Metabolic dialogues: regulators of chimeric antigen receptor T cell function in the tumor microenvironment.

Tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells have demonstrated remarkable success in the treatment of relapsed/refractory melanoma and hematological malignancies, respectively. These treatments have marked a pivotal shift in cancer management. However, as "living drugs," their effectiveness is dependent on their ability to proliferate and persist in patients. Recent studies indicate that the mechanisms regulating these crucial functions, as well as the T cell's differentiation state, are conditioned by metabolic shifts and the distinct utilization of metabolic pathways. These metabolic shifts, conditioned by nutrient availability as well as cell surface expression of metabolite transporters, are coupled to signaling pathways and the epigenetic landscape of the cell, modulating transcriptional, translational, and post-translational profiles. In this review, we discuss the processes underlying the metabolic remodeling of activated T cells, the impact of a tumor metabolic environment on T cell function, and potential metabolic-based strategies to enhance T cell immunotherapy.

CD3ζ ITAMs enable ligand discrimination and antagonism by inhibiting TCR signaling in response to low-affinity peptides.

The T cell antigen receptor (TCR) contains ten immunoreceptor tyrosine-based activation motif (ITAM) signaling sequences distributed within six CD3 subunits; however, the reason for such structural complexity and multiplicity is unclear. Here we evaluated the effect of inactivating the three CD3ζ chain ITAMs on TCR signaling and T cell effector responses using a conditional 'switch' mouse model. Unexpectedly, we found that T cells expressing TCRs containing inactivated (non-signaling) CD3ζ ITAMs (6F-CD3ζ) exhibited reduced ability to discriminate between low- and high-affinity ligands, resulting in enhanced signaling and cytokine responses to low-affinity ligands because of a previously undetected inhibitory function of CD3ζ ITAMs. Also, 6F-CD3ζ TCRs were refractory to antagonism, as predicted by a new in silico adaptive kinetic proofreading model that revises the role of ITAM multiplicity in TCR signaling. Finally, T cells expressing 6F-CD3ζ displayed enhanced cytolytic activity against solid tumors expressing low-affinity ligands, identifying a new counterintuitive approach to TCR-mediated cancer immunotherapy.

Co-op CARs for targeting acute myeloid leukemia.

Chimeric antigen receptor (CAR) T cell therapies are limited by antigen escape and on-target/off-tumor toxicity. In addressing these challenges, Haubner et al. develop an "IF-BETTER" strategy. Their combinatorial chimeric co-stimulatory receptor with an attenuated CAR enhances acute myeloid leukemia (AML) killing while protecting healthy progenitors, highlighting the potential to leverage cooperative CAR designs.

Apoptotic contraction drives target cell release by cytotoxic T cells.

Cytotoxic T lymphocytes (CTLs) fight intracellular pathogens and cancer by identifying and destroying infected or transformed target cells1. To kill, CTLs form a specialized cytotoxic immune synapse (IS) with a target of interest and then release toxic perforin and granzymes into the interface to elicit programmed cell death2-5. The IS then dissolves, enabling CTLs to search for additional prey and professional phagocytes to clear the corpse6. While the mechanisms governing IS assembly have been studied extensively, far less is known about target cell release. Here, we applied time-lapse imaging to explore the basis for IS dissolution and found that it occurred concomitantly with the cytoskeletal contraction of apoptotic targets. Genetic and pharmacological perturbation of this contraction response indicated that it was both necessary and sufficient for CTL dissociation. We also found that mechanical amplification of apoptotic contractility promoted faster CTL detachment and serial killing. Collectively, these results establish a biophysical basis for IS dissolution and highlight the importance of mechanosensory feedback in the regulation of cell-cell interactions.

Arginine metabolism regulates human erythroid differentiation through hypusination of eIF5A.

Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.

Universal antigen encoding of T cell activation from high-dimensional cytokine dynamics.

Systems immunology lacks a framework with which to derive theoretical understanding from high-dimensional datasets. We combined a robotic platform with machine learning to experimentally measure and theoretically model CD8+ T cell activation. High-dimensional cytokine dynamics could be compressed onto a low-dimensional latent space in an antigen-specific manner (so-called "antigen encoding"). We used antigen encoding to model and reconstruct patterns of T cell immune activation. The model delineated six classes of antigens eliciting distinct T cell responses. We generalized antigen encoding to multiple immune settings, including drug perturbations and activation of chimeric antigen receptor T cells. Such universal antigen encoding for T cell activation may enable further modeling of immune responses and their rational manipulation to optimize immunotherapies.

