To this end, we developed a series of multiparametric circulation cytometry panels to capture the functional phenotypes of T cells and myeloid cells (eFigure 4, links.lww.com/NXI/A676). Among major immune cell types, B cells Avarofloxacin appeared to be preferentially affected in PD. Proliferating B cell counts were decreased in patients with PD compared with controls. Proportions of B-cell subsets with regulatory capacity such as transitional B cells were preferentially reduced in the patients with PD, whereas proportions of proinflammatory cytokine-producing B cells increased, resulting in a proinflammatory shift of their B-cell functional cytokine responses. Unsupervised principal component analysis revealed increased expression of TNF and GM-CSF by both B cells and T cells of patients with PD. In addition, levels of follicular T cells, an important B-cell helper T-cell populace, decreased in the patients with PD, correlating with their B-cell abnormality. Conversation Our findings define a novel signature of peripheral immune cells and implicate aberrant Tfh:B-cell interactions in patients with PD. Parkinson disease (PD) is usually a neurodegenerative disease that affects more than 6 million people globally.1 The etiology of PD, which is characterized by a progressive loss of dopaminergic neurons, remains elusive.2,3 Emerging evidence suggests that immune system responses may be involved in PD pathogenesis.4 Genome-wide association studies (GWASs) relate haplotypes of the major histocompatibility complex (MHC) class II genes and other immune-related genes (e.g., Values of 0.05 or less were considered statistically significant. Data Availability Anonymized data will be shared by request from any qualified investigator. Results Immunophenotyping Major Immune Cell Subsets in Whole Blood of Patients With PD and Neurologically Normal Controls To broadly characterize the major circulating immune cell types of patients with PD and neurologically NCs we established a 15-color circulation cytometry panel to immune phenotype in Smad7 new whole blood: neutrophils (CD15+ CD16+), eosinophils (Siglec-8+), basophils (CD123high), monocytes (CD14+), dendritic cells (Lin? HLA-DR+), NK cells (CD14?, CD16+), T cells (CD3+), and B cells (CD19+), with the antibody panel and gating strategy summarized in eTable 4 and eFigure 1, respectively. Analysis of the (UPA) discovery cohort (eTables 1 and 2) suggested that complete B-cell counts Avarofloxacin were decreased in the blood of patients with PD compared with NCs (Physique 1A), with no apparent differences in other major cell types including granulocytes, myeloid cells, NK cells, and T cells. This observation was confirmed in an impartial (Columbia University or college) validation cohort (eTable 1 and eTable 2, links.lww.com/NXI/A676; Physique 1B), pointing to an abnormal B-cell immune compartment in patients with PD. Open in a separate window Physique 1 Whole Blood Immunophenotyping of Major Peripheral Immune Cell Types in Patients With PD and Neurologically Normal ControlsWhole blood samples were collected from 2 impartial cohorts of patients with well-characterized Parkinson disease (PD) and normal controls (NCs). New samples underwent reddish blood cell lysis and immediately stained with the indicated panel of antibodies to capture major immune cell types including neutrophils, eosinophils, basophils, monocytes, dendritic cells, NK cells, T cells, and B cells. (A) Complete counts of circulating B cells are decreased in patients with PD relative to NCs in the discovery (UPA) cohort (comprising n(NC) = 7, n(PD) = 13) and (B) confirmed in the validation Avarofloxacin (Columbia) cohort (n(NC) = 8, n(PD) = 21). ns = not significant, * 0.05. Comprehensive Immunophenotyping Within the B-Cell Compartment Reveals a Preferential Decrease of Transitional B-Cell Populations To analyze distinct subsets more comprehensively within the B-cell compartment in patients with PD, we next developed and applied 3 15-color multiparametric circulation cytometry panels to cryopreserved PBMCs isolated from patients and controls. These panels captured both phenotypic and functional profiles of B-cell subsets (eTable 4; eFigure 2A, links.lww.com/NXI/A676), including quantification of activation and proliferation markers, molecules associated with antigen-presenting function, and known B-cell cytokines previously implicated in immune activation (e.g., TNF, IL-6, and GM-CSF) and immune regulation (e.g., IL-10). Interpretation of the PBMC analyses and validity of comparisons across groups were optimized by applying strict standard operating procedures20 and routinely confirming high cell viabilities (eFigure 2B). We.