Fig. 1.

Generation of Aβ-specific Th1 cells retrovirally transduced to overexpress and secrete a biologically active BDNF.

(A) A scheme showing the generation of BDNF-producing, Aβ-specific Th1 cells. Two month-old mice were first vaccinated with Aβ1–42. After 7 d, CD4 T cells were purified from draining lymph nodes and stimulated with Aβ in the presence of antigen-presenting cells. The CD4 T cells were then polarized to a Th1 phenotype and transduced with the pMP71-EGFP-PRE and pMP71-BDNF-T2A-EGFP-PRE retroviruses to generate Th1-GFP and Th1-BDNF T cells, respectively. (B) Representative flow cytometry histograms showing EGFP mean fluorescence intensity (MFI) in Th1-GFP and Th1-BDNF T cells. (C) Secreted levels of BDNF measured by ELISA in supernatants of Th1-GFP and Th1-BDNF cells, 48 h after activation with anti-CD3/anti-CD28 Dynabeads. (D) WB analysis of BDNF expression in Th1-GFP and Th1-BDNF whole-cell lysates. Th1 cells producing GFP and BDNF were activated with anti-CD3/anti-CD28 Dynabeads and the cells were collected 48 h later. Recombinant BDNF (rBDNF) was used as a control. (E) Representative images of Th1-GFP and Th1-BDNF cells immunolabeled with anti-BDNF (red) and nuclei-labeled with DAPI (blue). (F) WB analysis of non-phosphorylated and phosphorylated TrkB (707/706) and ERK1/2 in lysates of HEK293T TrkB-transduced cells treated with Th1-GFP and Th1-BDNF cell supernatants (2.5 ng/ml BDNF) or with recombinant BDNF (rBDNF, 2.5 ng/ml) for 15, 30, 45, or 60 min, as indicated. Tubulin served as an internal loading control.