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.

Whole-exome sequencing and host cell reactivation assay lead to a diagnosis of xeroderma pigmentosum group D with mild ultraviolet radiation sensitivity.

A case of xeroderma pigmentosum (XP) group D in a 39-year-old Japanese man is reported. The patient had suffered from moderate to severe solar sensitivity and freckle-like pigmented macules in sun-exposed areas since 6 years of age, and developed skin malignancies such as squamous cell carcinoma, actinic keratosis, Bowen's disease and basal cell carcinoma. The minimal erythema dose for ultraviolet (UV) radiation was decreased with a delayed peak reaction. The level of unscheduled DNA synthesis of fibroblasts from the patient was 70% of normal, while they expressed POLH, a gene product responsible for the XP variant. Whole-exome sequencing indicated that the patient harbored a homozygous mutation of c.1802G>T, p.Arg601Leu in ERCC2. A genetic complementation test was carried out by host cell reactivation assay, which showed that the patient's fibroblasts recovered only when they were transfected with XPD cDNA, confirming the diagnosis of XP-D. Arg601Leu mutation in ERCC2 may be related to mild UV radiation sensitivity and moderate skin lesions.

Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients.

Late-onset inflammatory toxicities resembling hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) occur after chimeric antigen receptor T cell (CAR T cell) infusion and represent a therapeutic challenge. Given the established link between perforin deficiency and primary HLH, we investigated the role of perforin in anti-CD19 CAR T cell efficacy and HLH-like toxicities in a syngeneic murine model. Perforin contributed to both CD8+ and CD4+ CAR T cell cytotoxicity but was not required for in vitro or in vivo leukemia clearance. Upon CAR-mediated in vitro activation, perforin-deficient CAR T cells produced higher amounts of proinflammatory cytokines compared with WT CAR T cells. Following in vivo clearance of leukemia, perforin-deficient CAR T cells reexpanded, resulting in splenomegaly with disruption of normal splenic architecture and the presence of hemophagocytes, which are findings reminiscent of HLH. Notably, a substantial fraction of patients who received anti-CD22 CAR T cells also experienced biphasic inflammation, with the second phase occurring after the resolution of cytokine release syndrome, resembling clinical manifestations of HLH. Elevated inflammatory cytokines such as IL-1ß and IL-18 and concurrent late CAR T cell expansion characterized the HLH-like syndromes occurring in the murine model and in humans. Thus, a murine model of perforin-deficient CAR T cells recapitulated late-onset inflammatory toxicities occurring in human CAR T cell recipients, providing therapeutically relevant mechanistic insights.

Phosphate Transporter Profiles in Murine and Human Thymi Identify Thymocytes at Distinct Stages of Differentiation.

Thymocyte differentiation is dependent on the availability and transport of metabolites in the thymus niche. As expression of metabolite transporters is a rate-limiting step in nutrient utilization, cell surface transporter levels generally reflect the cell's metabolic state. The GLUT1 glucose transporter is upregulated on actively dividing thymocytes, identifying thymocytes with an increased metabolism. However, it is not clear whether transporters of essential elements such as phosphate are modulated during thymocyte differentiation. While PiT1 and PiT2 are both phosphate transporters in the SLC20 family, we show here that they exhibit distinct expression profiles on both murine and human thymocytes. PiT2 expression distinguishes thymocytes with high metabolic activity, identifying immature murine double negative (CD4-CD8-) DN3b and DN4 thymocyte blasts as well as immature single positive (ISP) CD8 thymocytes. Notably, the absence of PiT2 expression on RAG2-deficient thymocytes, blocked at the DN3a stage, strongly suggests that high PiT2 expression is restricted to thymocytes having undergone a productive TCRß rearrangement at the DN3a/DN3b transition. Similarly, in the human thymus, PiT2 was upregulated on early post-ß selection CD4+ISP and TCRαß-CD4hiDP thymocytes co-expressing the CD71 transferrin receptor, a marker of metabolic activity. In marked contrast, expression of the PiT1 phosphate importer was detected on mature CD3+ murine and human thymocytes. Notably, PiT1 expression on CD3+DN thymocytes was identified as a biomarker of an aging thymus, increasing from 8.4 ± 1.5% to 42.4 ± 9.4% by 1 year of age (p < 0.0001). We identified these cells as TCRγδ and, most significantly, NKT, representing 77 ± 9% of PiT1+DN thymocytes by 1 year of age (p < 0.001). Thus, metabolic activity and thymic aging are associated with distinct expression profiles of the PiT1 and PiT2 phosphate transporters.

