Asparagine restriction enhances CD8+ T cell metabolic fitness and antitumoral functionality through an NRF2-dependent stress response.

Robust and effective T cell immune surveillance and cancer immunotherapy require proper allocation of metabolic resources to sustain energetically costly processes, including growth and cytokine production. Here, we show that asparagine (Asn) restriction on CD8+ T cells exerted opposing effects during activation (early phase) and differentiation (late phase) following T cell activation. Asn restriction suppressed activation and cell cycle entry in the early phase while rapidly engaging the nuclear factor erythroid 2-related factor 2 (NRF2)-dependent stress response, conferring robust proliferation and effector function on CD8+ T cells during differentiation. Mechanistically, NRF2 activation in CD8+ T cells conferred by Asn restriction rewired the metabolic program by reducing the overall glucose and glutamine consumption but increasing intracellular nucleotides to promote proliferation. Accordingly, Asn restriction or NRF2 activation potentiated the T cell-mediated antitumoral response in preclinical animal models, suggesting that Asn restriction is a promising and clinically relevant strategy to enhance cancer immunotherapy. Our study revealed Asn as a critical metabolic node in directing the stress signaling to shape T cell metabolic fitness and effector functions.

The lactate dehydrogenase (LDH) isoenzyme spectrum enables optimally controlling T cell glycolysis and differentiation.

Isoenzyme divergence is a prevalent mechanism governing tissue-specific and developmental stage-specific metabolism in mammals. The lactate dehydrogenase (LDH) isoenzyme spectrum reflects the tissue-specific metabolic status. We found that three tetrameric isoenzymes composed of LDHA and LDHB (LDH-3/4/5) comprise the LDH spectrum in T cells. Genetically deleting LDHA or LDHB altered the isoenzyme spectrum by removing all heterotetramers and leaving T cells with LDH-1 (the homotetramer of LDHB) or LDH-5 (the homotetramer of LDHA), respectively. Accordingly, deleting LDHA suppressed glycolysis, cell proliferation, and differentiation. Unexpectedly, deleting LDHB enhanced glycolysis but suppressed T cell differentiation, indicating that an optimal zone of glycolytic activity is required to maintain cell fitness. Mechanistically, the LDH isoenzyme spectrum imposed by LDHA and LDHB is necessary to optimize glycolysis to maintain a balanced nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide hydrogen pool. Our results suggest that the LDH isoenzyme spectrum enables "Goldilocks levels" of glycolytic and redox activity to control T cell differentiation.

GAB functions as a bioenergetic and signalling gatekeeper to control T cell inflammation.

γ-Aminobutyrate (GAB), the biochemical form of (GABA) γ-aminobutyric acid, participates in shaping physiological processes, including the immune response. How GAB metabolism is controlled to mediate such functions remains elusive. Here we show that GAB is one of the most abundant metabolites in CD4+ T helper 17 (TH17) and induced T regulatory (iTreg) cells. GAB functions as a bioenergetic and signalling gatekeeper by reciprocally controlling pro-inflammatory TH17 cell and anti-inflammatory iTreg cell differentiation through distinct mechanisms. 4-Aminobutyrate aminotransferase (ABAT) funnels GAB into the tricarboxylic acid (TCA) cycle to maximize carbon allocation in promoting TH17 cell differentiation. By contrast, the absence of ABAT activity in iTreg cells enables GAB to be exported to the extracellular environment where it acts as an autocrine signalling metabolite that promotes iTreg cell differentiation. Accordingly, ablation of ABAT activity in T cells protects against experimental autoimmune encephalomyelitis (EAE) progression. Conversely, ablation of GABAA receptor in T cells worsens EAE. Our results suggest that the cell-autonomous control of GAB on CD4+ T cells is bimodal and consists of the sequential action of two processes, ABAT-dependent mitochondrial anaplerosis and the receptor-dependent signalling response, both of which are required for T cell-mediated inflammation.

Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation.

Effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By using complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fueled biosynthetic processes while controlling the ratio of succinate/α-ketoglutarate (α-KG) to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells caused proliferation and survival defects when the TCA cycle was truncated, blocking carbon flux to support nucleoside biosynthesis. Replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. SDH deficiency induced a proinflammatory gene signature in T cells and promoted T helper 1 and T helper 17 lineage differentiation. An increasing succinate/α-KG ratio in SDH-deficient T cells promoted inflammation by changing the pattern of the transcriptional and chromatin accessibility signatures and consequentially increasing the expression of the transcription factor, PR domain zinc finger protein 1. Collectively, our studies revealed a role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.

