
Naïve B cells are activated when an antigen associates with the BCR specific for that antigen, initiating downstream signaling. B cells can be activated by both T cell-independent (TI) and T cell-dependent (TD) antigens and differentiate into either antibody-secreting plasma cells or memory B cells. TD antigens must be accompanied by a second signal provided by Th cells to induce activation of the B cell. TI antigens are on the other hand sufficient for B cell
activation [81]. While central tolerance of B cells is regulated by receptor editing mechanisms and apoptosis, peripheral tolerance is mediated by Th cells, and is restricted to TD antigens, as lack of Th stimulation will lead to cell death by apoptosis [82].
To receive Th stimulation, B cells internalize TD antigens recognized by the BCR. The antigen in processed into a peptide antigen that is presented by the MHC II on the B cell surface (Figure 5). The MHC II-peptide complex acts as a ligand for the TCR and its CD4 co- receptor [44]. Recognition of the antigen by the TCR leads to the Th providing stimulatory signals to the B cell resulting in B cell activation. Such stimulation includes expression of
CD40L which binds to CD40 on the B cell surface, and secretion of cytokines including IL-4 and IL-21[11, 83]. IL-21 and IL-4 both play important roles in B cell activation. IL-21 activates the pro-survival, anti-apoptotic transcription factor STAT3 and IL-4 is associated with isotype switching to IgE [84, 85]. Upon activation of a B cell with a T cell-dependent antigen, binding of CD40L to CD40 activates the alternative NF-kB signaling pathway. In the alternative NF-kB pathway, NFkB2/p100, a subunit of nuclear factor kappa B (NF-kB), is phosphorylated by IKKa, a subunit of IKK. Phosphorylation leads to transition of
NFkB2/p100 into its active form, p52 [11, 86]. Active p52 is accompanied by RelB and translocate into the nucleus, where they target specific genes, leading to expression of the anti-apoptotic molecule Bcl-2 [11, 87].
Figure 5: Th dependent B cell activation. The antigen recognized by the BCR is internalized and processed into a peptide antigen displayed by MHC II which is recognized by the TCR and CD4. Antigen recognition by the TCR leads to expression of CD40L which binds to CD40 on the B cell, and secretion of cytokines (IL-4 and IL-21) which causes B cell activation through activation of the alternative NF-kB signaling pathway. Figure inspired from [11] and made in BioRender.com
Proliferative signals from Th cells leads to B cells differentiating into either plasmablasts, which are plasma cell precursors, and short-lived plasma cells, or memory B cells and high- affinity plasma cells. The latter two are the end-products of the germinal center reaction [88].
Germinal centers (GC) are specialized microenvironments in the secondary lymphoid follicles generated in response to an antigen, that serves as the major source of B cell proliferation and
differentiation [11, 89]. The germinal center reaction is a slow process of fine-tuning the specificity of immunoglobulins to provide the most optimal response toward the antigen [88].
In the meanwhile, initial antibody protection is provided by plasmablasts and short-lived plasma cells that produce immunoglobulins rapidly over a short period of time [18].
Short-lived plasma cells and plasmablast are generated both through activation by TI and TD antigens, while GC B cells (long-lived PCs and memory B cells) only are generated through activation by TD antigens [90, 91]. Long-lived PCs differ from memory B cells by secreting antibodies and by not having a proliferative potential, as they are terminally differentiated.
Memory B cells are in a resting state while they circulate throughout the body, awaiting antigen reencounter. When reactivated, memory B cells undergo clonal expansion. The clones then either differentiate into antibody-secreting plasma cells or face a new germinal center reaction [92].
