Vitamin A in the adaptive immune system

T cells are critical mediators of the adaptive immune system, and act by inducing cell-mediated immune responses. T cells are divided into distinct subpopulations based on expression of specific cellular markers and effector functions. Thus, T helper cells (Th) typically express CD4, and cytotoxic T cells (Tc) express CD8 (352;353). In contrast, Tregs

are characterized as cells expressing the transcription factor Foxp3 in addition to the surface markers CD4 and CD25 (354;355). Th cells can be further divided into Th1, Th2 and Th17 cells based on the cytokine profile (352;356). The balance between Tregs and Th17 cells is essential for maintaining immune tolerance and control of inflammation, as impaired balance between these cell types is implicated in autoimmune disease and excessive inflammation (357;358).

Vitamin A has roles in several aspects on T cell biology, where the effects are highly cell- and context dependent (287;359). In response to antigenic stimulation, naïve CD4⁺ T cells differentiate into effector cells such as Th1 and Th2 cells. Whereas Th1 cell produce IFNγ and mediates pro-inflammatory responses involved in killing of intracellular pathogens, Th2 cells secrete IL-4, IL-5 and IL-13 resulting in enhanced antibody secretion from B cells and eradication of extracellular pathogens (352). It is generally believed that RA modulates the balance between Th1 and Th2 cells by favoring the formation of Th2 cells concomitant with reduced development of Th1 cells (288;360). Whereas the inhibitory effect of RA on the formation of Th1 cells has been ascribed to downregulated expression of T-box expressed in T cell (T-bet), the enforced Th2 differentiation has been linked to the induction of GATA-binding protein-3 (GATA3) (360). Recent research has however challenged this somewhat simplistic view, since it has been shown in a murine model that RA also may promote Th1 differentiation by inducing stable expression of Th1 linage specific genes (361).

RA is also important for regulating the balance between Treg- and Th17 cells. Th17 are pro-inflammatory immune cells developed from CD4⁺ T cells. These are important in anti-microbial immunity, but are also linked to development of autoimmune disease (356).

RA has been shown to suppress the differentiation of the pro-inflammatory Th17 cells by inhibiting the expression of IL-6 receptor and IL-23 receptor and by antagonizing the activity of important Th17-spesific transcription factors (361;362). As described in section 1.4.3.2, RA produced by DCs facilitates the differentiation of Tregs by increasing the expression of Foxp3 (336;363-365). Tregs have suppressive roles against T cell responses and are important for maintaining peripheral tolerance (354;355). In particular, Tregs are essential for maintaining gut mucosal tolerance to commensal bacteria and food-based antigens (366). RA seems to generate a stable Treg phenotype that helps to maintain the numbers and functions of Tregs throughout the immune response (367). Studies on the mechanisms of action have

shown that RA promotes Foxp3 expression by markedly increasing extracellular signal-regulated kinase (ERK) 1/2 activity (363).

RA also affects the effector functions and location of differentiated T cells. Thus, RA promotes the homing of CD4+ cells to the site of infections (379). During inflammation, RA increases the functions of CD4⁺ T cells by enhancing the secretion of the pro-inflammatory cytokines IFN-γ and IL-17A by Th1 and Th17, respectively (368;369). RA affects not only CD4+ T cells, but also CD8+ T cells. Accordingly, the lack of RA-mediated signaling in mice resulted in reduced numbers, differentiation and migration of CD8⁺ effector T cells, whereas the development into CD8 memory T cells was enhanced (370). Our research group has previously shown that RA augments proliferation and inhibits spontaneous apoptosis of activated peripheral T cells by inducing the secretion of IL-2 (371-373).

RA in regulation of B cell responses

The primary stimulation of B cells during an immune response is via the antigen receptor (BCR). However, optimal stimulation also requires other receptors and costimulatory molecules such as CD38, CD40 and TLRs (27;374). Whereas numerous in vivo studies in mice have revealed an essential role of RA in B-cell proliferation and antibody production (375;376), the in vitro effects of RA on B-cell proliferation appear to be highly context-dependent. Early studies by our group demonstrated that RA inhibits BCR-mediated activation of B cells (377;378). The B cells were arrested in G0/G1 of the cell cycle via RA-mediated reduction of cyclin E and cyclin A levels concomitant with induction of the CKI p21^Cip (378). Later, other groups observed similar inhibitory effects of RA, demonstrating that RA inhibited proliferation of murine B cells activated through BCR, CD40 and CD38 (379;380). In one of the studies, it was reported that whereas RA generally inhibited the proliferation of B cells stimulated via BCR and CD38, RA simultaneously enriched a subgroup of differentiated B cells (380). This demonstrates differential effects of RA on various subgroups of B cells. In contrast to the inhibitory effects of RA reported on B cells stimulated via BCRs, our group has later shown that RA enhances the proliferation of B cells stimulated via TLR9 in the absence or presence of stimulation of the TLR RP105 (381;382).