A Simple and Robust Protocol for in vitro Differentiation of Mouse Non-pathogenic T Helper 17 Cells from CD4+ T Cells.

Functional and mechanistic studies of CD4+ T cell lineages rely on robust methods of in vitro T cell polarization. Here, we report an optimized protocol for in vitro differentiation of a mouse non-pathogenic T helper 17 (TH17) cell lineage. Most of the previously established protocols require irradiated splenocytes as artificial antigen presenting cells (APC) for TCR activation. The protocol described here employs plate-bound antibodies and a TH17-polarizing cytokine cocktail to activate and differentiate naïve CD4+ T (Tnai) cells, reflecting a simple and robust protocol for in vitro TH17n differentiation. Using T cells that are genetically engineered with an IL-17 reporter, this protocol may enable the rapid production of a pure population of IL17-expressing CD4+ T cells for system biology studies and high-throughput functional screening.

De novo synthesis and salvage pathway coordinately regulate polyamine homeostasis and determine T cell proliferation and function.

Robust and effective T cell-mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of biosynthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in extracellular polyamine uptake, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular polyamine pool, inhibits T cell proliferation, and suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine pool for managing inflammatory and autoimmune diseases.

Inosine is an alternative carbon source for CD8+-T-cell function under glucose restriction.

T cells undergo metabolic rewiring to meet their bioenergetic, biosynthetic and redox demands following antigen stimulation. To fulfil these needs, effector T cells must adapt to fluctuations in environmental nutrient levels at sites of infection and inflammation. Here, we show that effector T cells can utilize inosine, as an alternative substrate, to support cell growth and function in the absence of glucose in vitro. T cells metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase. We demonstrate that the ribose subunit of inosine can enter into central metabolic pathways to provide ATP and biosynthetic precursors, and that cancer cells display diverse capacities to utilize inosine as a carbon source. Moreover, the supplementation with inosine enhances the anti-tumour efficacy of immune checkpoint blockade and adoptive T-cell transfer in solid tumours that are defective in metabolizing inosine, reflecting the capability of inosine to relieve tumour-imposed metabolic restrictions on T cells.

Tolerogenic properties of CD206+ macrophages appeared in the sublingual mucosa after repeated antigen-painting.

The sublingual mucosa (SLM) in the oral cavity is utilized as the site for sublingual immunotherapy to induce tolerance against allergens. We previously reported that CD206+ round-type macrophage-like cells were induced in the SLM after repeated antigen (e.g. cedar pollen or fluorescein isothiocyanate (FITC))-painting. In this study, we examined the phenotypic and functional properties of CD206+ cells induced by repeated FITC-painting on the SLM. CD206+ cells after the repeated FITC-painting possessed a macrophage-like CD11b+Ly6C+ F4/80+CD64+ phenotype and expressed TIM-4, which was expressed in tolerogenic tissue-resident macrophages, at a high level. SLM CD206+ cells preferentially expressed molecules related to endocytosis and homeostatic processes, including the novel B7 family of immune checkpoint molecules, as assessed by microarray analyses. SLM CD206+ cells showed preferential expression of M2-related genes such as Fizz1, Aldh1a1 and Aldh1a2 but not Ym-1 and Arginase-1. A CD206+ cell-rich status inhibited OVA-specific CD4+ T-cell responses but reciprocally enhanced the proportion of both IL-10+CD4+ cells and Foxp3+ regulatory T-cells in regional lymph nodes. Co-culture of CD206+ cells with dendritic cells (DCs) showed that IL-12 production was suppressed in DCs concurrent with the decline of the MHC class IIhiCD86+ population, which was restored by neutralization of IL-10. These results demonstrate SLM CD206+ cells show the feature of tolerogenic macrophages and down-regulate the antigen-presenting cell function of mature DCs resulting in the inhibition of CD4+ T-cell responses.

A CS1-NKG2D Bispecific Antibody Collectively Activates Cytolytic Immune Cells against Multiple Myeloma.