1.5.2.1 The Germinal Center Reaction
B cells activated by TD antigens form a germinal center by presenting antigens that are recognized by Th cells and receive co-stimulatory signals from these cells [93]. Once the B cells have formed a germinal center, they undergo somatic hypermutations (SHM) and proliferation in the dark zone (Figure 6). SHM alters the variable region of immunoglobulin genes and hence the antigen specificity to generate B cells with BCRs that have higher affinity for antigen [93]. After SHM in the dark zone, B cells enter the light zone where they undergo affinity selection, competing for stimulation from follicular dendritic cells (FDC) and follicular Th cells (Tfh) [94]. B cells with low-affinity BCRs do not receive survival signals from follicular dendritic cells and therefore apoptose. Higher affinity B cells remain to compete for T cell help. The underlying principle of affinity selection is B cells expressing a BCR with high affinity for an antigen will take up and present more of that antigen to the Th cell on MHC II than a B cell with low BCR affinity. Thus, the higher affinity B cell will receive stronger Tfh cell help. As for FDC stimulation, B cells that do not receive Tfh cell survival signals will apoptose [95]. The B cells with highest affinity receive the most T cell help and are thought to exit the GC as long-lived plasma cells, while those who receive intermediate T cell help leave the GC as memory cells [96-98]. B cells that receive too little T cell help to leave the GC will re-enter the germinal center reaction for further affinity
maturation, making the GC reaction a cyclic process [89, 93]. B cells exiting the germinal
center first undergo class switch recombination of their immunoglobulin genes in the light zone, where a constant heavy chain region of the immunoglobulin is exchanged to that of another isotype. Class switching leads to altered effector functions whilst the antigen specificity remains the same as after SHM [11, 27].
Figure 6:The germinal center reaction. B cells activated by Th-cell dependent antigens can become short- lived antibody secreting plasmablasts (PB) and plasma cells (PC (SL)) or enter the dark zone of the germinal center where they undergo proliferation and somatic hypermutation (SHM) of the BCR genes to generate clones with diverse immunoglobulins. Following proliferation and SHM, the B cell enters the light zone where it undergoes affinity selection. B cells with high affinity receive stimulatory signals from follicular dendritic cells (FDC) and proceed to compete for selection by follicular Th cells (Tfh), while B cells with low affinity undergo apoptosis. Tfh cell signals in response to higher affinity BCRs enables B cells to leave the GC as either long- lived plasma cells (PC (LL)) or memory B cells (MBC). Alternatively, the B cell can undergo further affinity maturation by re-entering the dark zone. Lack of Tfh cell signals will lead to apoptosis. Figure inspired from [93, 95] and made in BioRender.com.
Even though the GC reaction is essential for establishing an effective immune response towards an antigen, genetic instability affecting the GC reaction can have unfortunate outcomes. The GC is a source of mutations mainly directed towards genes of the BCR.
However, other genes are also expressed during the GC reaction, and will occasionally be
subjected to mutations. This has been suggested as a mechanism underlying development of the B cell malignancy chronic lymphocytic leukemia (CLL) [88].
1.6 Chronic Lymphocytic Leukemia
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in western
countries and is characterized by accumulation of malignant CD5+CD19+ mature-like B cells in the lymphatic system and peripheral blood [99]. CLL cells are mainly arrested in the G0/G1 phase of the cell cycle, and therefore have weak proliferation [100]. Additionally, stimulatory signals from the microenvironment hinder the cells from undergoing apoptosis [101]. With a median diagnostic age of 72 years, this B cell malignancy most commonly occurs in elderly patients and more often in males than females [99]. The 5-year relative survival rate of patients diagnosed with CLL in the US is estimated to be 86.9% [102]. The clinical course of CLL is heterogeneous, dividing patients into three subgroups of prognostic outcomes according to the Rai staging system. These are either low, intermediate, or high-risk disease. Whereas the latter denotes a CLL clinical course that progresses rapidly, patients with low-risk disease are without need of treatment. Low-risk disease refers to patients with lymphocytosis of CLL cells in the blood and bone marrow [99]. Lymphocytosis is clonal expansion of lymphocytic cells leading to an increase in the lymphocyte count above normal levels (>4000 lymphocytes/µL) [103]. Patients with intermediate risk disease have enlarged lymph nodes in addition to lymphocytosis. High-risk disease is characterized by anemia, which is low hemoglobin levels (<11 g/dL) or thrombocytopenia, which is low platelet counts (<100 × 109/L). The Rai system of classification is one of two accepted clinical staging
systems. The other system is the Binet staging system and defines disease stages by number of enlarged lymph nodes (>1cm). In addition to these staging systems, various genetic and chromosomal aberrations can be used to predict prognostic outcome. Such prognostic factors include mutation or deletion of TP53, which arises from deletions in the short arm of
chromosome 17 [99]. TP53 is the gene encoding the tumor suppressor protein and cell cycle regulator p53 and is often seen inactivated in cancer cells to evade apoptosis [104]. Other negative prognostic markers include high expression of CD38 and the downstream signaling molecule ZAP-70. Additionally, immunoglobulin heavy chain variable (IGHV) mutations are used to predict prognostic outcome in CLL [99, 105].
