Dichotomous metabolic networks govern human ILC2 proliferation and function.

Group 2 innate lymphoid cells (ILC2s) represent innate homologs of type 2 helper T cells (TH2) that participate in immune defense and tissue homeostasis through production of type 2 cytokines. While T lymphocytes metabolically adapt to microenvironmental changes, knowledge of human ILC2 metabolism is limited, and its key regulators are unknown. Here, we show that circulating 'naive' ILC2s have an unexpected metabolic profile with a higher level of oxidative phosphorylation (OXPHOS) than natural killer (NK) cells. Accordingly, ILC2s are severely reduced in individuals with mitochondrial disease (MD) and impaired OXPHOS. Metabolomic and nutrient receptor analysis revealed ILC2 uptake of amino acids to sustain OXPHOS at steady state. Following activation with interleukin-33 (IL-33), ILC2s became highly proliferative, relying on glycolysis and mammalian target of rapamycin (mTOR) to produce IL-13 while continuing to fuel OXPHOS with amino acids to maintain cellular fitness and proliferation. Our results suggest that proliferation and function are metabolically uncoupled in human ILC2s, offering new strategies to target ILC2s in disease settings.

Tuft cell acetylcholine is released into the gut lumen to promote anti-helminth immunity.

Upon parasitic helminth infection, activated intestinal tuft cells secrete interleukin-25 (IL-25), which initiates a type 2 immune response during which lamina propria type 2 innate lymphoid cells (ILC2s) produce IL-13. This causes epithelial remodeling, including tuft cell hyperplasia, the function of which is unknown. We identified a cholinergic effector function of tuft cells, which are the only epithelial cells that expressed choline acetyltransferase (ChAT). During parasite infection, mice with epithelial-specific deletion of ChAT had increased worm burden, fitness, and fecal egg counts, even though type 2 immune responses were comparable. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. Finally, we demonstrated a direct effect of ACh on worms, which reduced their fecundity via helminth-expressed muscarinic ACh receptors. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infection provides an additional type 2 immune response effector function.

The transcription factor musculin promotes the unidirectional development of peripheral Treg cells by suppressing the TH2 transcriptional program.

Although master transcription factors (TFs) are key to the development of specific T cell subsets, whether additional transcriptional regulators are induced by the same stimuli that dominantly repress the development of other, non-specific T cell lineages has not been fully elucidated. Through the use of regulatory T cells (Treg cells) induced by transforming growth factor-ß (TGF-ß), we identified the TF musculin (MSC) as being critical for the development of induced Treg cells (iTreg cells) by repression of the T helper type 2 (TH2) transcriptional program. Loss of MSC reduced expression of the Treg cell master TF Foxp3 and induced TH2 differentiation even under iTreg-cell-differentiation conditions. MSC interrupted binding of the TF GATA-3 to the locus encoding TH2-cell-related cytokines and diminished intrachromosomal interactions within that locus. MSC-deficient (Msc-/-) iTreg cells were unable to suppress TH2 responses, and Msc-/- mice spontaneously developed gut and lung inflammation with age. MSC therefore enforced Foxp3 expression and promoted the unidirectional induction of iTreg cells by repressing the TH2 developmental program.

Targeting Glutamine Metabolism and PD-L1: A Novel Anti-tumor Pas de Deux.

In this issue of Molecular Cell, Byun et al. (2020) find that the dual targeting of glutamine metabolism and the PD-L1 checkpoint inhibitor augments anti-tumor immunity. Mechanistically, decreased glutamine availability attenuated S-glutathionylation of SERCA, resulting in an increase in cytosolic calcium, enhanced NF-κB activity, and upregulation of programmed death-ligand 1.

Single-Molecule DNA Methylation Reveals Unique Epigenetic Identity Profiles of T Helper Cells.

Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation in CpG context (5mCpG) and cytosine hydroxymethylation (5hmCpG) are DNA modifications that identify stable cell phenotypes, but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a method to profile Th cell identity using Cas9-targeted single-molecule nanopore sequencing. Targeting as few as 10 selected genomic loci, we were able to distinguish major in vitro polarized murine T cell subtypes, as well as intermediate phenotypes, by their native DNA 5mCpG patterns. Moreover, by using off-target sequences, we were able to infer transcription factor activities relevant to each cell subtype. Detection of 5mCpG and 5hmCpG was validated on intestinal Th17 cells escaping transforming growth factor ß control, using single-molecule adaptive sampling. A total of 21 differentially methylated regions mapping to the 10-gene panel were identified in pathogenic Th17 cells relative to their nonpathogenic counterpart. Hence, our data highlight the potential to exploit native DNA methylation profiling to study physiological and pathological transition states of Th cells.