Multiple myeloma (MM) is an incurable hematologic malignancy of plasma cells, with an estimated 30,000 new cases diagnosed each year in the United States, signifying the need for new therapeutic approaches. We hypothesized that targeting MM using a bispecific antibody (biAb) to simultaneously engage both innate and adaptive cytolytic immune cells could present potent antitumor activity. We engineered a biAb by fusing an anti-CS1 single-chain variable fragment (scFv) and an anti-NKG2D scFv (CS1-NKG2D biAb). Although NKG2D is a potent activation receptor ubiquitously expressed on mostly cytolytic immune cells including NK cells, CD8+ T cells, γδ T cells, and NKT cells, the CS1 tumor-associated antigen on MM represents a promising target. CS1-NKG2D biAb engaged human MM cell lines and NKG2D+ immune cells, forming immune synapses. In effector cells, CS1-NKG2D biAb triggered the phosphorylation of AKT, a downstream protein kinase of the activated NKG2D-DAP10 complex. The EC50 values of CS1-NKG2D biAb for CS1high and for CS1low MM cell lines with effector PBMCs were 10-12 and 10-9 mol/L, respectively. CS1-NKG2D biAb acted through multiple types of immune cells, and this induced cytotoxicity was both CS1- and NKG2D-specific. In vivo, survival was significantly prolonged using CS1-NKG2D biAb in a xenograft NOD-SCIDIL2γc-/- (NSG) mouse model engrafted with both human PBMCs and MM cell lines. Collectively, we demonstrated that the CS1-NKG2D biAb facilitated an enhanced immune synapse between CS1+ MM cells and NKG2D+ cytolytic innate and antigen-specific effector cells, which, in turn, activated these immune cells for improved clearance of MM. Cancer Immunol Res; 6(7); 776-87. ©2018 AACR.

The immune checkpoint molecule VISTA regulates allergen-specific Th2-mediated immune responses.

V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a novel immune checkpoint receptor and ligand that regulates T-cell activation. We investigated the functional involvement of VISTA in Th2 cell-mediated immune responses using an ovalbumin (OVA)-induced allergic asthma model. Treatment with an anti-VISTA monoclonal antibody (mAb) during allergen sensitization increased the production of antibodies, including total IgE, OVA-specific IgG1 and IgG2a and allergen-specific IL-5 and IL-13; it also increased the expression of IL-13 by splenic CD4+ T cells. However, treatment with the anti-VISTA mAb during sensitization did not accelerate asthmatic responses, including airway hyper-responsiveness (AHR) or the number of eosinophils in bronchoalveolar lavage (BAL) fluid. In contrast, treatment with the anti-VISTA mAb during allergen challenge significantly augmented AHR and BAL fluid eosinophilia. This treatment also increased the production of IL-5 and IL-13 in BAL fluid and the expression of IL-13 by CD4+ T cells in draining lymph nodes. These results suggest that VISTA is involved in the regulation of Th2 cell generation and Th2 cell-mediated antibody production and regulates asthmatic responses, especially in the effector phase.

Black Raspberries Enhance Natural Killer Cell Infiltration into the Colon and Suppress the Progression of Colorectal Cancer.

Natural killer (NK) cells are an essential component of innate immunity against cancer development. Many studies have been conducted to evaluate immune-modulating effects using dietary compounds. Our laboratory has been investigating the chemopreventive potential of black raspberries (BRBs) and previously demonstrated their beneficial modulation of genetic and epigenetic biomarkers in patients with colorectal cancer (CRC). The current study investigated their potential on modulating NK cells. To avoid the excessive inflammation caused by the dextran sulfate sodium (DSS) treatment that leads to colitis, we treated the mice with overnight DSS so that it would slightly irritate the colon but still promote colon carcinogenesis with 100% incidence in both the ApcMin/+ mice and azoxymethane (AOM)-treated mice. A significant decrease of tissue-infiltrating NK cells along the progression of microadenoma-to-adenoma and adenoma-to-adenocarcinoma was observed in the ApcMin/+ /DSS and AOM/DSS mice, respectively. Depletion of NK cells significantly promoted the development of CRC, suggesting a critical role of NK cells in combating CRC progression. BRBs significantly suppressed the CRC progression and increased the number of tissue-infiltrating NK cells in both mouse models. Moreover, we further determined BRBs' effects on NK cells in the human biopsy specimens collected from our previously completed clinical trial, in which CRC patients consumed BRBs for an average of 4 weeks during a presurgical window. We observed an increased number and an enhanced cytotoxicity of NK cells by BRB intervention. The current study provides evidence that BRBs have the potential to enhance the tumor immunesurveillance of NK cells that can be beneficial in the setting of CRC prevention and treatment.