The proliferative effect was more prominent in memory B cells than in naïve cells and involved induction of IL-10 (381;382).

RA is also known to augment antibody production (375;376). Animals deficient in vitamin A shows reduced antibody responses against TD and TI type II antigens, and vitamin A supplementation restores the antibody response in such animals (376). Thus, in combination with other stimuli, RA has the potential to enhance the primary response and induce a robust memory response in vivo (383;384). In vitro experiments have revealed that the effect of RA on Ig-secretion is mediated both by enhancing differentiation into plasma cells and by stimulating CSR (380;385). The ability of RA to facilitate differentiation of B cells into antibody secreting plasma cells seems to be a general feature. Thus, RA enhances plasma cell differentiation in murine B cells stimulated via BCR, CD38 or CD40 (379;380).

In addition, our group has shown that RA enhances plasma cell differentiation when combined with stimulation of TLR9 and RP105 (381;382;386). The differentiation of activated B cells into plasma cells and induction of antibody-secretion is regulated by a distinct set of genes (described in section 1.1.2.3). In order for B cells to differentiate into plasma cells, the expression of PAX5 and BCL6 needs to be downregulated (387), whereas BLIMP1 needs to be upregulated (26). Chen and Ross were able to show that RA decreases the expression of PAX5 in murine B cells stimulated via BCR, CD40 and CD38 (379;380).

Concomitantly, RA increased BLIMP1 expression and induced higher expression of sIgG1 and the plasma cell marker CD138 (380). In line with these results, our group has shown that when B cells are stimulated via TLR9 and RP105, RA enhances the expression of genes involved in plasma cell differentiation such as BLIMP1 and IRF4 (386). RA has also been shown to stimulate CSR and SHM by increasing the expression of AID (380;386). However, the context seems to be important for determining the outcome of RA-stimulation in terms of CSR. Hence, whereas RA in the presence of IL-5 and IL-6 acts as a highly specific switch factor for IgA (388;389), RA induces switching to IgG concomitant with reduced IgE secretion when the cells are co-stimulated with CD40 and IL-4 (379;390;391) This points to a regulatory role of RA in balancing the secretion of various antibody subclasses.

2 AIMS OF THE STUDY

The current project is part of a larger study with the overall aim to understand how vitamin A regulates the immune system – both in health and disease. By exploring the role of RA in survival and immune functions of both B cells from healthy donors, malignant B cells and CVID-derived B cells, we aim to increase the basic knowledge and identify potential therapeutic benefits of using RA in treatment of such diseases.

The specific aims were to:

1. Elucidate the effects of RA on immune functions and survival of CVID-derived B cells stimulated via TLR9 and/or RP105.

2. Reveal how RA regulates TLR9-mediated survival of normal and malignant B cells and study the mechanisms involved.

3. Explore how the levels of DNA lesions and DNA damage repsonses are affeceted by RA in the presence of TLR9 ligands in surviving B cells.

3 SUMMARY OF THE PAPERS

Paper I: 

Retinoic Acid improves defective TLR9/RP105-induced immune responses in Common variable immunodeficiency-derived B Cells. 

Impaired signaling from TLR9 as well as from other TLRs has been linked to the immune defects associated with CVID. Having previously shown that RA enhances TLR9/RP105-mediated proliferation and differentiation of normal B cells, we here elucidated the ability of RA to restore the defective TLR-signaling in CVID-derived B cells. To this end, B cells were isolated from 35 CVID patients and 35 healthy controls and stimulated via TLR9/RP105 in the presence or absence of RA. We demonstrated that RA nearly normalizes the diminished proliferation and IL-10 secretion in CVID-derived B cells. Furthermore, RA improved the defective IgG-secretion, although to a more limited extent. The defective IgG-secretion combined with normal secretion of IgM suggests that CVID B cells have retained their capability to undergo plasma cell differentiation, but that they exhibit defective isotype switching. This hypothesis was further verified by our finding that RA induced normal expression of the plasma cell markers CD38 and BLIMP1, whereas the RA-induced expression of AID was diminished in CVID-derived B cells. Despite the moderate levels of IgG induced in CVID B cells as compared to B cells from healthy controls, we still believe that the 6-fold induction of IgG obtained by RA in combination with TLR9/RP105-stimulation of CVID-derived B cells might be clinically relevant for these patients.