As mentioned above, CLL is often dived into subgroups based on somatic hypermutations in the IGHV region of the BCR. Low levels (<2%) of BCR IGHV mutations defines unmutated CLL (UM-CLL), a subgroup characterized by an aggressive disease state and a more
inducible level of BCR signaling. Patients with higher levels of BCR IGHV mutations (>2%) is defined as mutated CLL (M-CLL) and this subgroup is associated with a better prognostic outcome [106, 107]. Both UM-CLL and M-CLL are suggested to emerge from B cells that initially have autoreactive BCRs [108]. However, M-CLL is proposed to arise from B cells that have seeded the GC reaction, mutated the specificity of their BCR away from
autoreactivity, and exited the GC as memory B cells. As a counterpart, UM-CLL is thought to derive from naïve B cells which have not undergone a GC reaction. Thus, UM-CLL has a higher autoreactive potential and is more prone to activation than M-CLL [109].
1.6.1 Therapeutic Options
Generally, only patients with symptomatic disease require therapy. CLL related symptoms include weight loss, fatigue, fevers, and night sweats [99]. A variety of treatments are available for improving CLL patient outcome and alleviating symptoms, but there are currently no curative therapeutic options for CLL. Treatments include monotherapeutic options or combinational therapy using antibodies, inhibitors, and chemotherapy [110].
Some available treatments target CD20, a surface marker for pre-B cells and mature B cells.
Young (<65 years) and fit CLL patients are often treated with the medication rituximab, which is an anti-CD20 monoclonal antibody therapy [111]. Upon binding to CD20, rituximab induces cytotoxic responses towards both malignant and healthy B cells [112]. Other common treatments for CLL target BCR signaling and include inhibitors of PI3K (Idealisib, Duvelisib), Btk (Ibrutinib), and Bcl-2 (Venetoclax) [111].
Although it is not currently an available option, patients with high expression of the negative CLL prognostic marker CD38, in theory could be treated with anti-CD38 monoclonal antibodies such as Daratumumab or enzymatic inhibitors [1, 3]. Daratumumab induces complement dependent cytotoxicity in cells expressing CD38 and is an approved drug for treating multiple myeloma, a malignancy affecting plasma cells [113]. Currently, Daratumumab is in clinical trials to be approved for use in CLL [114]. The drug has the potential to be an effective therapeutic option for targeting severe disease upon a better
understanding of the role of CD38 in CLL progression, as it remains unclear why CD38 correlates to poor prognosis. Additionally, there are concerns regarding the safety of direct targeting of CD38, as it is expressed in various cell types and differentiation stages, including activated T cells [1].
1.6.2 The Negative Prognostic Marker CD38
CD38 is both a cell surface receptor and an ectoenzyme [1]. A high percentage (>20%) of CLL cells expressing surface CD38 is linked to a more aggressive clinical course and shorter life expectancy [115]. The CD38 ectoenzyme generates three 2nd messengers that impart biological function through changes in cytosolic Ca2+, these are: adenosine diphosphate ribose (ADPR), cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine
dinucleotide phosphate (NAADP). By hydrolysing NAD+, CD38 generates ADPR and, to a lesser extent, cADPR [116]. NAADP is synthesized by base-exchange reaction under acid conditions in the presence of nicotinic acid [117]. As a receptor, CD38 binds its ligand CD31, which initiates lipid raft movement, NF-κB signaling activation, and improves CLL cell survival [118, 119]. Additionally, CD31-CD38 ligation has been found to increase surface expression of CD38 through activation of Syk in CLL [120]. It is still not fully understood why CD38+ CLL disease correlates with worse prognosis, but it has been suggested that CD38 promotes CLL proliferation by being indirectly connected to BCR signaling [1]. CD38 has been found to co-localize with the BCR complex on lipid rafts and to be associated with CD19 in CLL cells, which could indicate involvement in BCR signaling [1, 119].
Supplementing this hypothesis, Th cell signals have been found to upregulate CD38
expression in CLL [9]. Thus, CD38 might not only be a negative prognostic marker in CLL, but could influence CLL function (e.g. proliferation and survival) through modulation of BCR signaling.
