PROJECT SUMMARY Parkinson’s disease (PD) is a devastating neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra, movement abnormalities, and in some cases the accumulation of α-synuclein (α-syn) as fibrils or Lewy bodies. It is well established that T-cells are involved in PD pathology, shaping the inflammatory cascade from the periphery into the brain, and should be the focus of additional studies. Immune alterations in PD are found in peripheral blood indicating a pro-inflammatory status during disease and as early as prodromal stages. However, specific alterations are disputed and must be clarified with a large-scale study and a carefully curated single-cell cohort that controls for biological heterogeneity. T-cell infiltration into the brain has been reported to parallel α-syn accumulation and neuronal death but exploring infiltrated T-cells to characterize the role of these cells in neurodegeneration is a challenge. Genome-wide association studies (GWAS) have identified human leukocyte antigen (HLA) class II PD risk loci, which suggests an unexplored important role for CD4 T-cells in their interaction with antigen presenting cells. Overall, this work evaluates T-cell biomarker potential by investigating their contribution to PD in different clinical phases and explores their mechanistic role in pathogenesis. In this proposed work, the role of T-cells, particularly antigen-specific ones, in PD pathogenesis will be explored by characterizing immune cell expression and repertoire in PD patients, prodromal patients (who have elevated risk of disease, including GBA/LRRK2 genetic carriers and patients with rapid eye movement (REM) sleep behavior disorder) and control donors. Further, the clinical relevance and antigen specificity of clonal T- cells will be determined by comparing with α-syn-specific cells identified in in-vitro stimulation experiments. Investigating prodromal patients with an elevated risk of developing PD will assess the importance of T-cells in disease development and their potential as biomarkers. Additionally, harnessing the human Living Brain Cohort data, I will compare gene expression and immune repertoire between circulating and infiltrated T-cells from the same donors at a single-cell level. Comparing the T-cell receptors (TCRs) in the blood and brain provides a critical link between T-cells and disease progression, and can reveal whether T-cells of particular specificities have infiltrated into the brain parenchyma. This work will also substantiate the relationship between risk/protective HLA alleles and T-cell activity in PD patients by investigating the association between HLA alleles and TCR gene usage and the hypervariable complementarity determining region 3. The results of these experiments will further our comprehension of the role of T-cells in PD, lead to mechanistic discovery and determine the potential for targeted immune-based therapeutic interventions or biomarker...