CD8 þ T cells are important in protective immunity against intracellular pathogens and tumors. In chronic infections or cancer, CD8 þ T cells are constantly exposed to antigens and inflammatory signals. Such excessive and constitutive signals lead to the deterioration of T cell function, called 'exhaustion'. Exhausted T cells are characterized by low proliferation in response to antigen stimulation, progressive loss of effector function (cytokine production and killing function), expression of multiple inhibitory receptors such as PD-1, Tim3, and LAG3, and metabolic alterations from oxidative phosphorylation to glycolysis. These dysfunctions are associated with altered transcriptional programs and epigenetic regulations and recent studies suggested that NR4a and TOX transcription factors are deeply involved in exhaustion phenotypes. However, an increase the early memory T cells including stem cell memory T (T SCM) cells is critical for T cell persistence and efficient tumor killing especially for adoptive cancer immunotherapy such as CART cell therapy. An increasing amount of evidence supports the therapeutic potential of targeting exhausted T cells and T SCM cells. We have begun to understand the molecular mechanisms of T cell exhaustion and early memory formation, and the clinical application of converting exhausted T cells to rejuvenated early memory T cells is the goal of our study.

Recent studies have shown that stem cell memory T (T SCM ) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor-engineered-T (CAR-T) cells. We previously reported that both human and murineactivated T cells are converted into stem cell memory-like T (iT SCM ) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCHmediated iT SCM reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into T SCM -like CAR-T, "CAR-iT SCM " cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iT SCM formation, which are essential for the properties of iT SCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iT SCM differentiation. Like NOTCH-induced CAR-iT SCM cells, FOXM1-induced CAR-iT SCM cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCHor FOXM1-driven CAR-iT SCM formation is an effective strategy for improving cancer immunotherapy.Significance: Manipulation of signaling and metabolic pathways important for directing production of stem cell memory-like T cells may enable development of improved CAR-T cells.

Inflammation and immune responses after tissue injury play pivotal roles in the pathology, resolution of inflammation, tissue recovery, fibrosis and remodeling. Regulatory T cells (Tregs) are the cells responsible for suppressing immune responses and can be activated in secondary lymphatic tissues, where they subsequently regulate effector T cell and dendritic cell activation. Recently, Tregs that reside in non-lymphoid tissues, called tissue Tregs, have been shown to exhibit tissue-specific functions that contribute to the maintenance of tissue homeostasis and repair. Unlike other tissue Tregs, the role of Tregs in the brain has not been well elucidated because the number of brain Tregs is very small under normal conditions. However, we found that Tregs accumulate in the brain at the chronic phase of ischemic brain injury and control astrogliosis through secretion of a cytokine, amphiregulin (Areg). Brain Tregs resemble other tissue Tregs in many ways but, unlike the other tissue Tregs, brain Tregs express neural-cell-specific genes such as the serotonin receptor (Htr7) and respond to serotonin. Administering serotonin or selective serotonin reuptake inhibitors (SSRIs) in an experimental mouse model of stroke increases the number of brain Tregs and ameliorates neurological symptoms. Knowledge of brain Tregs will contribute to the understanding of various types of neuroinflammation.

T cells with a stem cell memory (TSCM) phenotype provide long-term and potent antitumor effects for T-cell transfer therapies. Although various methods for the induction of TSCM-like cells in vitro have been reported, few methods generate TSCM-like cells from effector/exhausted T cells. We have reported that coculture with the Notch ligand–expressing OP9 stromal cells induces TSCM-like (iTSCM) cells. Here, we established a feeder-free culture system to improve iTSCM cell generation from expanded chimeric antigen receptor (CAR)-expressing T cells; culturing CAR T cells in the presence of IL7, CXCL12, IGF-I, and the Notch ligand, hDLL1. Feeder-free CAR-iTSCM cells showed the expression of cell surface markers and genes similar to that of OP9-hDLL1 feeder cell–induced CAR-iTSCM cells, including the elevated expression of SCM-associated genes, TCF7, LEF1, and BCL6, and reduced expression of exhaustion-associated genes like LAG3, TOX, and NR4A1. Feeder-free CAR-iTSCM cells showed higher proliferative capacity depending on oxidative phosphorylation and exhibited higher IL2 production and stronger antitumor activity in vivo than feeder cell–induced CAR-iTSCM cells. Our feeder-free culture system represents a way to rejuvenate effector/exhausted CAR T cells to SCM-like CAR T cells. Significance: Resting CAR T cells with our defined factors reprograms exhausted state to SCM-like state and enables development of improved CAR T-cell therapy.

Inflammation and immune responses after stroke, including ischemic cerebral infarction, play pivotal roles in the pathology, resolution of inflammation, and neurological recovery. Regulatory T (Treg) cells are the cells responsible for immune tolerance, usually activated by secondary lymphatic tissues, which subsequently regulate effector T cell activation and dendritic cell activation. Recently, Tregs that are present in tissues, called tissue Tregs, have been shown to exhibit tissue-specific functions in addition to immune regulation, contributing to the maintenance of homeostasis and tissue repair. We found that Tregs accumulate in the brain at the chronic phase of ischemic brain injury and control astrogliosis through the secretion of amphiregulin. Unlike other tissue Tregs, brain Tregs express a serotonin receptor (Htr7) that is characteristic of the nervous system, and are proliferated and activated by serotonin. Administering a serotonin or selective serotonin reuptake inhibitor (SSRI) in an experimental stroke mouse model increased the number of brain Tregs and improved neurological symptoms. Elucidation of the significance of brain Tregs should also contribute to the understanding of other types of neuroinflammation.

No abstract

<div>Abstract<p>Recent studies have shown that stem cell memory T (T_SCM) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor–engineered-T (CAR-T) cells. We previously reported that both human and murine-activated T cells are converted into stem cell memory-like T (iT_SCM) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iT_SCM reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into T_SCM-like CAR-T, “CAR-iT_SCM” cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iT_SCM formation, which are essential for the properties of iT_SCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iT_SCM differentiation. Like NOTCH-induced CAR-iT_SCM cells, FOXM1-induced CAR-iT_SCM cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCH- or FOXM1-driven CAR-iT_SCM formation is an effective strategy for improving cancer immunotherapy.</p>Significance:<p>Manipulation of signaling and metabolic pathways important for directing production of stem cell memory–like T cells may enable development of improved CAR-T cells.</p></div>

<p>Supplementary Figure S1. Metabolic analysis before or after the OP9-hDLL1 co-culture. Supplementary Figure S2. Metformin disrupts iTSCM induction Supplementary Figure S3. Anti-CD19 CAR-iTSCM cells possess superior antitumor ability in B-ALL models. Supplementary Figure S4. The effects of Notch intracellular domain(NICD) overexpression on T cell phenotypes. Supplementary Figure S5. NICDÎ"ANK-overexpression does not induce iTSCM cells Supplementary Figure S6. The regulation of FOXM1 expression by NICD or FOXM1 knockout. Supplementary Figure S7. FOXM1â^†N-overexpression induces iTSCM formation Supplementary Figure S8. NICD- and FOXM1-overexpressed TSCM-like cells are close to iTSCM cells induced by OP9-hDLL1 cells.</p>