The posttranscriptional modifications we examined were previously defined in the literature ( 17, 35, 37). The peptides included a set of nonmodified 16-mers overlapping by 8 aa that span the entire tau protein ( n = 55), as well as phosphorylated peptides ( n = 13) and one acetylated peptide (start position 169 Supplemental Table I). To determine whether tau peptides were recognized by T cells in our study participants ( Table I), PBMCs from the three cohorts (PD, HCam, and HC35) were stimulated for 14 d with tau-derived peptide pools containing 16 peptides each. Combinations of cytokine-producing cells were determined using Boolean gating. Frequencies of CD3 + T cells responding to tau megapool were quantified by determining the total number of gated CD3 + and cytokine + cells and background values subtracted (as determined from the medium alone control) using FlowJo X Software (FlowJo). Samples were acquired on a BD LSR II flow cytometer. Cells were stained for IFN-γ (anti–IFN-γ/APC), IL-17 (anti–IL-17/PECy7, eBio64DEC17 eBioscience), IL-4 (anti–IL-4/PE/Dazzle594), and IL-10 (anti–IL-10/AF488) in saponin buffer containing 10% FBS. After washing, cells were fixed using 4% paraformaldehyde and permeabilized using saponin buffer. After a total of 6 h, cells were harvested and stained for cell surface Ags CD4 (anti–CD4-APCEf780, RPA-T4 eBioscience), CD3 (anti–CD3-AF700, UCHT1 eBioscience), CD8 (anti–CD8-BV650, RPA-T8 BioLegend), CD14 (anti–CD14-V500, M5E2 BD Pharmingen), CD19 (anti–CD19-V500, HIB19 BD Pharmingen), and fixable viability dye eFluor 506 (eBiosciences). Unstimulated PBMCs were used to assess nonspecific/background cytokine production and PHA stimulation at 5 μg/ml was used as a positive control. After 2 h, 2.5 μg/ml each of brefeldin A and monensin was added for an additional 4 h at 37☌. PeptidesĪfter 14 d of culture, PBMCs were stimulated in the presence of 5 μg/ml tau peptide megapool for 2 h in complete RPMI 1640 medium at 37☌ with 5% CO 2. For the PD patients, the median age at onset was 58 y of age in the screening cohort and 65 y in the validation cohort. In the San Diego cohort, PD was self-reported. The Alabama cohort was recruited from the clinical practice of the UAB Movement Disorders Clinic. Parkinson’s Disease Brain Bank criteria, without excluding cases with a family history of PD ( 36). Blood samples were collected at the Columbia Center for Translational Immunology Human studies Core and approved by the CUMC Institutional Review Board. The New York cohort was recruited from the Center for Parkinson’s Disease at CUMC. We recruited a total of 59 participants with PD, 59 HCam without PD, and 60 HC35 without PD from the greater San Diego (PD, n = 10 and 14 for screening and validation, respectively HCam, n = 19 and 8 HC35, n = 21 and 36), New York City (PD, n = 12 and 17 HCam, n = 2 and 7 HC35, n = 0 and 0), and Alabama (PD, n = 0 and 6 HCam, n = 0 and 23 HC35, n = 0 and 3) areas. Thus, T cells recognizing tau epitopes escape central and peripheral tolerance in relatively high numbers, and the magnitude and nature of these responses are not modulated by age or PD disease. These results were verified in an independent set of PD and control donors (either age-matched or young controls). Some tau-derived epitopes, consisting of both unmodified and p-tau residues, were more highly represented in PD participants. The PD and control participants exhibited a similar magnitude and breadth of responses. All groups exhibited CD4 + T cell responses to tau-derived peptides, which were associated with secretion of IFN-γ, IL-5, and/or IL-4. We thus compared T cell responses to tau and p-tau–derived peptides between PD patients, age-matched healthy controls, and young healthy controls (<35 y old who are less likely to have high levels of tau aggregates). Although not known to produce autoimmune responses, we hypothesized that the appearance of aggregated tau and p-tau with disease could activate the immune system. Tau protein is found to be aggregated and hyperphosphorylated (p-tau) in many neurologic disorders, including Parkinson disease (PD) and related parkinsonisms, Alzheimer disease, traumatic brain injury, and even in normal aging.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |