Demyelination in the central nervous system (CNS) underlies many human diseases, including multiple sclerosis (MS). We report here the findings of our study of the CNS demyelination process using immune-induced [experimental autoimmune encephalomyelitis (EAE)] and chemical-induced [cuprizone (CPZ)] mouse models of demyelination. We found that necroptosis, a receptor-interacting protein 3 (RIP3) kinase and its substrate mixed lineage kinase domainlike protein (MLKL)-dependent cell death program, played no role in the demyelination process, whereas the MLKL-dependent, RIP3independent function of MLKL in the demyelination process initially discovered in the peripheral nervous system in response to nerve injury, also functions in demyelination in the CNS in these models. Moreover, a receptor-interacting protein 1 (RIP1) kinase inhibitor, RIPA-56, blocked disease progression in the EAE-induced model but showed no effect in the CPZ-induced model. It does so most likely at a step of monocyte elevation downstream of T cell activation and myelin-specific antibody generation, although upstream of breakdown of the blood-brain barrier. RIP1-kinase dead knock-in mice shared a similar result as mice treated with the RIP1 inhibitor. These results indicate that RIP1 kinase inhibitor is a potential therapeutic agent for immune-mediated demyelination diseases that works by prevention of monocyte elevation, a function previously unknown for RIP1 kinase.RIP1 kinase | myelin | demyelination | multiple sclerosis | MLKL M yelin is a lipid-rich (fatty) substance formed by glial cells called oligodendrocytes in the central nervous system (CNS) and by Schwann cells in the peripheral nervous system (PNS) that protects and nourishes neuron axons by wrapping the axons (1). Physiologically, the myelin structure speeds the transmission of electrical impulses called action potentials along myelinated axons by insulating the axon and reducing axonal membrane capacitance (2). The demyelination process occurs during nerve injury and happens in many human diseases, including, for example, in the PNS, Charcot-Marie-Tooth disease (3) and abdominal numbness brought about by diabetes (4), and in the CNS, multiple sclerosis (MS), during which the insulating covers of nerve cells in the brain and spinal cord are damaged by infiltrated immune cells, such as T cells and monocytes (5,6). Patients with MS are paralyzed gradually from the lower body to the upper body and eventually die; most of these patients are young adults ranging in age from their 20s-40s (7).The molecular mechanisms that underlie these demyelination diseases are largely unknown. A recent study indicated that preventing oligodendrocytes from necroptosis, a regulated form of necrotic cell death induced by ligands of the tumor necrosis factor (TNF) receptor family or Toll-like receptors, as well as certain viral infections, could decrease disease symptoms in MS disease models of experimental autoimmune encephalomyelitis (EAE) and a cuprizone (CPZ)-induced demyelination model (8). The TNF re...

CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics. Remarkably, CRISPR/Cas9-induced SVs persist and undergo clonal expansion in vivo after three weeks or even two months, evidenced by high enrichment and low junctional diversity of identified SVs post infusion. Specifically, we detected 128 expanded translocations, with 20 615 as the highest number of amplicons. The identified SVs are stochastically selected among different individuals and show an inconspicuous locus preference. Similar to SVs, viral DNA integrations are routinely detected in edited T cells and also undergo clonal expansion. The persistent SVs and viral DNA integrations in the infused T cells may constantly threaten genome integrity, drawing immediate attention to the safety of CRISPR/Cas9-engineered T cells mediated immunotherapy.