The effects of vitamin A in B cells from patients with the immunodeficiency disorder CVID

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The effects of vitamin A in B cells from patients with the immunodeficiency disorder

CVID

Master thesis by Ida Guttormsen

Department of Nutrition

Institute of Basic Medical Sciences Faculty of Medicine

UNIVERSITY OF OSLO

May 2018

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The effects of vitamin A in B cells: Importance for patients with the immunodeficiency disorder CVID

Ida Guttormsen

http://www.duo.uio.no/

Trykk: Reprosentralen, Universitetet i Oslo

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Acknowledgements

The work presented in this master thesis has been performed at the Department of

Biochemistry, Institute of Basic Medical Sciences at the University of Oslo from August 2017 to May 2018, in the laboratory of Professor Heidi Kiil Blomhoff.

First of all, I would like to express my gratitude to my supervisor Heidi Kiil Blomhoff for making me a part of your lab group. Thank you for introducing me to the field of vitamin A, the immune system and CVID, and for your guidance, advice, encouragement and enthusiasm throughout this period. Thank you for always being available to answer any questions. Also thanks to my co-supervisor, Rune Blomhoff, for your contribution to this master thesis.

Next, I would like to thank Kristine Lillebø Holm for a thorough introduction to the lab methods and good advice on both lab analyses and the written thesis. Thank you for being available even on maternity leave. Also thanks to Karin M. Gilljam, my office partner, for helping me throughout the whole year answering questions about lab methods, statistics and the project in general. I would also like to thank Siv Kjølsrud Bøhn for helping me with the statistics and Anne Randi Enget for collecting blood samples from the controls.

I am also grateful to the rest of Heidi’s lab group: Nina Richartz, Katrine Heggeset, Sampada Bhagwat and Eva Duthil, for welcoming me to your group and creating a friendly and

inspiring environment throughout my time here.

I would also like to thank Børre Fevang at Section of Clinical Immunology and Infection Medicine at Oslo University Hospital, Rikshospitalet, for outstanding collaboration on the CVID patients. Your effort in collecting blood samples from the patients made it possible for me to finish this master thesis on time.

Finally, I would like to give a special thanks to my parents, Ove and Gro Guttormsen, and my fiancé, Lars-Einar Haraldsen Osland, for love and support during this year.

Oslo, May 2018

Ida Guttormsen

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Summary

Introduction and aims: Common variable immunodeficiency (CVID) is a heterogenous primary immune deficiency disorder characterized by recurrent infections, defective B cell differentiation and decreased levels of switched immunoglobulins (Igs) like IgG and IgA.

Many CVID patients have vitamin A deficiency, which may contribute to their high infection risk. B cells from CVID patients also frequently present with defective toll like receptor (TLR) 9 signaling. TLR9 is part of the innate immune system, and is activated by microbial DNA, which is rich in unmethylated CpGs. In vitro, CpG oligodeoxynucleotides (CpG-ODN) are used to stimulate the receptor. RP105 also belongs to the TLR receptor family and

synergizes with TLR9-signaling. Our lab has previously demonstrated that the vitamin A metabolite retinoic acid (RA) is able to enhance the immunostimulatory effects of TLR9- and RP105-signaling, both in normal and CVID-derived B cells. They have also identified a subgroup of CVID patients with TLR9/RP105-stimulated B cells expressing high levels of IRF4 and low levels of AICDA combined with a poor response to RA in terms of enhanced expression of these plasma cell related genes.

The aims of this master thesis were 1. To confirm the existence of the CVID-subpopulation with IRF4^highAICDA^low-expressing B cells, and 2. To assess a possible correlation between the subgroup of CVID patients characterized by IRF4^highAICDA^low-expressing B cells, and the patients with B cells that respond poorly to RA in terms of enhanced TLR9/RP105-mediated secretion of IgG and/or IgA.

Methods: CD19+ B cells were isolated from whole blood collected from 10 healthy controls and 10 CVID patients, and the cells were stimulated with CpG-ODN and anti-RP105 with or without RA. ELISA assays were performed to analyze the effects of the stimulants on IgG and IgA secretion, and FAST rt-qPCR was performed to analyze the expression of the four plasma cell related genes AICDA, IRF4, BCL6 and PRDM1. The data from these two analyzes were then combined to reveal any correlations between the gene expression profiles and the secretion of IgG and IgA in response to RA-stimulation.

Results: RA was able to enhance TLR9/RP105-mediated IgG and IgA levels in both normal and CVID-derived B cells. The effects of RA varied considerably, in particular between B cells derived from different CVID patients. Results of the specific aims: 1. We identified the subpopulation of CVID patients with B cells expressing high IRF4 and low AICDA levels.

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The patients belonging to this group also had a poor response to RA in terms of enhancing the TLR9/RP105-mediated expression levels of IRF4 and AICDA. 2. There were no correlations between the B cells from the identified subpopulation of CVID patients, and CVID-derived B cells with the lowest response to RA in term of IgG/IgA secretions. Thus, we observed both relatively high and low/no RA-enhanced secretion of TLR9/RP105-induced IgG and IgA in the IRF4^highAICDA^low-expressing subpopulation of B cells from CVID patients.

Conclusions: The expression profiles of IRF4 and AICDA based on in vitro stimulation of CVID-derived B cells seem not to predict which patients that would benefit from

supplementation of vitamin A. However, the results of the present thesis support our previous suggestion that some CVID patients most likely will benefit from vitamin A supplementation to enhance their serum levels of IgG and/or IgA. In order to optimize the treatment of CVID patients, further studies should be conducted to distinguish the RA responding subpopulation of CVID patients from the non-responders.

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Abbreviations

3H Tritium

ADH Alcohol dehydrogenase

AGP α1-acid glycoprotein

AID Activation-induced cytidine deaminase APC Antigen-presenting cell

APR Acute-phase reaction

β-ME β-mercaptoethanol

BCL6 B-cell lymphoma 6

BCR B cell receptor

BLIMP1 B lymphocyte-induced maturation protein-1

C Constant (region of BCR)

cDNA Complementary DNA

CpG-ODN CpG oligodeoxynucleotide phosphorothionates CRAB-II Cellular retinoic acid binding protein II

CRBP-II Cellular retinol-binding protein type II CRP C-reactive protein

CSR Class switch recombination

CVID Common variable immunodeficiency EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay ERK Extracellular-signal-regulated kinase ESID European Society for Immunodeficiency

H Heavy (chain of BCR)

HRP Horseradish peroxidase

IFN Interferon

Ig Immunoglobulin

IL Interleukin

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VIII

IRF4 Interferon regulatory factor 4

L Light (chain of BCR)

LPS Lipopolysaccharide

LRAT Lecithin:retinol acyl transferase MAPK Mitogen-activated protein kinase MHC Major histocompatibility complex

µM Micro molar

MyD88 Myeloid differentiation primary response 88 NF-κB Nuclear factor kappa B

NK cell Natural killer cell

nM Nano molar

PAMP Pathogen-associated molecular patterns Pax5 Paired box gene 5

PBS Phosphate-Buffered Saline

PI3K Phosphatidylinositol 3-kinase

PKC Protein kinase C

PRR Pattern-recognition receptors

RA Retinoic acid

RAE Retinol activity equivalent

RALDH Retinal dehydrogenase

RAR Retinoic acid receptor

RARE Retinoic acid response element RBP Retinol binding protein

RP105 Radioprotective 105

Rt-qPCR Real-time quantitative polymerase chain reaction

RXR Retinoid X receptor

SHM Somatic hypermutation

TH cell T helper cell

TIR Toll/interleukin-1 receptor

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IX TLR Toll like receptor

TMB 3,3’,5,5’-tetramethylbenzidine TNF Tumor necrosis factor

Treg cell T regulatory cell

TTR Transthyretin

V Variable (region of BCR)

XBP-1 X-box binding protein 1

x g Times gravity

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X

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1 Introduction

This master thesis has focused on the putative effects of vitamin A on B cells from common variable immunodeficiency (CVID) patients in terms of gene regulation and immunoglobulin (Ig) secretion. CVID patients generally have impaired B cell functions, and hence have reduced Ig secretion and a weakened immune system resulting in an increased risk of

infections (1-4). The immune system is one of the many important targets of vitamin A in the body, and interestingly, many CVID patients are known to be vitamin A deficient (5-7). The vitamin A deficiency may therefore contribute to the high infection risk in these patients. In the present master thesis, we have explored whether a stimulatory effect of vitamin A on IgG- and IgA secretion in CVID-derived B cells is associated with a certain gene expression profile that our lab previously identified in a subgroup of CVID patients (8).

In the introduction, a general overview of vitamin A will be presented in section 1.1, followed by a brief description of the immune system in section 1.2. Next, the introduction will focus on B cells (section 1.3), the role of vitamin A in the immune system (section 1.4), and finally the CVID disease (section 1.5). Hopefully, the introduction will ease the interpretation of the results presented in the thesis.

1.1 Vitamin A

Vitamin A is a collective name for all compounds with the biological activity of retinol (7, 9).

In contrast, the term “retinoids” includes both naturally and synthetic analogues of retinol, with and without biological activity (10). Some naturally occurring retinoids are presented in Figure 1.

Vitamin A has several important functions in the body, e.g. in embryogenesis, in vision, for normal growth and differentiation, and for the immune system (5, 7, 11, 12). It was

discovered as an essential constituent of the diet already in 1913 (13), and the effects of vitamin A on the immune system have been documented since the 1920s (14). More details on the function of vitamin A in the immune system will be presented in section 1.4.

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Figure 1: Some naturally occurring retinoids. Modified from (15).

1.1.1 Requirements and dietary sources

The human body is not able of de novo synthesis of vitamin A (7), and it is therefore dependent on dietary intake, and/or supplements in cases where the diet is insufficient.

Vitamin A exists as provitamin A called carotenoids (e.g. α-carotene, β-carotene, and β- cryptoxanthin) in fruits and vegetables, and as preformed vitamin A as retinyl esters in animal sources (16). The international unit for vitamin A is named retinol activity equivalent (RAE), and 1 RAE equals the activity of 1 µg dietary or supplemental retinol. However, 12 µg of dietary β-carotene is needed to equal 1 RAE (9, 17). The biological activity of the different vitamin A compounds varies, but generally, vitamin A from animal sources is considered more biological active than vitamin A derived from plant sources (18). The requirements for vitamin A vary with age and sex, but in the range between 700 and 900 RAE per day for adults (10, 17).

The main dietary sources of vitamin A come in the form of carotenoids in colorful fruits and vegetables, and in the form of retinyl esters in food from animal sources, like dairy products, fatty fish, eggs and cod liver oil (11, 16). In Western diets, 25-75 % of the vitamin A intake is in the form of retinyl esters from animal sources (9, 19).

Because vitamin A is a fat-soluble vitamin, intakes above the need of the body will be stored in the liver (see section 1.1.2). Thus, excessive intake of vitamin A over a prolonged period of time will lead to accumulation of vitamin A, which is potentially harmful if toxic levels are

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17 reached (9, 19). This is in contrast to water-soluble vitamins, which are excreted through the urine when the intake exceeds the needs of the body.

1.1.2 Metabolism of vitamin A

After ingestion, vitamin A is absorbed in the proximal part of the small intestine. Vitamin A is a fat-soluble vitamin, and thus the absorption into the enterocytes and the subsequent

formation of chylomicrons is facilitated by the presence of dietary fat (20). Several retinoids are also to some extent soluble in fluids like plasma (9). The bioavailability and bioconversion of retinoids and carotenoids is affected by several factors in addition to the presence of dietary fat. These factors include alcohol, protein-energy malnutrition, zinc-deficiency and infections, as well as the degree of food processing (9).

Before absorption into the enterocytes of the intestine, essentially all retinyl esters and a portion of the carotenoids are enzymatically converted to retinol in the intestinal lumen by the enzymes pancreatic lipase and the brush-border retinyl ester hydrolase (16, 21). Retinol is primarily absorbed by carrier-mediated transport, but also by passive diffusion when

pharmacological doses are ingested. β-carotene is absorbed only by passive diffusion. 70-90

% of ingested retinoids are absorbed, while only 20-50 % of ingested β-carotene is

absorbed(19, 21). After diffusion into the enterocytes, β-carotene is mainly enzymatically converted to retinal and then further converted to retinol (9, 20).

In the enterocytes, retinol binds to long chain fatty acids and is re-esterified (see Figure 2).

This is facilitated by the binding of retinol to cellular retinol-binding protein type II (CRBP- II) via the enzyme lecithin:retinol acyl transferase (LRAT) (9). These are incorporated into chylomicrons that leave the enterocytes through the lymphatic circulation towards the liver (11, 16).

Chylomicrons are large lipoproteins that consist of aggregates of molecules of triacylglycerol and phospholipids together with retinyl esters, carotenoids, small amounts of retinol, a few specific lipoproteins, and cholesteryl esters (9). The chylomicrons leave the enterocytes via the lymphatic system and are transported to the circulatory system. Here they deliver fatty acids to fat- and muscle cells as they are converted to chylomicron remnants. The

chylomicron remnants containing the retinyl esters are primarily cleared by the liver parenchymal cells (i.e. hepatocytes) (9).

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Figure 2: Absorption of vitamin A in the intestine. Vitamin A may enter the body as retinol or carotenoids. These are absorbed into the enterocytes, where they are modified and packed into chylomicrons. The chylomicrons are transported in the lymph to the circulation. Modified from (22).

In the hepatocytes, the retinyl esters are hydrolyzed to retinol and then transferred to the hepatic stellate cells. There retinol is re-esterified to be stored in lipid droplets (20). In a vitamin A-sufficient state, most of the retinyl esters from the chylomicron remnants are stored in cytoplasmic lipid droplets in the liver stellate cells (9, 16). Up to 90 % of the body’s

vitamin A is stored in the liver (11, 16), and the normal reserve of retinyl esters represents a sufficient supply of vitamin A for an individual for several weeks to months (9). The ability of the stellate cells to store and mobilize retinol ensures that the blood plasma retinol levels always lie close to 2 µM, despite normal, daily variations in the intake of vitamin A (9, 16).

