Background and aims: Common variable immune deficiency (CVID) is a primary immune disease characterized by low levels of serum immunoglobulins (Igs) - in particular of the IgG class, recurrent infections, but also by increased radiosensitivity and risk of developing cancers. The etiology of CVID is complex, but mutations in genes related to B cell activation and differentiation have been identified. B cells from CVID patients have defective Toll like receptor 9 (TLR9)-signaling, and these patients are also frequently vitamin A deficient. TLR9 is expressed in the endolysosomes of B cells, and these receptors are considered as part of the innate immune system. The natural ligand for TLR9 is bacterial DNA rich in unmethylated CpG motifs, and CpG oligonucleotides are used to stimulate the receptors in vitro.
The innate receptor RP105 was first identified for its ability to protect murine B cells against γ-irradiation-induced cell death, but later it has been shown to synergize with TLR9-signaling. Recently our group has revealed that also the vitamin A metabolite retinoic acid (RA), which is important for a functional immune system, is able to enhance the immunostimulatory effects of TLR9-signaling. The overall aim of this thesis is to explore the possibility of using vitamin A to correct immune defects associated with CVID. Specific aims are to i. compare the effects of RA on proliferation and Ig-synthesis in TLR9/RP105-stimulated B cells from CVID patients and normal controls and ii. elucidate how the enhancing effect of RA on TLR9/RP105-stimulation affects γ-irradiation-induced cell death.
Methods: CD19+ B cells were isolated from whole blood collected from CVID patients and healthy controls, and the cells were stimulated with different combinations of CpG and anti-RP105 (in vitro ligands for TLR9 and RP105, respectively), in the presence or absence of RA. The effects of the stimulants were measured with respect to proliferation, as analyzed by measuring incorporation of [3H]-thymidine, and by ELISA for quantification of IL-10 and Ig-synthesis. In addition, normal B cells were exposed to γ-irradiation followed by analysis of DNA damage-induced cell death by flow cytometry.
Results: i. RA was able to enhance TLR9/RP105-mediated stimulation of B cells both from CVID patients and healthy controls, and was able to restore the diminished proliferation and IL-10 synthesis in the CVID-derived B cells. Although RA markedly enhanced TLR9/RP105-mediated IgG synthesis in normal B cells, its effect on IgG synthesis in B cells from CVID patients was more modest. ii. Stimulation of RP105 prevented γ-irradiation-induced cell death in normal B cells, and CpG enhanced the protection induced by RP105. Weak, but reproducible additive effects of RA were noted.
Conclusion: Despite a relatively modest effect of RA on TLR9/RP105-induced IgG synthesis in CVID-derived B cells, the ability of RA to correct the defects in TLR9/RP105-mediated proliferation and IL-10 production in B cells from CVID patients, suggests that RA could have a role in treatment of this disease. The tendency that RA also contributed to the protective effects of CpG and anti-RP105 on γ-irradiation-induced cell death gives further support to this notion.