Paper II:

Myeloid cell leukemia 1 has a vital role in retinoic acid-mediated protection of Toll-like receptor 9-stimulated B cells from spontaneous and DNA damage-induced apoptosis.

Survival of immune cells is important for maintaining a proper immune response. B cells are highly dependent on survival factors and are particularly sensitive to DNA damage-induced apoptosis. The goal of the present study was to unravel the impact of RA on B cell survival, both related to normal B-cell homeostasis and to the detrimental effects of DNA damaging

agents. We show that RA prevents spontaneous and DNA damage-induced apoptosis of TLR9-stimulated B cells. The effect of RA was mediated by the nuclear receptor RAR, and it involved transcriptional upregulation of the anti-apoptotic protein MCL1. The finding that RA in combination with TLR9-stimulation protects normal- and not malignant B cells from DNA damaging agents, suggests that these compounds could be used as adjuvants in cancer treatment to protect normal B cells from the detrimental effects of DNA damaging cancer treatment. The usefulness of these adjuvants in treatment of B-cell malignancies was underlined by the finding that TLR9-stimulation alone or in combination with RA in fact showed a tendency to increase the death of the malignant B cells.

Paper III:

TLR9-stimulation of B cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent of DNA strand breaks and DNA damage responses. Implications for Common variable immunodeficiency.

Any treatment that increases the survival of B cells treated with DNA-damaging therapy, could potentially result is cells surviving with unrepaired damage and thereby increase the risk of malignant transformation of the cells. We here explored the impact of RA and TLR9-stimulation on DNA damage in irradiated B cells, as measured by levels of DNA strand breaks and activation of various DDR components. Neither activation of DDR-related kinases nor measurement of DNA strand breaks by alkaline comet assay indicated effects of TLR9-stimulation and RA on the induction of DNA damage or the rate of repair of such lesions. The increased level of phosphorylated H2AX in irradiated cells stimulated via TLR9 was explained by increased levels of total H2AX. In addition, TLR9-stimulation increased the transcription of p53, resulting in low-level expression of inactive p53. The p53 molecules became phosphorylated and activate in response to irradiation, suggesting that the low-level expression of p53 could provide the B cells with a barrier against potential harmful consequences of enhanced proliferation and differentiation. We also found that RA in the presence of TLR9 ligands protect CVID-derived B cells from DNA damage-induced cell death. These findings are particularly relevant for CVID patients, known to have increased levels of spontaneous and induced apoptosis. In conclusion, there is no evidence pointing to B cells surviving with higher

levels of DNA lesions when stimulated with RA and TLR9 ligands, supporting the use of such compounds in treatment of CVID and other B cell related immune deficiencies.

4 DISCUSSION

4.1 Methodological considerations 4.1.1 Primary cell cultures

4.1.1.1 Peripheral blood B cells

The primary B cells used in paper I, II and III were isolated by positive selection from peripheral blood, using a method developed by Funderud and colleagues (392). As a source for isolating the B cells, we used buffy coats from normal blood donors admitted to Ullevål University Hospital. Buffy coats are prepared by density grade centrifugation of whole blood and mainly consist of leukocytes and platelets. The B cells were isolated from buffy coats by Dynabeads® CD19, which are superparamagnetic beads covalently coated with anti-human CD19 antibodies. After magnetic separation of the CD19⁺ B cells from the remaining cells, B cells were detached from the magnetic beads by two alternative methods. In paper I we used overnight detachment of the cells by simply letting the B cells gradually detach in culture due to internalization of the CD19-receptor (392). In papers II and III we utilized anti-Fab fragments in DETACHaBEADS® CD19 to detach the B cells from the magnetic beads by outcompeting the binding between Dynabeads® CD19 and the B cells (393). Both detachment methods resulted in isolated B cells with a purity of >98 %, as estimated by measuring the portion of cells with the pan B cell marker CD20. Although both methods are considered to produce viable (>95 %), non-activated B cells (392;393), the latter method appeared in our hands to be preferable when measuring the survival of B cells over time.

Independent of detachment method, the number of B cells isolated from buffy coats varied significantly from one donor to another, ranging between 5 and 50 million cells. We were therefore occasionally required to pool cells from several buffy coats to perform experiments.

We also experienced that the biological responses greatly differed between donors. This could in part be due to differential composition of various B cell subsets, such as the ratio between naïve and memory B cells. Thus, in order to draw statistically sound conclusions from research on normal peripheral blood B cells, one has to repeat experiments on cells from several donors – usually between 5 and 10.