Before secretion from the liver, retinyl esters in the stellate cells are hydrolyzed to retinol, which is transferred back to the hepatocytes (20). There retinol binds to retinol binding protein (RBP) to be secreted and transported to the tissues that need vitamin A (16, 19). All- trans retinoic acid (RA), which is the major active metabolite in target cells, is also present in peripheral blood bound to albumin, but at much lower concentrations than retinol,

approximately at concentrations of 5-10 nM (16, 23).

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1.1.3 Vitamin A – mechanisms of action

As mentioned in section 1.1.2, retinol is released from the stellate cells upon demand, bound to RBP. When bound to RBP, retinol becomes water-soluble. In the circulation, retinol and RBP are also bound to transthyretin (TTR), which reduces the filtration of retinol in the glomeruli of the kidneys (20). When a target cell is reached, TTR detaches from the complex.

The complex is then taken up via the RBP receptor of the cell. Inside the cell, RBP is released from retinol, and retinol is oxidized to retinal and then further to all-trans or 9-cis RA in a two-step process catalyzed by the enzymes alcohol dehydrogenase (ADH) and retinal dehydrogenase (RALDH) (9, 24). All-trans RA is the most common, active metabolite in target cells (9, 20). To be transported into the nucleus, RA binds to cellular retinoic acid binding protein II (CRAB-II), and in the nucleus RA binds to nuclear receptors that act as transcription factors for specific target genes (11, 12, 16) (see Figure 3). There are two families of nuclear receptors which RA binds to, the retinoid acid receptors (RARs) and the retinoid X receptors (RXRs) (23), both consisting of three isotypes (α, β, γ) (25, 26). The different isotypes are expressed in different cell types. All-trans RA binds to RARs, while 9- cis RA binds to both RARs and RXR (12, 25). After binding of the ligand, RAR and RXR form heterodimers. In addition, RXR can form homodimers (12). The receptors affect gene transcription of specific target genes by binding to retinoic acid response elements (RARE) in the DNA, resulting in either inhibitory or stimulatory effects (9). RA may also affect

transcription of target genes independently of RARE when bound to a RAR-RXR heterodimer (27). In addition, vitamin A may also bind to other target proteins than the nuclear receptors and regulate their activity independently of gene transcription. An example of such a target protein is protein kinase C (PKC) (9).

As mentioned previously, vitamin A plays an important role in many functions of the body, and all-trans RA seems to be the primary mediator of the functions of vitamin A (23, 25). 13- cis RA also plays important roles in the body, and is being used in vitamin A supplementation for treatment of for instance acne (28) and cancers (9). More than 500 genes are suggested to be affected by RA, either directly or indirectly. Of these, 27 genes are indisputably direct targets of the complexes formed by the binding of RAR, RXR and RARE (27). In addition, vitamin A is able to regulate target genes of some other nuclear receptors like LXR, PPAR and VDR, as they can form heterodimers with RXR. As part of such heterodimers, RXR may act as a silent or a hormone responsive partner (29).

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Vitamin A plays a significant role in immunity, but is also essential for maintenance of epithelial surfaces, for cellular differentiation, for normal growth, in embryogenesis and organogenesis, and for the vision (5, 11). In all these functions except for the vision process, RA is the active mediator. In the retina of the eye, 11-cis retinal is the active metabolite, serving as the chromophore of several visual pigments (9).

Figure 3: Cellular metabolism and mechanism of action of vitamin A. When retinol enters the cell, it is oxidized into the active metabolite RA. RA is then transported into the nucleus where it binds to and activates the nuclear receptors RAR and RXR. Modified from (22).

1.1.4 Vitamin A deficiency and toxicity

When the intake of vitamin A is lower than the needs of the body, vitamin A deficiency will develop. WHO estimates that more than 120 countries are severely affected by vitamin A deficiency, making it a moderate to severe health problem (30). Furthermore, it is estimated that 250 million children are affected by vitamin A deficiency (31). A poor diet is the main cause of vitamin A deficiency. As mentioned previously, vitamin A from animal sources is generally considered more biological active than vitamin A derived from plant-based food (18).

Vitamin A deficiency compromises the immune system and is associated with increased morbidity and mortality in children (25, 32). There is a vicious cycle between vitamin A deficiency and infectious diseases, as vitamin A deficiency increases the risk of infections and

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21 infections increase the risk of vitamin A deficiency. Infectious diseases contribute to vitamin A deficiency by increasing the metabolism in addition to reducing the intake and absorption of vitamin A. During infections, mobilization of hepatic reserves of retinol is also reduced, and urinary losses of vitamin A increase (11). More about vitamin A deficiency in relation to infections will be presented in section 1.4.

WHO defines the threshold for vitamin A deficiency to be serum concentrations less than 0.70 µmol/L (33). Clinical vitamin A deficiency is characterized by a generalized impaired

resistance to infections in addition to several ocular features (xeropthalmia) (9) that may lead to blindness. It is also associated with anemia, reduced or imbalanced numbers of

lymphocytes, a dysregulation of antibodies, and exacerbation of immunodeficiency (32).

Severe cases of measles, diarrhea and pneumonia are also associated with vitamin A deficiency (5). The risk of developing vitamin A deficiency is increased if the intake of vitamin A is reduced during nutritionally challenging periods of life, like for instance during infancy, childhood, pregnancy and lactation (33).

Toxic effects of vitamin A have generally been linked to preformed vitamin A (10).

Hypervitaminosis A can occur after excessive dietary intake of vitamin A or after intake of drugs containing large amounts of specific retinoids (9). Acute toxicity of vitamin A is rare, but chronic toxicity may be achieved if the daily intake of retinol in oil-based preparations reaches 2 mg/kg body weight over a period of several months to years (10). The risk of hypervitaminosis A due to intake of retinol-rich food over time is low. The risk of toxicity increases when larger doses of retinols from supplements or food fortified with retinol are consumed. Moreover, retinol antagonizes the effects of vitamin D and vice versa, which implies that people with vitamin D insufficiency have increased risk of vitamin A toxicity (10).

Classical symptoms of hypervitaminosis A typically occur in the skin, the nervous system, the peripheral circulation, internal organs and the musculo-skeletal system, in addition to the fetus (9). Hepatotoxicity as a consequence of hypervitaminosis A is linked to an overload of the storage capacity of vitamin A in the liver. This causes cellular toxicity and can eventually develop to fibrosis and cirrhosis of the liver. A possible association between hypervitaminosis A and reduced bone density has been observed, but most human studies conclude that there is no relation between intake of retinol and bone density (10).

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1.2 The immune system

The immune system defends the body against invading pathogens like viruses, microbes and fungi. The immune response involves several different cell types and tissues, which together protect the body by eliminating the invading pathogens.

The tissues of the immune system can be divided into primary and secondary lymphoid organs. The primary lymphoid organs are the thymus and the bone marrow. The bone marrow is the anatomical site for development of lymphocytes. Both B and T cells originate from lymphoid precursors in the bone marrow. The entire maturation of B cells occurs in the bone marrow, whereas immature T cells leave the bone marrow to finish their maturation in the thymus (34, 35). The secondary lymphoid organs include the lymph nodes, the spleen and Peyer’s patches in the small intestine, which all have organized structures with a division between the T cell and the B cell zone. The B cells are clustered in zones known as lymphoid follicles. Naïve B and T cells circulate through or reside in the secondary lymphoid organs, where they eventually are presented to antigens and an immune response is initiated (34, 35).

