Multiple sclerosis (MS) is a chronic inflammatory CNS disease with heterogeneous manifestation. Prognostic markers for early classification of MS are currently under investigation. Higher diagnostic resolution of cerebrospinal fluid (CSF) has the potential to contribute significantly to patient stratification, which should be especially important for a subgroup of patients with high risk to convert to a progressive disease course. This study aimed to determine whether spectral flow cytometry of CSF cells could identify pathogenic CD4+ T cell subset in MS. Using a two-step approach, we designed a marker panel informed by publicly available transcriptomic datasets from early human MS and our own single-cell RNA sequencing (scRNA-seq) in acute and chronic experimental autoimmune encephalomyelitis (EAE), a murine MS model. Notably, chronic ('phase') markers such as and (associated with memory T cells), (integrin beta-1) and anti-apoptotic genes like , and were enriched in EAE. These markers reflect pro-survival signalling and tissue-residency characteristics, including , and , which suggest an adaptation of CD4+ T cells towards persistent neuroinflammatory responses in chronic EAE. This phase-specific marker profile highlights CD4+ T cells as both indicators and contributors to disease progression in EAE. Translating these findings to MS datasets, we found an enrichment of phase-specific markers in CSF cells. Spectral flow cytometry in an independent MS cohort revealed distinct memory and effector T cell subsets, indicating unique CSF signatures in MS. This study underscores the heterogeneity and dynamic changes of CD4+ T cells detectable by spectral flow cytometry, enhancing diagnostic resolution of CSF cells and informing more precise therapeutic strategies for MS.