1.6.3 The CLL Microenvironment
CLL progression is strongly dependent on microenvironmental signaling as CLL cells spontaneously apoptose when cultured alone [121]. CLL cells recirculate between the lymphoid organs and peripheral blood and migrate from the blood into lymph nodes for clonal expansion [99, 106]. They are recruited to these proliferation sites by chemokines secreted by nurse-like cells (NLCs), which are CLL associated macrophages and stromal cells [105]. By
secreting chemokines, cytokines, and angiogenic factors that interact with surface receptors and adhesion molecules on the CLL cells, the surrounding microenvironment promotes CLL pathogenesis [99, 106]. T cells and NLCs play key roles in the microenvironment supporting CLL progression.
Stromal CD4+ Th-cells are important mediators in driving clonal expansion of CLL cells, as they express CD40L that binds to the CD40 receptor on the CLL cell surface. The CD40 receptor trimerizes upon binding to CD40L, recruiting tumor necrosis receptor associated factors (TRAFs) to the cytoplasmic domain of the receptor. Ligand binding leads to activation of signaling pathways involved in CLL proliferation, including the NF-kB signaling pathway [106]. Upon CD40 stimulation, malignant B cells recruit more CD4+ Th cells by secreting the T cell attracting chemokine CCL22, which binds to CCR4 receptors on T cells. This feedback mechanism enables constitutive active signaling of CLL cells [122]. Th cells also express CD31, the ligand for CD38, on their cell surface, which further promotes CLL survival and additionally is expressed by CLL cells themselves [123-125]. Also, T cells secrete the cytokines IL-4 and IL-21, as well as the chemokine stromal cell derived factor 1 (SDF- 1/CXCL12). All of these are involved in CLL activation and proliferation [126, 127].
Importantly, CXCL12/SDF-1 efficiently recruits CLL cells to proliferative sites in lymphoid organs by binding to the CXCR4 receptor expressed on CLL cells [105]. When bound to CXCR4, SDF-1 enables phosphorylation and activation of downstream targets in the MAPK/ERK signaling pathway [128]. Importantly, CLL cells have shown to be effective APCs and proliferate in response to Th cell signals [5]. As in normal B cells, IL-4 and IL-21 have been found to induce CLL proliferation in combination with CD40L. The IL-21 receptor on the CLL cell surface is upregulated upon CD40 stimulation, enabling greater IL-21
sensitivity. Increasing IL-21 sensitivity, when paired with IL-4 stimulation, produced increased CD40L mediated proliferation of CLL cells [129]. Lastly, Th cell secreted IFNγ promotes CD38 expression in CLL, which consequently could lead to enhanced proliferation upon crosslinking to CD31 [9].
By expressing a variety of proteins, NLCs serve as important constituents of the
microenvironment driving CLL survival [130]. Like T cells, NLCs express CD31 and thus could feasibly provide ligation for CD38 on CLL cells [123, 131]. NLCs secrete soluble CD14 that activates the canonical NF-kB pathway [132]. Active NF-kB heterodimers
translocate into the nucleus where they can alter gene expression. Additionally, NLCs secrete
chemokines such as SDF-1/CXCL12 and express BAFF and APRIL [123]. Together, BAFF and APRIL trigger the canonical NF-kB pathway, whereas BAFF in the absence of APRIL stimulates the alternative NF-kB pathway and cooperates with CD40L to promote CLL survival. BAFF and APRIL bind and signal through the TNF superfamily of receptors: B cell maturation antigen (BCMA), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and BLyS receptor 3 (BR3) (Figure 7) [11, 133, 134]. Furthermore, NLCs secrete Galectin-1, which is a modulator of both BAFF/APRIL expression and the BCR signaling pathway through CD45 [135, 136]
Figure 7: CLL cell interacting with cells of the microenvironment. CLL cells interact with NLCs and CD4+ Th cells. SDF-1/CXCL12 is secreted by both NLCs and Th cells and recruits CLL cells to proliferating sites.
TCR recognition of the antigen-MHC II complex expressed on the CLL cell activates the Th cell. In turn, the Th cell stimulates the CLL cell by secreting IL-4 and IL-21, as well as expressing cell surface CD31 and CD40L.
