TURNER, T. J. | bioRxiv Science

bioRxiv2025

Maintenance of chronic neuroinflammation in multiple sclerosis via interferon signaling and CD8 T cell-mediated cytotoxicity

Chronic neuroinflammation and neurodegeneration are critical but unresolved drivers of disability accumulation in progressive multiple sclerosis (MS). Chronic active white matter lesions (CAL), identifiable radiologically as paramagnetic rim lesions (PRL), indicate progression-relevant chronic neuroinflammation. Using single-cell transcriptomics (scRNAseq) and T-cell receptor sequencing (scTCR-seq), we profiled cerebrospinal fluid (CSF) and blood immune cells of 34 radiologically characterized adults with MS (17 untreated, 6 treated with B-cell-depletion) and 5 healthy controls. Coupled with proteomics, we found PRL-associated enrichment of interferon (IFN) signaling and upregulation of TCR signaling in CSF and blood. This was accompanied by clonal expansion of CD8+ T effector memory (TEM) cells, with the highly expanded clonal cells exhibiting T helper type 1 (TH1) and cytotoxic profiles. Validating the cytotoxic immune profile in blood using flow cytometry, we identified a cellular correlate of PRL exhibiting features of CD8+ TEMRA cells. Despite B-cell depletion, PRL-associated neuroinflammation, driven by myeloid activation and CD8+ T-cell cytotoxicity, persisted. Serum and CSF proteomic networks showed PRL-pertinent signatures, including networks unaffected by B-cell depletion. Using in silico perturbation, we nominated therapeutic targets, including MYD88, TNF, MYC, TYK2, JAK2, and BTK, for alleviating chronic neuroinflammation in MS. Our findings highlight mechanisms of chronic neuroinflammation in MS and point to potential biomarkers for monitoring disease progression.

immunology↗

bioRxiv2024

Comparative CNS Pharmacology of the Brutons Tyrosine Kinase (BTK) Inhibitor Tolebrutinib Versus Other BTK Inhibitor Candidates for Treating Multiple Sclerosis.

Tolebrutinib is a covalent BTK inhibitor designed and selected for potency and CNS exposure to optimize impact on BTK-dependent signaling in CNS-resident cells. We applied a translational approach to evaluate three BTK inhibitors in Phase 3 clinical development in MS with respect to their relative potency to block BTK-dependent signaling and exposure in the CNS, employing in vitro kinase, cellular activation assays, and pharmacokinetic sampling of cerebrospinal fluid (CSF) in the non-human primate cynomolgus to estimate the ability of these candidates (evobrutinib, fenebrutinib, and tolebrutinib) to block BTK-dependent signaling inside the CNS. In vitro kinase assays demonstrated that tolebrutinib reacted with BTK 65-times faster than evobrutinib, while fenebrutinib, a classical reversible antagonist with a Ki value of 4.7 nM and slow off-rate (1.54 x 10-5 s-1), also had an association rate 1760-fold slower (3.28 x 103 M-1 * s-1). Estimates of cellular potency were largely consistent with the in vitro kinase assays, with an estimated IC50 of 0.7 nM for tolebrutinib against 34.5 nM for evobrutinib and 2.9 nM for fenebrutinib. We then observed that evobrutinib, fenebrutinib, and tolebrutinib achieved similar levels of exposure in non-human primate CSF after oral doses of 10 mg/kg. However, tolebrutinib CSF exposure (4.8 ng/mL) (kp,uu CSF=0.40) exceeded the IC90 (the estimated concentration inhibiting 90% of kinase activity) value, while evobrutinib (3.2 ng/mL) (kp,uu CSF=0.13) and fenebrutinib (12.9 ng/mL) (kp,uu CSF=0.15) failed to reach the estimated IC90 values. We conclude that tolebrutinib is the only candidate that attained relevant CSF exposure in non-human primates. DISCLOSURESTT, PB, DO: Employees of Sanofi (may hold shares and/or stock options in the company). RGnone.

pharmacology and toxicology↗