The immune system provides resistance to pathogens via the innate and the adaptive immune system. The innate immune system is inherent, and provides a broad but general immunity.

The adaptive immune system is not fully developed at birth, but matures during life in response to exposure to pathogens. In contrast to the innate immune system, the adaptive immune system provides highly specific protection against pathogens (36). Hematopoietic pluripotent stem cells are the precursor cells of all cells in the immune system. As the cells develop, they gain specialized functions required for the immune response. Some are specialized to detect and present antigens to other cells in the immune system, which are specialized effector cells that eradicate the pathogen (35).

1.2.1 Innate immunity

As mentioned in section 1.2, the innate immune system provides a generalized defense against pathogens. It is also important for presenting pathogens and thereby instructing the adaptive immune system to respond (36, 37). In contrast to the adaptive immune system, the innate immune system is considered unspecific, because it recognizes structures common for several pathogens rather than a specific antigen (35). The innate immune system consists of epithelial

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23 barriers, circulating phagocytes like neutrophils and macrophages, and cytotoxic cells like natural killer (NK) cells. In addition, inflammation-induced proteins like acute-phase proteins, as well as certain serum proteins like complement factors, are also a part of the innate immune system (26). Regulation of the innate immune system is conducted by pro-inflammatory cytokines such as interleukin (IL) 1, tumor necrosis factor (TNF) α, IL-6 and IL-12, primarily produced by macrophages and neutrophils, in addition to anti-inflammatory cytokines such as IL-10 (26).

Dendritic cells belong to the group of antigen-presenting cells (APCs) due to their ability to present antigens derived from pathogens for adaptive immune cells. After ingesting and degrading the pathogens, dendritic cells present protein- and polysaccharide sequences derived from the pathogen to other cells of the immune system. This presentation occurs in the secondary lymphoid organs, through which naïve B- and T cells circulate until they recognize an antigen presented by an APC. The protein sequences derived from the degraded pathogen are presented on the cell surface of the APC bound to major histocompatibility complex (MHC) class II molecules. Lymphocytes recognizing these antigens will in turn initiate an adaptive immune response (35).

Dendritic cells as well as phagocytes like macrophages and neutrophils have receptors called pattern-recognition receptors (PRRs), which bind conserved molecular structures that are typical for pathogens. These are called pathogen-associated molecular patterns (PAMPs), and are important constituents of pathogens, like e.g. proteoglycans from bacterial cell walls or nucleic acids from DNA (37, 38). A pathogen may activate several PRRs, and this combined activation of lymphocytes results in a stronger and more specific immune response (39). Upon binding of PAMPs to the PRRs, inflammatory cytokines and interferons (IFNs) affecting other cells of the immune system are induced (37). Cells of the adaptive immune system, like for instance B cells, also express PRRs (40), and these will be the focus in section 1.3.

Toll like receptors (TLRs)

One of the most widely expressed groups of PRRs is the TLRs (1). So far, 12 members of the TLR-family have been identified in mice (39, 41), and 10 TLR family members have been identified in humans (37, 42). Also the TLR-homolog radioprotective 105 (RP105) belongs to this group of PRRs. The TLRs are type 1 integral membrane glycoproteins. The extracellular

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N-terminal is leucine-rich, and the intracellular C-terminal domain is known as the Toll/IL-1 receptor (TIR) domain. The TIR domain is required for the interaction and recruitment of adaptor molecules to activate the downstream signaling pathway (37, 41). TLRs can function both as homodimers and as heterodimers, and their activation induces transcription of

inflammatory genes mediated by nuclear factor kappa B (NF-κB) (41, 43). The expression of the TLRs is cell-type specific (36), and individual TLRs identify different PAMPs. PAMPs are highly expressed in infectious microorganism, but rarely in host cells (44). All TLRs apart from TLR3 rely on the adaptor protein Myeloid differentiation primary response 88 (MyD88) to mediate their signals (37, 45). MyD88 is recruited to the TIR after association between the TLR and PAMP, and subsequently activates the IRAK1-TRAF6-TAK1 pathway (44). TLR4 is the only TLR that can signal both through an MyD88-dependent and an MyD88-

independent pathway (45).

TLR9

TLR9 in humans is expressed abundantly in B cells and plasmacytoid dendritic cells (1, 43), as well as in macrophages. It is located in the membranes of endolysosomes (37, 41). In vivo, TLR9 is triggered by unmethylated CpG DNA (43, 46). CpG dinucleotides are generally present at a frequency of 1 per 16 dinucleotides in microbial DNA, which is 4 times the frequency found in DNA from vertebrates. In addition, the cytosines in CpG dinucleotides are highly methylated in DNA from vertebrates but not in microbial DNA (47). Unmethylated CpG oligodeoxynucleotides (CpG-ODN) modified by phosphorothionated backbones mimic microbial CpGs, and such CpG-ODN are widely used to activate TLR9 in vitro (47).

Throughout this master thesis, we have used CpG-ODN to stimulate the B cells via TLR9.

CpG-ODN are taken up by cells via endocytosis, and as endocytotic vesicles fuse with lysosomes, endolysosomes containing CpG-ODN are formed (depicted in Figure 4). In the endolysosomes, TLR9 is cleaved by cathepsins B, K and L and by an asparagine

endopeptidase. The cleaved form of TLR9 recognizes the CpG-ODN, which triggers MyD88- mediated activation of p38, NF-κB and mitogen-activated protein kinases (MAPKs) (37, 48).

The result is induction of type I IFNs and pro-inflammatory cytokines (37). The signaling pathways activated by ligation of TLR9 in B cells lead to proliferation, isotype class switching and differentiation into Ig secreting plasma cells (40).

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25 Figure 4: Signaling pathway of TLR9. TLR9 and CpG-ODN interact in the endolysosome and activate signaling pathways ultimately leading to initiation of transcription. Modified from (49).

RP105

RP105, also known as CD180, is a type I transmembrane protein located in the plasma

membrane. The protein is primarily, but not exclusively, expressed on B cells (50-52). RP105 was first identified by its radioprotective role, as it had the ability to protect murine B cells against cell death induced by irradiation (46, 53). The expression of RP105 is approximately 3-fold higher on memory B cells than on naïve B cells (42). The protein is part of the TLR family of proteins, with homology to TLR4 (51). RP105 has structural similarities with the TLRs, but is different from the typical TLRs in that it lacks the intracellular TIR domain (51, 52, 54), which is important for the signaling of TLRs.

The ligand of RP105 is still unknown, but in vitro activation is possible through cross-linking the receptors with monoclonal anti-RP105 antibodies (55). RP105 associates and forms a complex with the glycoprotein MD1, which plays a role in regulating the cell surface

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expression of RP105 (56). Upon ligation with anti-RP105, the RP105-MD1 complex can transmit a signal resulting in survival and proliferation in B cells (50).

