Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease

Abstract

Alzheimer disease (AD) is characterized by the progressive deposition of the 42-residue amyloid beta protein (Abeta) in brain regions serving memory and cognition. In animal models of AD, immunization with Abeta results in the clearance of Abeta deposits from the brain. However, a trial of vaccination with synthetic human Abeta1-42 in AD resulted in the development of meningoencephalitis in some patients. We measured cellular immune responses to Abeta in middle-aged and elderly healthy subjects and in patients with AD. A significantly higher proportion of healthy elderly subjects and patients with AD had strong Abeta-reactive T cell responses than occurred in middle-aged adults. The immunodominant Abeta epitopes in humans resided in amino acids 16-33. Epitope mapping enabled the identification of MHC/T cell receptor (TCR) contact residues. The occurrence of intrinsic T cell reactivity to the self-antigen Abeta in humans has implications for the design of Abeta vaccines, may itself be linked to AD susceptibility and course, and appears to be associated with the aging process.

Figure 1

Aβ1–42 is more immunogenic than Aβ1–40. PBMCs were isolated from nine subjects (three adults, three older healthy subjects, and three patients with clinically mild or moderate AD) and cultured in U-bottom 96-well plates in the presence of human Aβ1–40 or Aβ1–42 (30 wells per peptide) as described in Methods. Cells were then restimulated at day 10 in the presence or absence of the peptide used initially. T cell proliferation was measured by [³H]thymidine incorporation 72 hours after the secondary stimulation. The percentage of positive wells was obtained according to the number of wells (out of 30) with SIs (cpm in the presence of antigen divided by cpm in the absence of antigen) of at least 2.5 and a Δcpm of at least 2,000.

Figure 2

Aβ T cell reactivity is increased in elderly subjects and patients with AD. PBMCs were isolated from adult and elderly healthy subjects and patients with AD as described in Methods. Aβ-induced proliferation was measured after secondary stimulation in the split-well assay. (a) Percentage of positive wells was calculated for each individual as in Figure 1. (b) Average SIs ± SEM were calculated for each group on the basis of the average SIs for each individual (sum of positive SIs [each at least 2.5 with a Δcpm of > 2,000] divided by the number of positive wells). Two-tailed P values were calculated using the alternate Welch t test, assuming Gaussian populations with unequal SDs.

Figure 3

Epitope specificity and cytokine profile of Aβ-reactive T cell lines. Split-well assays were performed as described in Methods. To generate Aβ-reactive T cell lines, positive wells were restimulated in the presence of irradiated PBMCs and Aβ and then maintained with 10 U/ml IL-2. To determine Aβ and epitope specificity, T cell lines were stimulated with Aβ1–42 and two overlapping peptides, Aβ1–28 or Aβ15–42, followed by their stimulation with nested peptides of the Aβ15–42 region. To measure Aβ-induced cytokine production, supernatants were collected 48 hours after stimulation and examined by ELISA. T cell proliferation of three representative lines is shown: (a–c) induced by Aβ1–42 (a and c) Aβ15–42, and (b) Aβ1–28. T cell proliferation was also induced by (d) Aβ16–30 (e) Aβ19–33, and (f) Aβ28–42. Aβ-induced secretion of IFN-γ and IL-13 is shown in g through l.

Figure 4

Activated Aβ-reactive T cells exhibit Th1, Th2, and Th0 phenotypes after stimulation with autologous PBMCs and Aβ peptides. Aβ-reactive T cells were established from a secondary stimulation assay as described in Methods. Resting cells were cultured with autologous PBMCs alone or stimulated in the presence of Aβ1–42, Aβ15–42, or Aβ1–28 and harvested after 30 hours. Cells were stained with Cy-chrome–labeled anti-CD4 and FITC-labeled anti-CD69 (a) followed by intracellular staining with phycoerythrin-labeled anti–IFN-γ, –IL-13, –IL-5, –IL-10, and –IL-12 (b). Cells also were stained with Cy-chrome–labeled anti-CD4 followed by intracellular staining with FITC-labeled anti–IFN-γ and phycoerythrin-labeled anti–IL-13 or anti–IL-5 (c). Positive cells were not observed when intracellular staining was performed with isotype controls (data not shown). For (b) and (c), analysis was gated on CD4⁺ cells. No Ag., no antigen.