Site-specific regulation of oral mucosa-recruiting CD8+ T cells in a mouse contact allergy model.

Contact allergy is a T cell-mediated, delayed-type hypersensitivity generated by contact exposure of an allergen to the skin and mucosal surface. The clinical manifestations of allergic responses between the skin and oral mucosa vary and the differences in immunopathology have not been clarified. We generated hapten-induced contact hypersensitivity (CH) of the buccal mucosa (BM) in parallel studies with ear skin (ES) CH, and observed several characteristic findings of BM CH. The BM challenge induced more rapid and more severe inflammation than the ES challenge, with abundant granulocyte and CD8+ T cell infiltration. However, these inflammatory responses diminished quickly. Recruiting CD8+ T cells in the BM had higher ratios of CD62L-CD44low-hi memory-type cells, and showed impaired IFN-γ, greater PD-1, and comparable Ki-67 expression, suggesting that the recruiting-proliferating CD8+ T cells were unable to differentiate into effector T cells and converted into exhausted T cells at the local site. This finding may explain the rapid recovery of the BM from severe inflammation. Preferentially greater expression of PD-1 ligand (B7-H1), was observed in the BM epithelium under the peak inflammation, and the absence of B7-H1 further accelerated CH responses, suggesting the occurrence of PD-1:B7-H1-mediated immune regulation at the local site. Our results may facilitate the understanding of the unique features of contact allergies in the oral mucosa, and guide the development of new strategies for control of contact allergy.

Repeated antigen painting and sublingual immunotherapy in mice convert sublingual dendritic cell subsets.

The sublingual mucosa (SLM) is utilized as the site for sublingual immunotherapy (SLIT) to induce tolerance against allergens. The contribution of SLM-dendritic cells (SLM-DCs) has not been clarified. The aim of this study was to examine the dynamics and phenotype of SLM-DCs after topical antigen painting and SLIT. SLM-DCs were histologically evaluated after FITC painting. A novel murine Japanese cedar pollinosis (JCP) model was generated and change in SLM-DCs after SLIT was examined. The density of SLM-DCs was clearly lower compared with the buccal mucosa and dorsal surface of the tongue. Topical FITC painting on the SLM induced maximal recruitment of submucosal DCs (smDCs) at 6h, but most smDCs had vanished at 24h. Repeated painting on the SLM induced exhaustion and conversion of the smDC phenotype. CD206(high)CD11c(low) round-type cells with fewer dendrites and less lymph node migration capacity became dominant. In the murine model of JCP, SLIT efficiently inhibited clinical symptoms and allergen-mediated immunological responses. SLIT markedly reduced the number of SLM-DCs, converted to the round-type dominant phenotype and inhibited the activation of regional lymph node DCs. Topical antigen painting on the SLM induced rapid exhaustion and conversion of smDCs. The unique dynamics of SLM-DCs may contribute to tolerance induction in SLIT.

Synergistic effects of bone mesenchymal stem cells and chondroitinase ABC on nerve regeneration after acellular nerve allograft in rats.

This study aimed to evaluate whether combination therapy of bone marrow stromal cells (BMSCs) transplantation and chondroitinase ABC (ChABC) treatment further enhances axonal regeneration and functional recovery after acellular nerve allograft repair of the sciatic nerve gap in rats. Eight Sprague-Dawley rats were used as nerve donors, and 32 Wistar rats were randomly divided into four groups: Group I: acellular rat sciatic nerve (ARSN) group; Group II: ChABC treatment; Group III: BMSCs transplantation; and Group IV: ChABC treatment and BMSCs transplantation. The results showed that compared with ARSN control group, BMSC transplantation promoted axonal regeneration, the secretion of neural trophic factors NGF, BDNF and axon angiogenesis in nerve graft. ChABC treatment degraded chondroitin sulfate proteoglycans in ARSN in vitro and in vivo and improved BMSCs survival in ARSN. The combination therapy caused much better beneficial effects evidenced by increasing sciatic function index, nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, and myelinated nerve number, but did not further boost the therapeutic effects on neurotrophic factor production, axon angiogenesis, and sensory functional recovery by BMSC transplantation. Taken together, for the first time, we demonstrate the synergistic effects of BMSC transplantation and BMSCs treatment on peripheral nerve regeneration, and our findings may help establish novel strategies for cell transplantation therapy for peripheral nerve injury.