Cross talk between Paneth and tuft cells drives dysbiosis and inflammation in the gut mucosa.

Gut microbiota imbalance (dysbiosis) is increasingly associated with pathological conditions, both within and outside the gastrointestinal tract. Intestinal Paneth cells are considered to be guardians of the gut microbiota, but the events linking Paneth cell dysfunction with dysbiosis remain unclear. We report a three-step mechanism for dysbiosis initiation. Initial alterations in Paneth cells, as frequently observed in obese and inflammatorybowel diseases patients, cause a mild remodeling of microbiota, with amplification of succinate-producing species. SucnR1-dependent activation of epithelial tuft cells triggers a type 2 immune response that, in turn, aggravates the Paneth cell defaults, promoting dysbiosis and chronic inflammation. We thus reveal a function of tuft cells in promoting dysbiosis following Paneth cell deficiency and an unappreciated essential role of Paneth cells in maintaining a balanced microbiota to prevent inappropriate activation of tuft cells and deleterious dysbiosis. This succinate-tuft cell inflammation circuit may also contribute to the chronic dysbiosis observed in patients.

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.

Glutamine promotes the generation of B10+ cells via the mTOR/GSK3 pathway.

Alterations in cell metabolism can shift the differentiation of immune cells toward a regulatory or inflammatory phenotype, thus, opening up new therapeutic opportunities for immune-related diseases. Indeed, growing knowledge on T- cell metabolism has revealed differences in the metabolic programs of suppressive Tregs as compared to inflammatory Th1 and Th17 cells. In addition to Tregs, IL-10-producing regulatory B cells are crucial for maintaining tolerance, inhibiting inflammation, and autoimmunity. Yet, the metabolic networks regulating diverse B-lymphocyte responses are not well known. Here, we show that glutaminase blockade decreased downstream mTOR activation and attenuated IL-10 secretion. Direct suppression of mTOR activity by rapamycin selectively impaired IL-10 production by B cells whereas secretion was restored upon Glycogen synthase kinase 3 (GSK3) inhibition. Mechanistically, we found mTORC1 activation leads to GSK3 inhibition, identifying a key signalling pathway regulating IL-10 secretion by B lymphocytes. Thus, our results identify glutaminolysis and the mTOR/GSK3 signalling axis, as critical regulators of the generation of IL-10 producing B cells with regulatory functions.

Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3(-/-) mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.

IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(-) effector T cells.

Transcription factor Foxp3 is critical for generating regulatory T cells (T(reg) cells). Transforming growth factor-beta (TGF-beta) induces Foxp3 and suppressive T(reg) cells from naive T cells, whereas interleukin 6 (IL-6) inhibits the generation of inducible T(reg) cells. Here we show that IL-4 blocked the generation of TGF-beta-induced Foxp3(+) T(reg) cells and instead induced a population of T helper cells that produced IL-9 and IL-10. The IL-9(+)IL-10(+) T cells demonstrated no regulatory properties despite producing abundant IL-10. Adoptive transfer of IL-9(+)IL-10(+) T cells into recombination-activating gene 1-deficient mice induced colitis and peripheral neuritis, the severity of which was aggravated if the IL-9(+)IL-10(+) T cells were transferred with CD45RB(hi) CD4(+) effector T cells. Thus IL-9(+)IL-10(+) T cells lack suppressive function and constitute a distinct population of helper-effector T cells that promote tissue inflammation.

CAR T-cell therapy of solid tumors.

The potential for immunotherapy as a treatment option for cancer is clear from remarkable responses of some leukemia patients to adoptive cell transfer using autologous T cells genetically modified to express chimeric antigen receptors (CARs). However, the vast majority of cancers, in particular the more common solid cancers, such as those of the breast, colon and lung, fail to respond significantly to infusions of CAR T cells. Solid cancers present some formidable barriers to adoptive cell transfer, including suppression of T-cell function and inhibition of T-cell localization. In this review, we discuss the current state of CAR T-cell therapy in solid cancers, the variety of concepts being investigated to overcome these barriers as well as approaches aimed at increasing the specificity and safety of adoptive cell transfer.

Recurrence of melanoma following T cell treatment: continued antigen expression in a tumor that evades T cell recruitment.