CD19-targeted CAR regulatory T cells suppress B cell pathology without GvHD.

Regulatory T cells (Tregs) play essential roles in maintaining immunological self-tolerance and preventing autoimmunity. The adoptive transfer of antigen-specific Tregs has been expected to be a potent therapeutic method for autoimmune diseases, severe allergy, and rejection in organ transplantation. However, effective Treg therapy has not yet been established because of the difficulty in preparing a limited number of antigen-specific Tregs. Chimeric antigen receptor (CAR) T cells have been shown to be a powerful therapeutic method for treating B cell lymphomas, but application of CAR to Treg-mediated therapy has not yet been established. Here, we generated CD19-targeted CAR (CD19-CAR) Tregs from human PBMCs (hPBMCs) and optimized the fraction of the Treg source as CD4+CD25+CD127loCD45RA+CD45RO-. CD19-CAR Tregs could be expanded in vitro while maintaining Treg properties, including high expression of the latent form of TGF-ß. CD19-CAR Tregs suppressed IgG antibody production and differentiation of B cells via a TGF-ß-dependent mechanism. Unlike conventional CD19-CAR CD8+ T cells, CD19-CAR Tregs suppressed antibody production in immunodeficient mice that were reconstituted with hPBMCs, reducing the risk of graft-versus-host disease. Therefore, the adoptive transfer of CD19-CAR Tregs may provide a novel therapeutic method for treating autoantibody-mediated autoimmune diseases.

In Vitro Generation of Stem Cell Memory-Like T Cells from Activated T Cells.

Adoptive T-cell therapy is an attractive strategy for cancer immunotherapy. The transfer of in vitro expanded tumor-associated antigen (TAA)-specific T cells from patients may effectively fight against the original tumor cells. The chimeric antigen receptor-engineered T (CAR-T) cells are also shown to be a promising therapy for hematologic malignancies. However, one of the limitations of these T-cell-based therapies is a rapid acquisition of tolerant (anergy, deletion, dysfunctional and/or exhausted) phenotypes of T cells during activation in vitro and/or after transfer in vivo. We and others found that stem cell memory T (TSCM) cells are strongly resistant against such tolerance, showing strong expansion and persistence in vivo, and provide long-lasting antitumor effects. Here we describe a protocol for the generation of phenotypically TSCM-like cells (iTSCM cells), which can be induced by simple co-culture of activated T cells with OP9 stroma cells expressing a Notch ligand. We also showed the methods of cancer immunotherapy by using NSG mice.

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.

In Vitro Conversion of Activated T Cells into Stem Cell Memory-Like T Cells.

Adoptive T cell therapy is an attractive strategy in tumor immunotherapy. The transfer of in vitro expanded tumor-associated antigen (TAA)-specific T cells from patients may effectively destroy the original tumor cells. One of the limitations is a rapid acquisition of tolerant (anergy, deletion, dysfunctional, and/or exhausted) phenotypes. We and others found that stem cell memory T (TSCM) cells are strongly resistant to tolerance, showing strong expansion and persistence in vivo and providing long-lasting antitumor effects. We previously established that phenotypically TSCM cells (iTSCM) can be induced using a simple coculture of activated T cells with OP9 stroma cells expressing a Notch ligand. Here, we describe a defined protocol for generating human iTSCM cells, including reagents, culture setting, and procedure.

Brain regulatory T cells suppress astrogliosis and potentiate neurological recovery.

In addition to maintaining immune tolerance, FOXP3+ regulatory T (Treg) cells perform specialized functions in tissue homeostasis and remodelling1,2. However, the characteristics and functions of brain Treg cells are not well understood because there is a low number of Treg cells in the brain under normal conditions. Here we show that there is massive accumulation of Treg cells in the mouse brain after ischaemic stroke, and this potentiates neurological recovery during the chronic phase of ischaemic brain injury. Although brain Treg cells are similar to Treg cells in other tissues such as visceral adipose tissue and muscle3-5, they are apparently distinct and express unique genes related to the nervous system including Htr7, which encodes the serotonin receptor 5-HT7. The amplification of brain Treg cells is dependent on interleukin (IL)-2, IL-33, serotonin and T cell receptor recognition, and infiltration into the brain is driven by the chemokines CCL1 and CCL20. Brain Treg cells suppress neurotoxic astrogliosis by producing amphiregulin, a low-affinity epidermal growth factor receptor (EGFR) ligand. Stroke is a leading cause of neurological disability, and there are currently few effective recovery methods other than rehabilitation during the chronic phase. Our findings suggest that Treg cells and their products may provide therapeutic opportunities for neuronal protection against stroke and neuroinflammatory diseases.

