Increased Number of Plasma B Cells Producing Autoantibodies Against Aβ42 Protofibrils in Alzheimer's Disease

Abstract

The Alzheimer's disease (AD)-related peptide amyloid-β (Aβ) has a propensity to aggregate into various assemblies including toxic soluble Aβ protofibrils. Several studies have reported the existence of anti-Aβ antibodies in humans. However, it is still debated whether levels of anti-Aβ antibodies are altered in AD patients compared to healthy individuals. Formation of immune complexes with plasma Aβ makes it difficult to reliably measure the concentration of circulating anti-Aβ antibodies with certain immunoassays, potentially leading to an underestimation. Here we have investigated anti-Aβ antibody production on a cellular level by measuring the amount of anti-Aβ antibody producing cells instead of the plasma level of anti-Aβ antibodies. To our knowledge, this is the first time the anti-Aβ antibody response in plasma has been compared in AD patients and age-matched healthy individuals using the enzyme-linked immunospot (ELISpot) technique. Both AD patients and healthy individuals had low levels of B cells producing antibodies binding Aβ40 monomers, whereas the number of cells producing antibodies toward Aβ42 protofibrils was higher overall and significantly higher in AD compared to healthy controls. This study shows, by an alternative and reliable method, that there is a specific immune response to the toxic Aβ protofibrils, which is significantly increased in AD patients.

Keywords: Amyloid-β; amyloid-β protofibrils; anti-amyloid-β antibodies; enzyme-linked immunospot assay.

Fig.1

B cell ELISpot. A) PBMCs were collected by Ficoll-Paque separation, stimulated with R848 and hIL-2 to start their antibody production and B) added to ELISpot wells coated with anti-IgG capture antibodies. C) Biotinylated Aβ binds to the captured anti-Aβ antibodies secreted from the activated B cells. D) The dots representing cells secreting anti-Aβ antibodies were visualized by Streptavidin-ALP.

Fig.2

Characterization of PBMCs and validation of the antigen. A) Total IgG production from activated B cells. Number of spots representing IgG producing cells detected in the ELISpot. AD individuals (n = 39) (■) had higher number of spots than healthy controls (n = 30) (•) (p≤0.05). PBMCs from individuals failing activation (spots <20) (AD patients (n = 7), healthy controls (n = 10)) were excluded from the study (below horizontal line). B) B cell levels in AD patients and healthy individuals. The proportion of B cells in PBMCs was defined with flow cytometry by determining the number of CD19+ cells. There was no significant difference in the percentage of B cell in AD patients compared to healthy controls (p = 0.085). C) Aβ₄₂ protofibril evaluation with protofibril specific ELISA. No considerable difference was noticed comparing biotinylated Aβ₄₂ protofibrils with non-biotinylated Aβ₄₂ protofibrils.

Fig.3

B cell production of anti-Aβ₄₀ monomer and anti-Aβ₄₂ protofibril antibodies. A) Number of spots representing cells producing antibodies to Aβ₄₀ monomers (■) and Aβ₄₂ protofibrils (•). Looking at the whole study material (n = 63), antibodies detected Aβ₄₂ protofibrils to a higher degree than Aβ₄₀ monomers (p = <0.0001). B) B cell production of anti-Aβ₄₀ monomer antibodies. Number of spots representing cells producing antibodies to Aβ₄₀ monomers in AD patients (n = 37) (■) and healthy individuals (n = 26) (•) No significant difference was seen when comparing AD patients with healthy controls (p <  0.23). C) Number of spots representing cells producing antibodies detecting Aβ₄₂ protofibrils in AD individuals (n = 37) (■) and healthy individuals (n = 26) (•). There was a significant difference in number of spots comparing AD patients with healthy individuals (p <  0.05). D) In AD patients, the number of spots representing B cells producing antibodies binding to Aβ₄₂ protofibrils correlated weakly with the number of spots representing the total number of IgG producing B cells (Spearman r = 0.59, p <  0.001).

Fig.4

Anti-Aβ₄₂ protofibril antibody response correlated with mini mental state. No correlation was seen when comparing the levels of anti-Aβ₄₂ protofibril selective antibody responses to the state of cognition in AD patients (n = 22).