Clinical therapy with T cells shows promise for cancer patients, but is currently challenged by incomplete responses and tumor relapse. The exact mechanisms that contribute to tumor relapse remain largely unclear. Here, we treated mouse melanomas with T cell receptor-engineered T cells directed against a human peptide-major histocompatibility complex antigen in immune-competent mice. T cells resulted in significant tumor regression, which was followed by relapse in about 80-90% of mice. Molecular analysis revealed that relapsed tumors harbored nonmutated antigen genes, not silenced by promoter methylation, and functionally expressed surface antigen at levels equal to nontreated tumors. Relapsed tumors resisted a second in vivo T cell treatment, but regained sensitivity to T cell treatment upon retransplantation in mice. Notably, relapsed tumors demonstrated decreased levels of CD8 T cells and monocytes, which were substantiated by downregulated expression of chemoattractants and adhesion molecules. These observations were confirmed when using T cells specific for a less immunogenic, endogenous mouse melanoma antigen. We conclude that tumors, when exposed to T cell treatment, can relapse without loss of antigen and develop a milieu that evades recruitment of effector CD8 T cells. Our findings support the concept to target the tumor milieu to aid T cell therapy in limiting tumor relapse.

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.

Ras/MAPK signalling intensity defines subclonal fitness in a mouse model of hepatocellular carcinoma.

Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to secondary, metastatic sites, despite the apparent evolutionary trade-off of increased apoptotic death in the liver that correlated with high Ras dosage. Analysis of the immune tumour microenvironment at the two locations suggests that fast peritoneal tumour growth in the immunocompromised setting is abrogated in immunocompetent animals due to efficient antigen presentation by peritoneal dendritic cells. Furthermore, our data indicate that, in contrast to the metastatic-like outgrowth, strong MAPK signalling is required in the primary liver tumours to resist elimination by NK (natural killer) cells. Overall, this study describes a quantitative aspect of tumour heterogeneity and points to a potential vulnerability of a subtype of hepatocellular carcinoma as a function of MAPK Erk signalling intensity.

Mitochondria Transfer from Mesenchymal Stem Cells Confers Chemoresistance to Glioblastoma Stem Cells through Metabolic Rewiring.

Glioblastomas (GBM) are heterogeneous tumors with high metabolic plasticity. Their poor prognosis is linked to the presence of glioblastoma stem cells (GSC), which support resistance to therapy, notably to temozolomide (TMZ). Mesenchymal stem cells (MSC) recruitment to GBM contributes to GSC chemoresistance, by mechanisms still poorly understood. Here, we provide evidence that MSCs transfer mitochondria to GSCs through tunneling nanotubes, which enhances GSCs resistance to TMZ. More precisely, our metabolomics analyses reveal that MSC mitochondria induce GSCs metabolic reprograming, with a nutrient shift from glucose to glutamine, a rewiring of the tricarboxylic acid cycle from glutaminolysis to reductive carboxylation and increase in orotate turnover as well as in pyrimidine and purine synthesis. Metabolomics analysis of GBM patient tissues at relapse after TMZ treatment documents increased concentrations of AMP, CMP, GMP, and UMP nucleotides and thus corroborate our in vitro analyses. Finally, we provide a mechanism whereby mitochondrial transfer from MSCs to GSCs contributes to GBM resistance to TMZ therapy, by demonstrating that inhibition of orotate production by Brequinar (BRQ) restores TMZ sensitivity in GSCs with acquired mitochondria. Altogether, these results identify a mechanism for GBM resistance to TMZ and reveal a metabolic dependency of chemoresistant GBM following the acquisition of exogenous mitochondria, which opens therapeutic perspectives based on synthetic lethality between TMZ and BRQ. Significance: Mitochondria acquired from MSCs enhance the chemoresistance of GBMs. The discovery that they also generate metabolic vulnerability in GSCs paves the way for novel therapeutic approaches.

Tim-3/galectin-9 pathway: regulation of Th1 immunity through promotion of CD11b+Ly-6G+ myeloid cells.

IFN-gamma plays a central role in antitumor immunity. T cell Ig and mucin domain (Tim-3) is expressed on IFN-gamma-producing Th1 cells; on interaction with its ligand, galectin-9, Th1 immunity is terminated. In this study, we show that transgenic overexpression of Tim-3 on T cells results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Molecular characterization of CD11b(+)Ly-6G(+) cells reveals a phenotype consistent with granulocytic myeloid-derived suppressor cells. Accordingly, we find that modulation of the Tim-3/galectin-9 (Gal-9) pathway impacts on tumor growth. Similarly, overexpression of Tim-3 ligand, Gal-9, results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Loss of Tim-3 restores normal levels of CD11b(+)Ly-6G(+) cells and normal immune responses in Gal-9 transgenic mice. Our data uncover a novel mechanism by which the Tim-3/Gal-9 pathway regulates immune responses and identifies this pathway as a therapeutic target in diseases where myeloid-derived suppressor cells are disadvantageous.

IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells.

