Neurotrophic factor Neuritin modulates T cell electrical and metabolic state for the balance of tolerance and immunity.

The adaptive T cell response is accompanied by continuous rewiring of the T cell's electric and metabolic state. Ion channels and nutrient transporters integrate bioelectric and biochemical signals from the environment, setting cellular electric and metabolic states. Divergent electric and metabolic states contribute to T cell immunity or tolerance. Here, we report that neuritin (Nrn1) contributes to tolerance development by modulating regulatory and effector T cell function. Nrn1 expression in regulatory T cells promotes its expansion and suppression function, while expression in the T effector cell dampens its inflammatory response. Nrn1 deficiency causes dysregulation of ion channel and nutrient transporter expression in Treg and effector T cells, resulting in divergent metabolic outcomes and impacting autoimmune disease progression and recovery. These findings identify a novel immune function of the neurotrophic factor Nrn1 in regulating the T cell metabolic state in a cell context-dependent manner and modulating the outcome of an immune response.

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response.

Regulatory T cells (Treg) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment-naive non-small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Only one subset selectively expresses high levels of TNFRSF4 (OX40) and TNFRSF18 (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in Treg suppression, including LAG3. Functionally, the OX40hiGITRhi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-Treg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-Treg-expressing T cell receptors that are specific for TAA fully develop a distinct TH1-like signature over a 2-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFNG, and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific TH1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1-responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to antitumor responses.

TIGIT signaling and its influence on T cell metabolism and immune cell function in the tumor microenvironment.

One of the key challenges for successful cancer therapy is the capacity of tumors to evade immune surveillance. Tumor immune evasion can be accomplished through the induction of T cell exhaustion via the activation of various immune checkpoint molecules. The most prominent examples of immune checkpoints are PD-1 and CTLA-4. Meanwhile, several other immune checkpoint molecules have since been identified. One of these is the T cell immunoglobulin and ITIM domain (TIGIT), which was first described in 2009. Interestingly, many studies have established a synergistic reciprocity between TIGIT and PD-1. TIGIT has also been described to interfere with the energy metabolism of T cells and thereby affect adaptive anti-tumor immunity. In this context, recent studies have reported a link between TIGIT and the hypoxia-inducible factor 1-α (HIF1-α), a master transcription factor sensing hypoxia in several tissues including tumors that among others regulates the expression of metabolically relevant genes. Furthermore, distinct cancer types were shown to inhibit glucose uptake and effector function by inducing TIGIT expression in CD8+ T cells, resulting in an impaired anti-tumor immunity. In addition, TIGIT was associated with adenosine receptor signaling in T cells and the kynurenine pathway in tumor cells, both altering the tumor microenvironment and T cell-mediated immunity against tumors. Here, we review the most recent literature on the reciprocal interaction of TIGIT and T cell metabolism and specifically how TIGIT affects anti-tumor immunity. We believe understanding this interaction may pave the way for improved immunotherapy to treat cancer.

Pleiotropic effects of antibiotics on T cell metabolism and T cell-mediated immunity.

T cells orchestrate adaptive and innate immune responses against pathogens and transformed cells. However, T cells are also the main adaptive effector cells that mediate allergic and autoimmune reactions. Within the last few years, it has become abundantly clear that activation, differentiation, effector function, and environmental adaptation of T cells is closely linked to their energy metabolism. Beyond the provision of energy equivalents, metabolic pathways in T cells generate building blocks required for clonal expansion. Furthermore, metabolic intermediates directly serve as a source for epigenetic gene regulation by histone and DNA modification mechanisms. To date, several antibiotics were demonstrated to modulate the metabolism of T cells especially by altering mitochondrial function. Here, we set out to systematically review current evidence about how beta-lactam antibiotics, macrolides, fluoroquinolones, tetracyclines, oxazolidinones, nitroimidazoles, and amphenicols alter the metabolism and effector functions of CD4+ T helper cell populations and CD8+ T cells in vitro and in vivo. Based on this evidence, we have developed an overview on how the use of these antibiotics may be beneficial or detrimental in T cell-mediated physiological and pathogenic immune responses, such as allergic and autoimmune diseases, by altering the metabolism of different T cell populations.

Distinct roles of ORAI1 in T cell-mediated allergic airway inflammation and immunity to influenza A virus infection.

