NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment.

T cell exhaustion impairs tumor immunity and contributes to resistance against immune checkpoint inhibitors. The nuclear receptor subfamily 4 group A (NR4a) family of nuclear receptors plays a crucial role in driving T cell exhaustion. In this study, we observe that NR4a1 and NR4a2 deficiency in CD8+ tumor-infiltrating lymphocytes (TILs) results in potent tumor eradication and exhibits not only reduced exhaustion characteristics but also an increase in the precursors/progenitors of exhausted T (Pre-Tex) cell fraction. Serial transfers of NR4a1-/-NR4a2-/-CD8+ TILs into tumor-bearing mice result in the expansion of TCF1+ (Tcf7+) stem-like Pre-Tex cells, whereas wild-type TILs are depleted upon secondary transfer. NR4a1/2-deficient CD8+ T cells express higher levels of stemness/memory-related genes and illustrate potent mitochondrial oxidative phosphorylation. Collectively, these findings suggest that inhibiting NR4a in tumors represents a potent immuno-oncotherapy strategy by increasing stem-like Pre-Tex cells and reducing exhaustion of CD8+ T cells.

SOCS3 deletion in effector T cells confers an anti-tumorigenic role of IL-6 to the pro-tumorigenic cytokine.

Interleukin (IL)-6 is abundantly expressed in the tumor microenvironment and is associated with poor patient outcomes. Here, we demonstrate that the deletion of the suppressor of cytokine signaling 3 (SOCS3) in T cells potentiates anti-tumor immune responses by conferring the anti-tumorigenic function of IL-6 in mouse and human models. In Socs3-deficient CD8+ T cells, IL-6 upregulates the expression of type I interferon (IFN)-regulated genes and enhances the anti-tumor effector function of T cells, while also modifying mitochondrial fitness to increase mitochondrial membrane potential and reactive oxygen species (ROS) levels and to promote metabolic glycolysis in the energy state. Furthermore, Socs3 deficiency reduces regulatory T cells and increases T helper 1 (Th1) cells. SOCS3 knockdown in human chimeric antigen receptor T (CAR-T) cells exhibits a strong anti-tumor response in humanized mice. Thus, genetic disruption of SOCS3 offers an avenue to improve the therapeutic efficacy of adoptive T cell therapy.

Rejuvenating Effector/Exhausted CAR T Cells to Stem Cell Memory-Like CAR T Cells By Resting Them in the Presence of CXCL12 and the NOTCH Ligand.

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.

Memory T cell, exhaustion, and tumor immunity.

CD8+T cells are important in protective immunity against intracellular pathogens and tumors. In chronic infections or cancer, CD8+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 (TSCM) cells is critical for T cell persistence and efficient tumor killing especially for adoptive cancer immunotherapy such as CAR-T cell therapy. An increasing amount of evidence supports the therapeutic potential of targeting exhausted T cells and TSCM 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.

The NOTCH-FOXM1 Axis Plays a Key Role in Mitochondrial Biogenesis in the Induction of Human Stem Cell Memory-like CAR-T Cells.

Recent studies have shown that stem cell memory T (TSCM) 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 (iTSCM) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iTSCM reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into TSCM-like CAR-T, "CAR-iTSCM" cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iTSCM formation, which are essential for the properties of iTSCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iTSCM differentiation. Like NOTCH-induced CAR-iTSCM cells, FOXM1-induced CAR-iTSCM cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCH- or FOXM1-driven CAR-iTSCM 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.

Tissue regulatory T cells and neural repair.

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.