CD31 binds to the dimeric receptor CD38 and CD40L to trimeric CD40 on the CLL cell surface. These signals accompany antigen recognition by the BCR to promote survival and proliferation. NLCs express CD31, APRIL, and BAFF. These bind to the appropriate receptors on the CLL cell surface, providing the cell with survival
1.6.4 BCR signaling in CLL
The entirety of CLL pathogenesis is yet to be described, but it is known that the
microenvironment, such as Th cells and triggering of BCR signaling plays a fundamental role [137]. Th cell signals enhance BCR signaling have been found to drive CLL survival and proliferation [5, 6, 42]. CD38, whose high expression indicates poor prognosis in CLL, has been found to be upregulated in CLL in response to stimulation by Th cells [9]. Additionally, indications suggest that CD43 and Galectin-1 modulate the phosphatase activity of CD45, which is a known regulator of BCR signaling (Imbery et al., under revision, British J Haematol 2022) [6].
Our group has previously shown that in healthy B cells, Galectin-1 is transcribed in response to CD40L binding to CD40 and once expressed, associates with CD45 to enhance its
phosphatase activity [6]. Galectin-1 is known to have several binding partners other than CD45 [138]. CD43, a possible binding partner of Galectin-1, is typically not expressed in normal mature B cells, but is expressed in most cases of CLL [138, 139]. Our group has found that CD43 is upregulated in CLL in response to Th cell signals (Imbery et al., under revision, British J Haematol 2022). Interestingly, Galectin-1 has been found to co-cluster CD43 and CD45 in dendritic cells, leading to Syk-mediated signaling and cell activation [8].
Unpublished observations from our group show co-localization of CD43 and CD45 in
response to B cell activation, suggesting a connection between the two glycoproteins (Imbery et al., under revision, British J Haematol 2022).
We hypothesize that CD43 regulates CD45 activity, and that Galectin-1 mediates this regulation by bridging between the two glycoproteins, allowing them to interact with each other and in this way enhance CD45 activity. Additionally, we hypothesize that the negative prognostic marker CD38, whose expression is similarly upregulated upon T cell help and may contribute to BCR signaling, positively modulates CD45 activity by regulating Galectin-1 and CD43 expression. Altogether we believe that this interplay is important for mediating CLL BCR signaling and proliferation (Figure 8).
Figure 8: Hypothesized BCR Signaling in CLL. Antigen binding to the BCR leads to phosphorylation and activation of the Igα/β chains and CD19 by SFKs, which are positively regulated by CD45. Transcription of Galectin-1 (Gal-1) is induced by CD40-CD40L and CD38-CD31 signaling. CD38-CD31 ligation additionally leads to CD43 transcription. Galectin-1 bridges CD43 and CD45, allowing for positive regulation of CD45 activity. Increased CD45 activity leads to increased activity of SFKs. Subsequently, Syk and PI3K are recruited to phosphorylated ITAM motifs and CD19, respectively. Downstream targets become activated, and together with Th cell signaling (CD40L and IL4), induces CLL cell activation. Figure inspired from [6, 10] and made in BioRender.com.
2 Thesis Aims
This master thesis aims to further describe the mechanisms driving CLL survival and
proliferation. Specifically, the aim is to determine a potential connection between CD38 and CD45 phosphatase activity regulation in CLL. We hypothesize that CD38 modulates the phosphatase activity of CD45 through regulating expression of the CD45 regulators CD43 and Galectin-1. Achieving this aim can ultimately contribute to development of personalized therapeutic options for patients suffering from CLL with high CD38 expression.
To conduct the thesis aim, we will test the CD38 enzymatic inhibitor 78c and monoclonal anti-CD38 blocking antibody AT1 for an antiproliferative effect in CLL cells and investigate the impact these pharmaceuticals have on CD45 activity and the expression of its regulators, CD43 and Galectin-1.
3 Materials and methods
3.1 Patient Material
The Regional Ethics Committee for Medical and Health Research Ethics approved the project (ethical approval number 2016-947, 2016-1466). Blood samples from patients diagnosed with CLL (Table 1) were obtained after informed consent at the hematological outpatient clinic at The Oslo University Hospital, Rikshospitalet and Ullevål, Norway. Cryopreserved blood samples from CLL patients were used. Patient information provided in Table 1.