Generation and application of human induced-stem cell memory T cells for adoptive immunotherapy.

Adoptive T-cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen-specific stem cell memory T (TSCM ) cells is expected to overcome this shortcoming as TSCM cells are close to naïve T cells, but are also highly proliferative, long-lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into TSCM -like cells (iTSCM ) by coculturing with OP9 cells expressing Notch ligand, Delta-like 1 (OP9-hDLL1). Here we show the methodological parameters of human CD8+ iTSCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti-CD3/CD28 antibodies or by antigen-presenting cells, human iTSCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9-hDLL1 cells, interleukin (IL)-7 and IL-15 (but not IL-2 or IL-21) could efficiently generate iTSCM cells. Epstein-Barr virus-specific iTSCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein-Barr virus transformed-tumor model mice. Thus, adoptive T-cell therapy with iTSCM offers a promising therapeutic strategy for cancer immunotherapy.

Inhibition of Nr4a Receptors Enhances Antitumor Immunity by Breaking Treg-Mediated Immune Tolerance.

Enhanced infiltration of regulatory T cells (Treg) into tumor tissue is detrimental to patients with cancer and is closely associated with poor prognosis as they create an immunosuppressive state that suppresses antitumor immune responses. Therefore, breaking Treg-mediated immune tolerance is important when considering cancer immunotherapy. Here, we show that the Nr4a nuclear receptors, key transcription factors maintaining Treg genetic programs, contribute to Treg-mediated suppression of antitumor immunity in the tumor microenvironment. Mice lacking Nr4a1 and Nr4a2 genes specifically in Tregs showed resistance to tumor growth in transplantation models without exhibiting any severe systemic autoimmunity. The chemotherapeutic agent camptothecin and a common cyclooxygenase-2 inhibitor were found to inhibit transcriptional activity and induction of Nr4a factors, and they synergistically exerted antitumor effects. Genetic inactivation or pharmacologic inhibition of Nr4a factors unleashed effector activities of CD8+ cytotoxic T cells and evoked potent antitumor immune responses. These findings demonstrate that inactivation of Nr4a in Tregs breaks immune tolerance toward cancer, and pharmacologic modulation of Nr4a activity may be a novel cancer treatment strategy targeting the immunosuppressive tumor microenvironment.Significance: This study reveals the role of Nr4a transcription factors in Treg-mediated tolerance to antitumor immunity, with possible therapeutic implications for developing effective anticancer therapies. Cancer Res; 78(11); 3027-40. ©2018 AACR.

Generation of allo-antigen-specific induced Treg stabilized by vitamin C treatment and its application for prevention of acute graft versus host disease model.

Antigen-specific regulatory T cells (Tregs) possess the potential to reduce excess immune responses in autoimmune diseases, allergy, rejection after organ transplantation and graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation. Although in vitro-expanded antigen-specific induced Tregs (iTregs) have been considered to be a promising therapeutic agent against such excessive immune reactions, the instability of iTregs after transfer is a fundamental problem in their clinical application. In this study, we searched for the optimal way to generate stable iTregs for the prevention of the murine GVHD model, in which conventional iTregs are reported to be inefficient. Allo-antigen-specific iTregs were generated by co-culturing naive T cells with allogenic dendritic cells in the presence of TGF-ß and retinoic acid. By examining various agents and genes, we found that vitamin C stabilized Foxp3 expression most effectively in adoptively transferred iTregs under a GVHD environment. Vitamin C treatment caused active DNA demethylation specifically on the conserved non-coding sequence 2 (CNS2) enhancer of the Foxp3 gene locus in allo-antigen-specific iTregs and reduced iTreg conversion into pathogenic exFoxp3 cells. Vitamin C-treated iTregs suppressed GVHD symptoms more efficiently than untreated iTregs. Vitamin C also facilitated induction of a FOXP3high iTreg population from human naive T cells, which was very stable even in the presence of IL-6 in vitro. The treatment of vitamin C for iTreg promises innovative clinical application for adoptive Treg immunotherapy.