In addition to the limitations of low numbers and donor-specific variations, isolated primary peripheral blood B cells cannot be cultured for more than a few days in vitro, as the B

cells are gradually undergoing spontaneous apoptosis (394). A more prosaic disadvantage is that isolation of primary B cells is time consuming and expensive. However, despite of all limitations, there are major advantages of using primary peripheral blood B cells. Cell lines are generally the alternative to research on primary cells, and B cell lines are established either from malignant cells or by EBV-infection. Accordingly, results acquired from primary B cells can be considered as being more physiologically relevant than results obtained from cell lines. Furthermore, B cells from peripheral blood are naturally synchronized in the resting G0 phase of the cell cycle. It is therefore possible to study the processes of B cell activation, proliferation and differentiation in a more physiological manner. It should be noted that B cells isolated from peripheral blood generally are more resting, i.e. have lower numbers of in vivo activated cells, compared to primary B cells isolated from lymphoid organs such as lymph nodes and tonsils (395;396).

4.1.1.2 CVID-derived B cells

In papers I and III, B cells were isolated from whole blood samples collected from CVID patients and healthy donors. Blood samples from CVID patients were obtained after written consent from patients admitted to Oslo University Hospital, Rikshospitalet, for routine follow-up controls. For patients on intravenous Ig-replacement therapy blood samples were drawn immediately prior to infusion. Patients with ongoing infections or on immunosuppressive therapy were excluded from the study. Approximately 35 ml of whole blood was collected from both CVID-patients and healthy donors, and the B cells were isolated by positive selection using Dynabeads® CD19 by following the same principle as for isolation of cells from buffy coats. The number of B cells obtained from each patient ranged between 0.1 and 4 million, and the purity was >98 %. The amount of blood collected from each patient was limited by ethical considerations (see below), and the low numbers of B cells obtained from such small amounts of blood limited the number- and type of experiment that could be performed on each patient sample. Accordingly, in some of the experiments presented in paper I, we could only use donors with high numbers of B cells. This could potentially result in a selection bias, favoring results not necessarily representing all CVID patients.

4.1.1.3 Malignant B cells

In paper II, we used malignant B cells isolated by density gradient centrifugation of tumor biopsies from patients with various forms of B-cell malignancies admitted to Oslo University

Hospital, Radiumhospitalet. The purity of the tumor samples was between 70-90 %. We obtained approximately 20 million frozen cells from each patient. To avoid interference with the treatment protocols, all patients were untreated for their B-cell malignancy at the time of collecting the biopsy. The malignant B cells turned out to be more fragile than their normal counterparts, and the viability of the cells after thawing was generally low (on average approximately 50 %). A recent study showed that freezing of peripheral blood mononuclear cells also affects gene expression profiles with upregulation of genes involved in cellular stress responses (397). Hence, both the freezing process and the fact that the fraction of normal cells in the tumor biopsies is not accounted for, may potentially affect the results obtained on malignant cells in paper II.

4.1.1.4 Ethical considerations associated with collection of primary cells

There are few ethical problems connected to collecting blood samples from patients and healthy donors. In our study, normal B cells were isolated from buffy coats prepared from whole blood of healthy blood donors admitted to Oslo University Hospital, Ullevål. The buffy coasts are byproducts from the sampling of red blood cells and plasma used in blood transfusions, and it is considered as ethically sound to use these products in research. Blood samples from CVID-patients were collected during their routine follow-up controls after written consent. As healthy controls, whole blood samples from volunteers among students and staff at Institute of Basic Medical Science were collected. Both patients and controls volunteered and signed an informed consent prior to blood donation. Generally, collection of peripheral blood is considered as harmless and is well tolerated by both patients and healthy donors. Nevertheless, any procedure breaking the natural barrier, such as punctuation of the skin, increases the risk of infections. This could be harmful for CVID-patients, who have

There are few ethical problems connected to collecting blood samples from patients and healthy donors. In our study, normal B cells were isolated from buffy coats prepared from whole blood of healthy blood donors admitted to Oslo University Hospital, Ullevål. The buffy coasts are byproducts from the sampling of red blood cells and plasma used in blood transfusions, and it is considered as ethically sound to use these products in research. Blood samples from CVID-patients were collected during their routine follow-up controls after written consent. As healthy controls, whole blood samples from volunteers among students and staff at Institute of Basic Medical Science were collected. Both patients and controls volunteered and signed an informed consent prior to blood donation. Generally, collection of peripheral blood is considered as harmless and is well tolerated by both patients and healthy donors. Nevertheless, any procedure breaking the natural barrier, such as punctuation of the skin, increases the risk of infections. This could be harmful for CVID-patients, who have