1.2.2 Adaptive immunity

The adaptive immune system is highly specialized and is mediated by circulating

lymphocytes. As mentioned in section 1.2, the adaptive immune system is not fully developed at birth and matures as the body is presented to different pathogens during life. The specific, adaptive immune response can be divided into primary and secondary immune responses (presented in Figure 5). The first time a pathogen enters the body and is presented to the lymphocytes, the primary immune response is induced. If the same pathogen reenters the body at a later point in life, a secondary immune response is initiated. After an adaptive immune response, some of the lymphocytes persist in the body and provide a long-lasting immunological memory of the invading pathogen (26, 35). These are called memory cells.

Induction of immunological memory to a pathogen is the purpose of vaccines, so that an infection later in life will initiate a secondary immune response which terminates the infection with minimal illness (35). Adaptive immunity provided by immunological memory is also called acquired immunity. Due to memory cells, the secondary immune responses are stronger and faster than the primary immune responses (35, 57) (see Figure 5).

Both B and T cells originate from the bone marrow, but whereas B cells complete their maturation in the bone marrow, T cells leave the bone marrow while still immature and complete their maturation in the thymus (34, 35).

The adaptive immune responses are initiated in secondary lymphoid organs, where the lymphocytes are presented to pathogens by APCs of the innate immune system. Adaptive immunity can be mediated through a humoral or a cell-mediated immune response. Humoral immunity is mediated by B cells and their secreted Igs, while the cell-mediated immune response is conducted by T cells, which contribute to the killing of infected cells (35).

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27 Figure 5: The primary and secondary immune responses. The primary immune response after the first exposure to a pathogen is characterized by IgM production and differentiation of B cells into memory B cells. Upon re-exposure to the pathogen, these memory B cells will initiate a stronger, more specific and more efficient immune response. Modified from (58).

The T lymphocytes can be divided into T helper (TH) cells (CD4⁺) and T cytotoxic (CD8⁺) cells. Antigens in the presence of MHC class II complexes are recognized by CD4⁺ T cells (25). These may be peptides presented by B cells, in which case the CD4+ T cell will contribute to the activation of the B cell (35). TH cells can further be divided into several subgroups. TH I cells recognize and respond to intracellular pathogens like viruses, by producing IFN-γ and IL-2, while TH II cells can produce IL-4, IL-5 and IL-10 in response to extracellular pathogens. They contribute by stimulating B cell help (e.g. for IgG, IgE and IgA), enhancing mast cell and eosinophil development, and deactivating macrophages (26).

Several other groups of T cells also exist. Cells with MHC class I complexes are recognized by CD8⁺ T lymphocytes (25), which will deliver cytotoxins that kill the infected cell (35).

T cells only recognize and are activated by a peptide antigen in complex with an MHC molecule. B cells, on the other hand, are able to recognize various macromolecules, such as protein or carbohydrate epitopes on the surface of a microorganism. Whereas the B cell receptor (BCR) is specific for only one antigen epitope, the TLRs also present in B cells are able to bind several types of pathogens that contain PAMPs (35). The TLRs are part of the

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innate immune system, but as their activation can initiate immune responses of the adaptive immune system, they provide a link between innate and adaptive immunity (36).

1.3 B-cells

B cells are responsible for the execution of humoral immune responses to remove

extracellular pathogens (35). Their primary role is to produce Igs, which contribute to the immune response by attaching to the pathogen and thereby recruit immune effector cells which then destroy the pathogen (25, 35). Igs are produced by plasma cells, which represent the end-point of B cell differentiation (59). Based on their activation and degree of

differentiation, B cells can belong to different subtypes, which circulate in the peripheral blood. Before B cells encounter their specific antigens, they are called naïve B cells. Billions of new, naïve B cells are exported from the bone marrow into the peripheral circulation each day, but the B cells are likely to die within a few weeks unless they encounter their specific antigen. B cells that are activated by their antigen differentiate into Ig-secreting plasma cells.

Memory B cells are activated cells which have differentiated into resting cells ready to mount an immune response against future exposure to the same pathogen (35).

The following sections will focus on the development and activation of B cells (sections 1.3.1 and 1.3.2, respectively) and the role of plasma cells in immune responses (section 1.3.4).

1.3.1 Development of B cells in the bone marrow

B cells originate from and are fully developed into mature B cells in the bone marrow, and this process continues throughout life (60). The development begins as pluripotent

hematopoietic stem cells give rise to lymphoid progenitor cells. These have the ability to produce both B and T cells. The lymphoid progenitor cells that develop into precursor cells committed to becoming B cells will further develop into pro-B cells (see Figure 6). The cells undergo several steps of rearranging the genes coding for the BCR. The BCR contains two heavy (H) and two light (L) chains, which each has a variable (V) and a constant (C) region.

The ordered gene rearrangements result in cells expressing one L-chain together with the µ-H chain to form surface IgM. To form a functional BCR, IgM associates with Igα and Igβ in the

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29 endoplasmic reticulum. The receptor is then transported to the cell surface, and the cell

develops into an immature B cell (35, 60, 61).

Figure 6: B cell development. See section 1.3.1 for more explanation. Modified from (62).

During B cell development, a control system is necessary to prevent dysfunctional B cells from leaving the bone marrow. After each step in the development, the B cells must pass a checkpoint before they can continue developing. If the appropriate criteria are not met, the B cell undergoes apoptosis. B cells with BCRs that react against an individual’s own cells or tissues are defined as self-reactive or autoreactive. The antigens these cells bind to are termed self-antigens. Autoreactive immature B cells that encounter their self-antigens are prevented from further development into mature B cells by negative selection. The remaining immature B cells will undergo alternative mRNA splicing of H-chain gene transcripts resulting in production of a δ-H chain in addition to the µ-H chain. This leads to the formation of IgD, which is expressed on the cell surface together with IgM. The immature B cells enter secondary lymphoid organs, where they enter primary follicles to complete the maturation.

During this last maturation process, the BCRs are enabled to generate positive signals upon binding of their cognate antigens (35, 60).

1.3.2 Activation of B cells

B cells are primarily activated in the secondary lymphoid organs. They can be activated both upon binding of antigens to the BCRs and through binding of PAMPs to PRRs. Upon

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activation, the B cells initiate proliferation and differentiation into Ig producing plasma cells (40).

Activation of B cells through the B cell receptor

Activation of B cells through the BCR can be T cell dependent or T cell independent. T cell dependent antigens are proteins, and these antigens generally enter the secondary lymphoid organ presented by APC (63). As the naïve B cell recognizes and binds to its specific antigen, the receptors become cross-linked, and the proteins Igα and Igβ initiate intracellular signaling pathways leading to changes in the gene expression in the nucleus. These signals are

necessary, but not sufficient to activate the naïve B cell. Another set of signals are provided by B cell co-receptors (CD19, CD21 and CD81) associating with the BCR. The simultaneous ligation of BCR and the B cell co-receptor increases the signal up to 10 000 fold, thereby increasing the sensitivity of the B cell to the antigen (35). Several other B cell surface

molecules, e.g. CD38 and CD40, also function as co-stimulators or accessory receptors in the activation process (32).

