Shinji Oki

In addition to disease-associated microglia (DAM), microglia with MHC-II and/or IFN-I signatures may form additional pathogenic subsets that are relevant to neurodegeneration. However, the significance of such MHC-II and IFN-I signatures remains elusive. We demonstrate here that these microglial subsets play intrinsic roles in orchestrating neurotoxic properties of neurotoxic Eomes⁺ Th cells under the neurodegeneration-associated phase of experimental autoimmune encephalomyelitis (EAE) that corresponds to progressive multiple sclerosis (MS). Microglia acquire IFN-signature after sensing ectopically expressed long interspersed nuclear element-1 (L1) gene. Furthermore, ORF1, an L1-encoded protein aberrantly expressed in the diseased central nervous system (CNS), stimulated Eomes⁺ Th cells after Trem2-dependent ingestion and presentation in MHC-II context by microglia. Interestingly, administration of an L1 inhibitor significantly ameliorated neurodegenerative symptoms of EAE concomitant with reduced accumulation of Eomes⁺ Th cells in the CNS. Collectively, our data highlight a critical contribution of new microglia subsets as a neuroinflammatory hub in immune-mediated neurodegeneration.

Pathogenic T helper cells (Th cells) that respond to self‐antigen cannot be easily distinguished from beneficial Th cells. These cells can generate systemic autoimmune disease in response to widely expressed self‐antigens. In this study, we have identified neuropilin‐1 (NRP1) as a cell surface marker of self‐reactive Th cells. NRP1⁺ Th cells, absent in non‐regulatory T cell subsets in normal mice, appeared in models of systemic autoimmune disease and strongly correlated with disease symptoms. NRP1⁺ Th cells were greatly reduced in Nr4a2 cKO mice, which have reduced self‐reactive responses but showed normal responses against exogenous antigens. Transfer of NRP1⁺ Th cells was sufficient to initiate or accelerate systemic autoimmune disease, and targeting NRP1‐expressing Th cells therapeutically ameliorated SLE‐like autoimmune symptoms in BXSB‐Yaa mice. Peripheral NRP1⁺ Th cells were significantly increased in human SLE patients. Our data suggest that self‐reactive Th cells can be phenotypically distinguished within the Th cell pool. These findings offer a novel approach to identify self‐reactive Th cells and target them to treat systemic autoimmune disease.

Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5′-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research.

Significance: Multiple sclerosis (MS) can transition from a relapsing-remitting form (RRMS) into a chronic progressive form (secondary progressive MS, SPMS). SPMS pathogenesis remains poorly understood with diagnosis based entirely on retrospective clinical monitoring. We show that T helper cells expressing the transcription factor Eomes (Eomes ⁺ Th cells) were significantly increased in peripheral blood from SPMS patients versus healthy subjects or RRMS and other progressive MS patients. Moreover, Eomes ⁺ Th cells expressing granzyme B were found infiltrating brain tissues in SPMS autopsy samples, providing support for a pathogenic role. Analysis of clinical status on follow-up indicated the value of measuring Eomes ⁺ Th cells for SPMS diagnosis and prognostic monitoring. This study may contribute to future development of biomarker-assisted “precision medicine” for MS.

Significance: We have previously demonstrated that induction of pathogenic eomesodermin-positive CD4 ⁺ T cells (Eomes ⁺ T helper [Th] cells) is associated with transition from an acute stage to a later stage of experimental autoimmune encephalomyelitis (EAE). In the late phase of EAE, B cells and non-B cell antigen-presenting cells (APCs) recruited to the central nervous system strikingly up-regulate prolactin (PRL). The PRL-producing APCs have the potential to promote generation of Eomes ⁺ Th cells from naïve T cells in an MHC class II-restricted manner, and therapies inhibitory for PRL production suppress the induction of Eomes ⁺ Th cells and ameliorate clinical signs of EAE. Our study highlights the unexpected role of extrapituitary PRL in the development of persistent neuroinflammation.