The development of T helper (T(H))17 and regulatory T (T(reg)) cells is reciprocally regulated by cytokines. Transforming growth factor (TGF)-beta alone induces FoxP3(+) T(reg) cells, but together with IL-6 or IL-21 induces T(H)17 cells. Here we demonstrate that IL-9 is a key molecule that affects differentiation of T(H)17 cells and T(reg) function. IL-9 predominantly produced by T(H)17 cells, synergizes with TGF-beta1 to differentiate naïve CD4(+) T cells into T(H)17 cells, while IL-9 secretion by T(H)17 cells is regulated by IL-23. Interestingly, IL-9 enhances the suppressive functions of FoxP3(+) CD4(+) T(reg) cells in vitro, and absence of IL-9 signaling weakens the suppressive activity of nT(regs) in vivo, leading to an increase in effector cells and worsening of experimental autoimmune encephalomyelitis. The mechanism of IL-9 effects on T(H)17 and T(regs) is through activation of STAT3 and STAT5 signaling. Our findings highlight a role of IL-9 as a regulator of pathogenic versus protective mechanisms of immune responses.

T-bet, a Th1 transcription factor regulates the expression of Tim-3.

T-cell immunoglobulin, mucin domain-3 (Tim-3) is a membrane protein expressed at late stages of IFN-gamma secreting CD4(+) Th1 cell differentiation and constitutively on DC. Ligation of Tim-3 on Th1 cells terminates Th1 immune responses. In addition, Tim-3 plays a role in tolerance induction, although the mechanism by which this is accomplished has yet to be elucidated. While it is clear that Tim-3 plays an important role in the immune system, little is known regarding the molecular pathways that regulate Tim-3 expression. In the current study, we examine the role of Th1-associated transcription factors in regulating Tim-3 expression. Our experiments reveal that Tim-3 expression is regulated by the Th1-specific transcription factor T-bet. This introduces a novel paradigm into the generation of a Th1 response, whereby a transcription factor responsible for effector Th1 cell differentiation also increases the expression of a specific counter-regulatory molecule to ensure appropriate termination of pro-inflammatory Th1 immune responses.

Th1, Th17, and Th9 effector cells induce experimental autoimmune encephalomyelitis with different pathological phenotypes.

Experimental autoimmune encephalomyelitis (EAE) is a model of human multiple sclerosis induced by autoreactive Th cells that mediate tissue inflammation and demyelination in the CNS. Initially, IFN-gamma-producing Th1 cells and, more recently, IL-17-producing Th17 cells with specificity for myelin Ags have been implicated in EAE induction, but whether Th17 cells are encephalitogenic has been controversial. Moreover, a new effector T cell subset, Th9 cells, has been identified; however, the ability of this T cell subset to induce EAE has not been investigated. Here, we have developed protocols to generate myelin oligodendrocyte glycoprotein-specific Th17, Th1, Th2, and Th9 cells in vitro, so that we could directly compare and characterize the encephalitogenic activity of each of these subsets upon adoptive transfer. We show that myelin oligodendrocyte glycoprotein-specific Th1, Th17, and Th9 cells but not Th2 cells induce EAE upon adoptive transfer. Importantly, each T cell subset induced disease with a different pathological phenotype. These data demonstrate that different effector T cell subsets with specificity for myelin Ags can induce CNS autoimmunity and that the pathological heterogeneity in multiple sclerosis lesions might in part be due to multiple distinct myelin-reactive effector T cells.

IL-6 controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells into Foxp3+ regulatory T cells.

The conditions leading to the induction of adaptive Foxp3(+) regulatory T cells (T-regs) from peripheral T cells in vivo are incompletely understood. Here, we show that unresponsiveness of T cells to IL-6 by T cell-selective deletion of gp130 or immunization of wild-type mice with antigen in incomplete Freund's adjuvant (IFA), which fails to induce IL-6, promotes the conversion of peripheral CD4(+) T cells into adaptive Foxp3(+) T-regs. Thus, both T cell-conditional gp130 knockout (KO) mice immunized with MOG35-55 in complete Freund's adjuvant (CFA) and wild-type mice immunized with MOG35-55 in IFA develop overwhelming antigen-specific T-reg responses and are protected from experimental autoimmune encephalomyelitis (EAE). Depletion of T-regs restores T helper (Th)17 responses and clinical EAE in MOG/CFA-immunized T cell-conditional gp130 KO mice, but not in MOG/IFA-immunized wild-type mice. We conclude that in the absence of T-regs, IL-6 signaling is dispensable for the induction of Th17 cells, and alternative pathways exist to induce Th17 cells and EAE in the absence of IL-6 signaling. However, IL-6 signaling is dominant in inhibiting the conversion of conventional T cells into Foxp3(+) T-regs in vivo, and in the absence of IL-6 signaling, no other cytokine can substitute in inhibiting T-reg conversion. These data identify IL-6 as an important target to modulate autoimmune responses and chronic inflammation.