Single cell RNAseq in the CNS and blood reveals T-cell states in healthy and diseased individuals

Jenna Pappalardo, David van Dijk, Le Zhang, Maggie Pecsok, Smita Krishnaswamy and David A. Hafler

Departments of Neurology, Immunobiology, and Genetics, Yale School of Medicine, New Haven, CT

The peripheral immune system is becoming increasingly appreciated as an integral component of tissue homeostasis. Tissues develop unique immune states through the regulated recruitment and retention of specific subsets of immune cells which then become further specialized based on surrounding tissue cues. Here, we explore the continuum of T cell states present in the peripheral blood and cerebrospinal fluid (CSF) of healthy individuals to define how the central nervous system (CNS) shapes T cell profiles and function using Seq-Well as a platform for massively-parallel single-cell RNA sequencing. We leveraged the natural progression of the single-cell data represented by Potential of Heat-diffusion for Affinity-based Trajectory Embedding (PHATE) to generate a tissue score that provided the basis for determining the acquisition of tissue-specific characteristics. We validated that this approach mirrored current knowledge about the state of T cells in CSF, including the increased expression of the brain-homing trafficking molecules ITGA4 and CXCR3. Moreover, we have identified metabolic pathways and other pathways necessary for maintaining brain homeostasis are enriched in CSF T cells. Further in vitro modeling of factors in the CNS that may shape T cell function are elucidating how these alterations converge on altering T cell function. Finally, this atlas of healthy CSF allows us to examine perturbation of the system by CNS autoimmune disease and how those perturbations can be corrected by immunotherapy. Thus, these findings will inform our understanding of immune-tissue interactions in the healthy brain as a critical basis for contextualizing perturbations in neuroinflammatory and neurodegenerative disease.