Table 1. Patient Characteristics
Patient Identifier
Gender/age (y)
IGHV CD38 (%)
CLL 106 F/56 M 1
CLL 116 M/83 M 2%
CLL 142 F/37 M 2.5
CLL 143 M/94 M <1
CLL 145 M/53 M 1
CLL 149 M/48 UM 1.7
CLL 153 M/48 UM 49
CLL 156 M/44 UM 43
CLL 179 M/45 M 22
CLL 185 F/53 M <1
CLL 195 F/79 M NA
CLL 207 M/61 M 40
CLL 222 F/43 M NA
CLL 223 F/53 M 3
CLL239 M/64 M 1
Age: Age at inclusion
IGHV: mutational status of immunoglobulin heavy chain variable. M=mutated UM=unmutated
CD38: Percentage of CD38+ CLL cells compared to isotype control NA: Not available
3.2 PBMC isolation
Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation of patient derived blood samples (LymphoprepTM, Alere Technologies) after being diluted 1:1 in PBS. The resultant PBMC containing layer was harvested, washed three times in PBS, and depleted of red blood cells by treatment with an ammonium chloride potassium lysing buffer.
The isolated PBMCs were put directly in growth media RPMI-1640 (Gibco, ThermoFischer Scientific) supplemented with 10% fetal bovine serum (Gibco, ThermoFischer Scientific), 1 mM sodium pyruvate, 1X non-essential amino acids, 50 nM 1-thioglycerol (an antioxidant), and 12 µg/mL gensumycin (an abiotic). Cultured PBMCs were bio-banked and
cryopreserved for later use. Following thawing, PBMCs were put directly in growth media.
3.3 Cell Counting
Brightfield counts were performed to find the number of live cells in a cell suspension using the Invitrogen Countess 3 Automated Cell Counter (Thermo Fischer Scientific) in accordance with the manufacturers protocol. Pre-diluted cell samples were diluted in 1:1 Trypan blue and applied to a chamber slide of which was inserted into the cell counter.
3.4 CD8+ cytotoxic T cell depletion and activation of CLL-derived PBMCs
PBMCs were depleted of CD8+ cytotoxic T lymphocytes using CD8+ Dynabeads (Invitrogen).
Two separate conditions were set for the CD8-depeleted lymphocytes. CD3/CD28 activating beads (Gibco, ThermoFischer Scientific) (6µL per 106 cells) and 20U interleukin-2 (Sigma- Aldrich) were added to PBMCs to create a «stimulated» condition, whereas cells that did not receive activating beads or IL2 are referred to as «unstimulated». Alternatively, cells were stimulated with human recombinant IFN-γ (ImmunoTools) (0.5µg/mL) instead of IL2 and CD3/CD28 activating beads for confocal microscopy4 and certain flow cytometry
experiments5. Isolated6 or co-cultured CLL cells were plated into 96-well plates (volume 200 µL) and 24-well plates (1 mL), respectively. The plates were incubated at 37oC for 72-96 hours before addition of the indicated concentrations of pharmacological inhibitor, antibody,
4 Confocal microscopy explained in section 3.11
5 Experiment referred to explained in section 3.8.2 Anti-CD38 mAb AT1
or its respective control7. For q-RT PCR experiments, positive isolation of CD38+ CLL cells occurred prior to pharmacological treatment8.
3.5 Positive CLL cell isolation
Magnetic cell separation was performed to positively select CD19+ CLL-cells using CD19 antibody conjugated magnetic microbeads (Miltenyi Biotec). The procedure was performed in accordance with the manufacturer’s protocol. Briefly, CLL cells were first magnetically labeled with the CD19 microbeads and the entire PBMC suspension was passed through a column attached to a manual MACS separator (Figure 9). Unlabeled cells flowed through, while CD19+ CLL cells were retained by the magnet. CD19+ CLL cells were released from the magnet, resulting in a cell suspension containing purified CD19+ CLL cells.
Figure 9: Positive CD19+ cell isolation. A) CD19+ cells (red, circular) labeled with CD19 magnetic microbeads (red, rectangular) in culture with other non-labeled PBMCs (green). B) When passed through a MACS separator, CD19+ labeled cells attach to the magnet while non-labeled PBMCS flow through the column.
C) CD19+ cells were released from the magnet, which resulted in a pure CLL culture. Figure made in BioRender.com.
3.6 Negative CLL cell isolation
Magnetic cell separation was performed to negatively select CLL cells using the B-CLL cell isolation kit (Miltenyi Biotec). The procedure was performed in accordance with the
7 Pharmacology explained in section 3.8