Upon binding of the antigen, the B cell endocytoses and processes the BCR-antigen complex to present the antigen peptides on MHC class II molecules on the cell surface. Activation of the chemokine receptor CCR7 is induced, which results in the B cell being drawn to the boundary between the B- and T-cell zones. Here, a TH cell activated by the same pathogen recognizes the antigen presented by the activated B cell, and form a conjugate pair with the B cell. The connection between the TH cell and the B cell drives the differentiation of the B cell into an Ig secreting plasma cell (35, 63). This initial activation of the naïve B cell leads to IgM secretion. However, somatic hypermutation (SHM) and class switch recombination (CSR) may lead to the production of antibodies that are able to bind more tightly to the pathogen and more efficiently recruit effector cells. SHM and CSR occur in the germinal centers in follicles of secondary lymphoid organs (35, 57). The amount of antigen and T cell help needed to initiate an effector response is lower for memory B cells than for naïve B cells, enabling memory B cells to respond to some stimuli that are incapable of initiating responses in naïve B cells (57).

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31 Activation of B cells through TLR9 and RP105

In addition to activation through the BCR, B cells may also be activated via PRRs. B cells express several different PRRs, including TLR1, TLR6-10 and RP105 (40). When activated by PAMPs, signaling via these receptors may modulate B cell responses like antibody secretion and antigen presentation (45).

As described in section 1.2.1, TLR9 is activated in vivo by unmethylated CpG-DNA found in microbial DNA (43). The receptor is primarily expressed on B cells, and contributes to proliferation and differentiation into Ig producing plasma cells (40). Human B cells undergo proliferation and initially produce IgM upon stimulation of TLR9 by CpG-DNA, but studies have also found that TLR9 is also able to initiate IgG production. For IgG production, the signaling requires autocrine production of IL-10 in addition to MyD88 (64). Memory B cells express higher levels of TLR9, and they also show a stronger response to CpG-DNA.

Furthermore, whereas memory B cells elicit an immune response to CpG-DNA alone, naïve B cells require co-stimulation elicited for instance by BCR. Stimulation via TLR9 results in polyclonal activation of memory B cells and plays an important role in maintaining immunological memory (43, 45, 46, 57).

As mentioned previously, RP105 is a TLR homolog originally identified as a radioprotective protein (46, 53). Like TLR9, RP105 is expressed at higher levels in memory B cells than in naïve B cells (42, 43). Activation of B cells by RP105 alone has only a small effect on proliferation and Ig secretion, but the effect increases when the B cells are co-stimulated via other receptors. The TLR9-mediated immune response and survival of B cells is potentiated by RP105, and in addition, both the surface and intracellular expression of TLR9 increases (46). The synergy between TLR9 and RP105 affecting the survival of B cells involves increased Akt phosphorylation and NF-κB activation, while neither of the two receptors are able to perform this alone. Activation of Akt is important in cell growth and survival, and activation of NF-κB prolongs cell survival by inhibition of apoptosis (46).

1.3.3 Proliferation of B cells

B cells proliferate in response to signals from both the innate and the adaptive immune system. During cell proliferation, the B cells double their size before they eventually divide

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into two identical cells. The cell cycle consists of four highly controlled phases. These are the first gap phase (G1), the DNA replication phase (S), the second gap phase (G2), and finally the mitosis phase (M). Before proliferation is initiated, the B cell resides in the resting phase called G0 until it is activated and can enter the G1 phase.

The proliferation of B cells in vivo may require several different factors, including CD38 and CD40 combined with cytokines like IL-2, IL-4, IL-10, IL-13 and IL-21 (12, 45, 65). As described in section 1.3.2, ligation of TLR9 both in the presence or absence of RP105 results in proliferation of B cells in vitro.

1.3.4 Differentiation of B cells into plasma cells

Mature, naïve B cells circulate and reside in secondary lymphoid organs like lymph nodes, the spleen and Peyer’s patches in the intestine until they encounter their specific antigen and are activated. Several hundred genes are involved in the differentiation of activated B cells into plasma cells (35). Some genes, like the transcription factors paired box gene 5 (Pax5) and B- cell lymphoma 6 (BCL6) are silenced for the differentiation to proceed, whereas plasma cell specific genes are induced. Various transcription factors, like interferon regulatory factor 4 (IRF4), B lymphocyte-induced maturation protein-1 (BLIMP1) and X-box binding protein 1 (XBP1), contribute to guide the development into plasma cells. IRF4 is highly expressed and essential for the differentiation, partly due to its regulation of the gene PRDM1, which encodes BLIMP1 (66).

Some of the activated B cells in lymph nodes or the spleen immediately proliferate and differentiate into plasma cells, which secrete IgM antibodies. This is due to a change in the processing of the H-chain mRNA of the BCR resulting in the synthesis of the secreted form of the Ig rather than the membrane-bound form. The plasma cells are dedicated to secrete

antibodies, and they cease to proliferate and to express cell surface Ig and MHC class II molecules. This makes plasma cells unresponsive to antigens and interaction with T cells (35).

In addition to IgM, there are four other classes of Igs, i.e. IgA, IgD, IgE and IgG. Not all B cells immediately differentiate into IgM secreting plasma cells. Some activated B cells migrate to a primary follicle which in turn develops into a secondary lymphoid follicle with a germinal center. In the germinal center the B cell undergoes SHM (35, 57). During this

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33 process the V-domains of the H- and L-chains of the BCR undergo SHM in the form of an almost random introduction of point mutations at a rate much higher than an ordinary mutation rate for a gene (35). The enzyme activation-induced cytidine deaminase (AID) is responsible for the SHM. AID is produced only by proliferating B cells and is encoded by the gene AICDA (32, 35, 67). B cells undergo SHM to produce a high-affinity repertoire of antigen-specific B cells (57). Antibodies of progressively higher affinity are produced as the adaptive immune response proceeds. This phenomenon is called affinity maturation (35).

After SHM, further recombination of the DNA enables the V-region sequences to combine with other H-chain C-region genes. This process is called isotype switching, class switching (35) or CSR and leads to the production of Igs of the IgG, IgA and IgE classes. CSR, like SHM, requires the enzyme AID (35, 67).

During the primary immune response, some of the activated B cells differentiate into memory B cells, which become resting cells waiting to be re-exposed to the antigen. Memory B cells have undergone SHM and isotype switching, but do not secrete antibodies. Their

immunoglobulin molecules are expressed on the cell surface. Memory B cells can be

characterized by the expression of CD27, and they have higher affinity antigen receptors than naïve B cells. The memory B cells can persist for many years after the primary infection or vaccination, and will produce a stronger and faster immune response upon activation than naïve B cells (35).

As mentioned previously, the primary function of B cells in the immune system is the

production and secretion of antibodies. Neutralizing antibodies are able to directly inactivate a pathogen, thereby preventing them from interacting with the cells of the body. Other

antibodies may opsonize the pathogen, marking it for ingestion by phagocytes. The antibodies are constructed similarly to the BCR, and consist of two H-chains and two L-chains. The isotype of the C region of the H-chain determines the class of the Igs. Cα gives rise to IgA, Cδ to IgD, Cε to IgE, Cγ to IgG, and Cµ gives rise to IgM. Secreted and membrane-bound Igs originate from the same DNA sequence, but a difference in the processing of the mRNA results in the production of two different forms (35). As a result of increased mRNA stability and transcription, the amount of Ig RNA increases a 100-fold when B cells differentiate into plasma cells (68). The structure of an antibody is presented in Figure 7.

