Article Figures & Data
Figures
- Figure 1 Whole Blood Immunophenotyping of Major Peripheral Immune Cell Types in Patients With PD and Neurologically Normal Controls
Whole blood samples were collected from 2 independent cohorts of patients with well-characterized Parkinson disease (PD) and normal controls (NCs). Fresh samples underwent red 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) Absolute 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 (Columbia) cohort (n(NC) = 8, n(PD) = 21). ns = not significant, *p < 0.05.
- Figure 2 B-Cell Subset Analysis in Blood of Patients With Parkinson Disease and Normal Controls
Cryopreserved peripheral blood mononuclear cells were thawed and stained with B cell–related markers. (A–G) Among all B-cell subsets, transitional B cells are particularly affected in patients with Parkinson disease (PD) (A–C) resulting in reduced ratios of their transitional to mature B cells (E). ns = not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. Discovery cohort (comprising n(NC) = 12, n(PD) = 17) and validation cohort (n(NC) = 18, n(PD) = 18). NC = normal control.
- Figure 3 B-Cell Activation, Proliferation, and Costimulation in Parkinson Disease and Normal Controls
Cryopreserved peripheral blood mononuclear cells were thawed and stained with B cell–related markers to capture their activation/proliferation states and their costimulation capacity. Although the activation marker CD71 did not show consistent changes in the 2 cohorts (A), patients with Parkinson disease (PD) harbor decreased counts of circulating B cells expressing the proliferation-associated marker Ki-67 (B), and the decreased proliferation correlates with their decreased B-cell counts (C). A decrease in CD80+ B cells was only observed in the discovery cohort but not in the validation cohort (D). Levels of both CD11c+ CD86+ atypical memory B cells (E) and CD43+ CD27+ B1 cells (F) are decreased in the circulation of patients with PD. ns = not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. Discovery cohort (comprising n(NC) = 12, n(PD) = 17) and validation cohort (n(NC) = 18, n(PD) = 18). NC = normal control.
- Figure 4 Cytokine Profiles of B Cells in Normal Controls and Parkinson Disease
Cryopreserved peripheral blood mononuclear cells were thawed and rested overnight before stimulation with PMA and ionomycin in the presence of GolgiStop for 4 hours. B-cell expression of IL-10, GM-CSF, TNFα, and IL-6 was detected by flow cytometry using intracellular cytokine staining. IL-10–producing B-cell counts are reduced in patients with Parkinson disease (PD) (A), whereas GM-CSF and TNFα-expressing B-cell frequencies are increased in patients with PD (B), which together results in decreases of both GM-CSF/IL-10 ratios (C) and TNFα/IL-10 ratios (D) ratios of cytokine-expressing B cells in PD compared with normal control (NC). ns = not significant, *p < 0.05, and **p < 0.01. Discovery cohort (comprising n(NC) = 12, n(PD) = 17) and validation cohort (n(NC) = 18, n(PD) = 18).
- Figure 5 Decreased Counts of Follicular T Cells Correlate With Decreased Counts of Ki-67+ B Cells in Patients With Parkinson Disease
(A–D) Cryopreserved peripheral blood mononuclear cells were thawed and stained with panels of T cell–related phenotypic and functional markers (eTable 4 and Figure 6) and then analyzed by flow cytometry in the discovery cohorts and validation cohorts of patients with Parkinson disease (PD) and normal controls (NCs). (A) Proliferating T cells (Ki-67+ T cells) are reduced in patients with PD. (B) Within major T-cell subsets, counts of central memory T cells are decreased in patients with PD. (C and D) Decreased counts of follicular T (Tfh) cells are seen in patients with PD, and these reduced Tfh cell counts correlate with the decreased Ki-67–expressing B cells (E and F). ns = not significant, *p < 0.05 and **p < 0.01. Discovery cohort (comprising n(NC) = 12, n(PD) = 17) and validation cohort (n(NC) = 18, n(PD) = 18).
- Figure 6 Identification of the Immune Signature of Peripheral Immune Cells in Patients With Parkinson Disease Using Unbiased PC Analysis
Principal component (PC) analysis was applied to the complete data set containing 198 features comprising both counts and frequencies of different immune cell subset/functional markers. (A) Percent variance accounted for by each of the first 10 principal components. (B) Plot of PC1 vs PC2 showing separation between Parkinson disease (PD) and normal control (NC) along PC2. (C) Boxplot of PC2 values grouped by disease status and cohort. (D) ROC curve of PC2 values (area under the curve (AUC): 0.88, p < 0.0001). (E) Top 30 features within the PC2 loadings. (F and G) TNFα and GM-CSF expressing B-cell and T-cell subsets are increased in patients with PD. ns = not significant, ***p < 0.001 and ****p < 0.0001. Discovery cohort (comprising n(NC) = 12, n(PD) = 17) and validation cohort (n(NC) = 18, n(PD) = 18).
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