IgM is the first antibody produced during an antibody response and is secreted as a pentamer by plasma cells in the bone marrow, the spleen and lymph nodes. IgM has 10 antigen binding

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sites and binds strongly to the pathogen. This quickly activates the complement system, leading to the production of chemokines and opsonins, which attract immune cells and facilitate phagocytosis, respectively. A disadvantage of IgM is that it is too large to leave the bloodstream and penetrate infected tissues (35).

As B cells undergo SHM and CSR, IgG and IgA are produced. These Igs are smaller and hence are better suited to reach infected tissues. IgG is the most abundant Ig in the internal body fluids and has also a longer half-life than most plasma proteins. IgG, like IgM, has the ability to neutralize the pathogen and activate the complement cascade, but is also capable of binding to the pathogen and working as an opsonin and of activating NK cells. These two additional features lead to the activation of phagocytosis and to the killing of the pathogen, respectively. IgM, IgG and monomeric IgA provide antigen defense within the fluids and tissues of the body. IgA exists both as monomeric IgA and dimeric IgA, and dimeric IgA is important for the protection of surfaces of the mucosal epithelium which communicates with the external environment (35).

Figure 7: Structure of antibody. Antibodies consist of two heavy and two light chains, which are divided into constant and variable regions. The variable regions form the Fab region, while the constant region of the heavy chain forms the Fc region. The hinge region is of importance for flexibility in the Fab region. The membrane Ig is converted into its secreted form upon rearrangements of the H-chain. Modified from (69).

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1.4 Vitamin A and the immune system

As already mentioned, vitamin A plays an important role in regulating the immune system (14, 23). Both vitamin A deficiency and hypervitaminosis A seem to have negative effects on immunity (70), and both the innate and the adaptive immune system are affected (26).

Appropriate levels of vitamin A are therefore important for maintaining proper immune functions.

Malaria, diarrhea and pneumonia are the principal causes of deaths from infectious diseases worldwide, and cause about one third of deaths among children under the age of 5 (71).

Vitamin A supplementation of children has been proven to contribute to reduce morbidity and mortality from such infectious diseases (26, 30, 71), even among children without clinical signs of vitamin A deficiency (11).

The main vitamin A metabolite in the immune system is RA (23, 25). RA is required for proper differentiation of epithelial cells and thus affects the number of mucus-producing goblets cells in the epithelial linings (26, 72). Vitamin A is also important for ensuring proper levels of IgA produced by B cells in mucosal epithelial barriers, and reduced levels of

secretory IgA in the saliva of vitamin A deficient children have been reported (73, 74). Thus, mucosal immunity seems to be compromised in vitamin A deficient individuals (11), making them vulnerable to infections by affection of epithelial barriers of the eye in addition to the respiratory, gastrointestinal and urogenital tracts (26).

RA is important for mediating T cell help through production of cytokines, and a hallmark for vitamin A deficiency is the inability to mount an antibody defense against T cell dependent antigens (25). In addition to reduced antibody production and T cell responses, decreased levels of vitamin A also has a negative impact on the phagocytosis of pathogens (6).Vitamin A contributes to the immune response by enhancing the activity of T cytotoxic cells, NK cells and macrophages (25, 26). RA also affects the cytokine production in macrophages, resulting in a shift in the activity of TH 1 cells and TH 2 cells towards the ant-inflammatory effects of TH 2 cells (7, 75). Vitamin A deficiency causes an imbalance in TH cells, resulting in an excess of TH 1 cells and insufficient TH 2 activity (5, 75). This leads to excess production of IFN-γ and impaired antibody responses (7). RA also promotes the induction of CD4+ T regulatory (Treg) cells (76) and inhibits the formation of pro-inflammatory TH 17 cells (7), which play a role in the downregulation of the immune response and the induction of tolerance to self-antigens (77).

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1.4.1 Vitamin A and B-cells

While the stimulatory effects of vitamin A on T cells is well established (23, 78), the effects of vitamin A on B cells have been controversial. Although stimulatory effects of vitamin A on B cell differentiation (79) and antibody responses (80) have been reported, we and others have documented inhibitory effects of vitamin A on the proliferation of B cells (23, 32, 70). Later, our group showed that the effect of RA on peripheral blood B cells was dependent on the type of antigen used to stimulate the B cells and of the subtype of B cells that were stimulated (81).

It was demonstrated that whereas the proliferation of memory B cells stimulated via TLR9 is enhanced by RA, naïve B cells stimulated via BCR are inhibited. Our research group also demonstrated a synergy between RA and TLR9-mediated stimulation in the presence of RP105 ligation (82, 83).

Vitamin A has also been shown to promote the differentiation of B cells into Ig-producing plasma cells both in vivo (84, 85) and in vitro (85, 86). Animal studies have found that RA stimulates SHM and CSR by increasing the expression of AID (32). Our research group has also demonstrated that RA augments the production of IgM and IgG in memory B cells stimulated through TLR9 (81). This suggests that vitamin A may be required for maintaining a repertoire of circulating Ig producing plasma cells.

1.5 CVID

CVID is a primary immune deficiency disorder first described in 1953 (4). The disease is characterized by hypogammaglobulinemia and recurrent infections (3-5, 87). The prevalence of CVID in Norway is 1 in 25 000 (88). Diagnostic criteria established by the European Society for Immunodeficiency (ESID) include a marked decrease of IgG and IgA with or without low IgM levels, diagnosis established after the 4^th year of life, exclusion of secondary causes of hypogammaglobulinemia, no evidence of profound T cell deficiency, poor antibody response to vaccines (and/or absent isohemagglutinins) or low levels of switched memory B cells. In addition, at least one of the following must apply: increased susceptibility to

infection, autoimmune manifestations, granulomatous disease, unexplained polyclonal

lymphoproliferation or an affected family member with antibody deficiency (89). The primary treatment of CVID is antibody replacement therapy, in addition to symptomatic treatment of infections, autoimmunity and other complications associated with the disease (90).

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37 CVID patients have disturbed humoral immunity owing to defects in the differentiation of B cells into functional plasma cells (1, 4), and have a poor or missing antibody response to vaccination (3). As previously mentioned, CVID patients are also frequently vitamin A deficient (5-7), which may contribute to their poor immune responses. A higher risk of complication is associated with the reduced vitamin A status among these patients, and often are higher doses of vitamin A supplements needed to restore their vitamin A status (6).

Chronic respiratory infections, especially bronchiectasis, are common among CVID patients, and they may suffer from gastrointestinal diseases resulting in diarrhea, malabsorption and weight loss. Autoimmune diseases like autoimmune hemolytic anemia and rheumatoid

arthritis are also relatively common, in addition to an increased incidence of cancer, especially of B cell lymphomas (4). The increased risk of cancer may be associated with an increased chromosomal instability in cells of CVID patients (91).

As the clinical range of CVID is wide, onset of symptoms may not become obvious until adult life (4), delaying the point of diagnosis. The high morbidity among CVID patients leads to increased mortality, but the increased mortality is mainly due to the many non-infectious complications. The prevalence of CVID is equal between males and females, but females tend to be older at the time of diagnosis (4, 92) and have a higher risk of developing lymphomas (92).

Mutations in more than 20 genes are known to cause or be associated with CVID (93, 94).

However, as only 10-20 % of the patients are diagnosed at a molecular level (93, 95), a large number of additional genetic defects most likely exists. Missense mutations or deletions of genes involving ICOS, TACI, IKZF1, CTLA4, NF-κB1 and NF-κB2 are known underlying causes in some patients (93, 94). Other genes demonstrated to be mutated in CVID patients are BAFF-R, CD19 (94, 96), CD20 and CD81 (94, 97). These gene defects involve both B and T cell functions (97). Interestingly, although the main feature of CVID is reduced levels of Igs, patients generally have normal B cell counts (89), but tend to have lower levels of plasma cells, as well as lower numbers of circulating memory- and switched memory B cells (1, 2).

Defects in SHM are a common feature of CVID and are associated with both infectious and non-infectious complications (89, 97).

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1.5.1 TLR9 and RP105 in relation to CVID

TLR9 activation of B cells has in several studies been shown to be impaired in CVID patients (1, 65). CVID-derived B cells express lower levels of intracellular TLR9 than B cells derived from healthy controls (1). Thus, B cells activated via TLR9 show less proliferation and differentiation in addition to impaired secretion of Igs, IL-6, IL-10 and IFN-α (1). The immunophenotype of CVID patients has significance for the in vitro response to CpG-ODN, which is poorer in patients with the lowest levels of switched memory B cells (98). Patients with the lowest levels of switched B cells also tend to have the lowest expression of AID, indicating a reduced capacity for switching of Ig isotypes (99). Our research group has previously shown that both TLR9- and RP105-mediated proliferation and differentiation are weaker in CVID-derived B cells than in B cells derived from healthy control subjects (8, 100).

Furthermore, our group has shown that RA is able to restore the proliferation of CVID- derived B cells to nearly normal levels (100). Although the effects of RA on IgG production in CVID-derived B cells were generally marginal, significant stimulating effects on IgG production were noted in B cells from some of the CVID patients that were analyzed (8, 100).

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2 Aims of the study

In our research group we had previously identified a subpopulation of CVID patients

characterized by B cells with a high expression of IRF4 and a low expression of AICDA (8).

When co-stimulated with CpG-ODN and anti-RP105, this group (IRF4^highAICDA^low) of B cells had a poor response to RA in terms of expression of plasma-cell related genes.

Furthermore, our group had shown that although the response of CVID-derived B cells to RA in terms of IgG-production generally was low, B cells from some patients responded better than others (8, 100). Based on these findings, it was proposed that B cells from patients belonging to the newly identified CVID-subpopulation could be the ones with the lowest RA- mediated IgG response (8). Furthermore, it was suggested that in vitro studies on isolated B cells might predict which patients that would or would not benefit from vitamin A

supplementation.

The previously identified subgroup of CVID patients was based only on 7 patients and 7 healthy controls.

The specific aims of the present study were therefore to

1. Extend the study group of CVID patients and healthy controls to confirm the existence of a CVID subpopulation with IRF4^highAICDA^low-expressing B cells and low response to RA in terms of enhancing the expressions of these plasma cell related genes.

2. Assess whether B cells from the subgroup of CVID patients identified in aim 1 are the ones with low in vitro response to RA in terms of IgG/IgA secretion.

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3 Materials

3.1 Chemicals

Chemicals Manufacturer Location

2N Sulfuric acid solution (H2SO4) Sigma-Aldrich Missouri, USA

[³H]-thymidine Perkin Elmer Massachusetts, USA

Activation-induced cytidine deaminase Invitrogen California, USA (AID) (QT00024864)

All-trans retinoic acid (RA) Sigma-Aldrich Missouri, USA

β-mercaptoethanol (β-ME) Sigma-Aldrich Missouri, USA

Beta-2-microglobulin (B2M) Qiagen Hilden, Germany

(QT00088935)

Bovine Serum Albumin (BSA) Sigma-Aldrich Missouri, USA

Buffer RLT Plus Qiagen Hilden, Germany

Buffer RPE Qiagen Hilden, Germany

Buffer RW1 Qiagen Hilden, Germany

CpG oligonucleotides phosphorothionate Enzo Life Science New York, USA ELISA Blocking Buffer (for IgG ELISA) Bethyl Laboratories Texas, USA ELISA Coating Buffer (for IgG ELISA) Bethyl Laboratories Texas, USA ELISA Enzyme Substrate (TMB-solution) Bethyl Laboratories Texas, USA ELISA Horseradish Peroxidase (HRP) Bethyl Laboratories Texas, USA conjugate (for IgG ELISA)

ELISA Sample Diluent (for IgG ELISA) Bethyl Laboratories Texas, USA ELISA Standard IgG Bethyl Laboratories Texas, USA ELISA Wash Solution (for IgG ELISA) Bethyl Laboratories Texas, USA Ethanol (70 % solution) University of Oslo Oslo, Norway Ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich Missouri, USA 0.5 M

Fetal Bovine Serum (FBS) Bionordika Oslo, Norway

iScript Reaction Mix Bio-Rad California, USA

iScript Reverse Transciptase Bio-Rad California, USA

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Chemicals Manufacturer Location

Microscint™ 0 Perkin Elmer Massachusetts, USA

MilliQ water Merck Millipore Massachusetts, USA

Phosphate buffered saline (PBS) tablet Sigma-Aldrich Missouri, USA

PRDM1 (QT00060494) Qiagen Hilden, Germany

RNAse free water Qiagen Hilden, Germany

RPMI 1640 Medium Lonza Basel, Switzerland

SsoFast™EvaGreen® Supermix Bio-Rad California, USA

TaqMan Fast Advanced Master Mix Thermo Fisher Scientific Massachusetts, USA

TATA binding protein (TBP) Qiagen Hilden, Germany

(QT00000721)

Tween 20 Sigma-Aldrich Missouri, USA

3.2 Equipment

Equipment Manufacturer Location

0.2 ml Skirted 96-well PCR Plate Thermo Fisher Scientific Massachusetts, USA 0.2 ml Thin-wall 8-tube strip BIOplastics BV Landgraaf, The

Netherlands 96 well Microtiter Plate (for IgG ELISA) Bethyl Laboratories Texas, USA

Ambion® RNAse-Free 1.5 mL Microfuge Thermo Fisher Scientific Massachusetts, USA Tubes

Biohit Proline Single Channel Electronic Sartorius/Biohit Göttingen, Germany Pipette (0.01-0.5 mL)

Buffy coat Ullevål University Oslo, Norway

Hospital, OUS

Cell culture flask (75 cm³) Thermo Fisher Scientific Massachusetts, USA Cell culture plate (6, 12, 24, 96 wells) Thermo Fisher Scientific Massachusetts, USA

Collection tube, 1.5 mL Qiagen Hilden, Germany

Collection tube, 2 mL Qiagen Hilden, Germany

Counting slide Bio-Rad California, USA

Eppendorf tube, 1.5 mL BrandTech Connecticut, USA

The effects of vitamin A in B cells from patients with the immunodeficiency disorder CVID