T cell activation and function depend on Ca2+ signals mediated by store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels formed by ORAI1 proteins. We here investigated how SOCE controls T cell function in pulmonary inflammation during a T helper 1 (TH1) cell-mediated response to influenza A virus (IAV) infection and TH2 cell-mediated allergic airway inflammation. T cell-specific deletion of Orai1 did not exacerbate pulmonary inflammation and viral burdens following IAV infection but protected mice from house dust mite-induced allergic airway inflammation. ORAI1 controlled the expression of genes including p53 and E2F transcription factors that regulate the cell cycle in TH2 cells in response to allergen stimulation and the expression of transcription factors and cytokines that regulate TH2 cell function. Systemic application of a CRAC channel blocker suppressed allergic airway inflammation without compromising immunity to IAV infection, suggesting that inhibition of SOCE is a potential treatment for allergic airway disease.

Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge.

Memory CD8+ T cells are characterized by their ability to persist long after the initial antigen encounter and their capacity to generate a rapid recall response. Recent studies have identified a role for metabolic reprogramming and mitochondrial function in promoting the longevity of memory T cells. However, detailed mechanisms involved in promoting their rapid recall response are incompletely understood. Here, we identify a role for the initial and continued activation of the trifunctional rate-limiting enzyme of the de novo pyrimidine synthesis pathway CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) as critical in promoting the rapid recall response of previously activated CD8+ T cells. We found that CAD was rapidly phosphorylated upon naïve T cell activation in an mTORC1-dependent manner, yet remained phosphorylated long after initial activation. Previously activated CD8+ T cells displayed continued de novo pyrimidine synthesis in the absence of mitogenic signals, and interfering with this pathway diminished the speed and magnitude of cytokine production upon rechallenge. Inhibition of CAD did not affect cytokine transcript levels but diminished available pre-rRNA (ribosomal RNA), the polycistronic rRNA precursor whose synthesis is the rate-limiting step in ribosomal biogenesis. CAD inhibition additionally decreased levels of detectable ribosomal proteins in previously activated CD8+ T cells. Conversely, overexpression of CAD improved both the cytokine response and proliferation of memory T cells. Overall, our studies reveal a critical role for CAD-induced pyrimidine synthesis and ribosomal biogenesis in promoting the rapid recall response characteristic of memory T cells.

Restoration of TET2 Function Blocks Aberrant Self-Renewal and Leukemia Progression.

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer. PAPERCLIP.

Targeting Autocrine CCL5-CCR5 Axis Reprograms Immunosuppressive Myeloid Cells and Reinvigorates Antitumor Immunity.

The tumor-promoting potential of CCL5 has been proposed but remains poorly understood. We demonstrate here that an autocrine CCL5-CCR5 axis is a major regulator of immunosuppressive myeloid cells (IMC) of both monocytic and granulocytic lineages. The absence of the autocrine CCL5 abrogated the generation of granulocytic myeloid-derived suppressor cells and tumor-associated macrophages. In parallel, enhanced maturation of intratumoral neutrophils and macrophages occurred in spite of tumor-derived CCL5. The refractory nature of ccl5-null myeloid precursors to tumor-derived CCL5 was attributable to their persistent lack of membrane-bound CCR5. The changes in the ccl5-null myeloid compartment subsequently resulted in increased tumor-infiltrating cytotoxic CD8+ T cells and decreased regulatory T cells in tumor-draining lymph nodes. An analysis of human triple-negative breast cancer specimens demonstrated an inverse correlation between "immune CCR5" levels and the maturation status of tumor-infiltrating neutrophils as well as 5-year-survival rates. Targeting the host CCL5 in bone marrow via nanoparticle-delivered expression silencing, in combination with the CCR5 inhibitor Maraviroc, resulted in strong reductions of IMC and robust antitumor immunities. Our study suggests that the myeloid CCL5-CCR5 axis is an excellent target for cancer immunotherapy. Cancer Res; 77(11); 2857-68. ©2017 AACR.

IFN-γ-induced macrophage antileishmanial mechanisms in mice: A role for immunity-related GTPases, Irgm1 and Irgm3, in Leishmania donovani infection in the liver.

In C57BL/6 mice, Leishmania donovani infection in the liver provoked IFN-γ-induced expression of the immunity-related GTPases (IRG), Irgm1 and Irgm3. To gauge the antileishmanial effects of these macrophage factors in the liver, intracellular infection was analyzed in IRG-deficient mice. In early- (but not late-) stage infection, Irgm3(-/-) mice failed to properly control parasite replication, generated little tissue inflammation and were hyporesponsive to pentavalent antimony (Sb) chemotherapy. Observations limited to early-stage infection in Irgm1(-/-) mice demonstrated increased susceptibility and virtually no inflammatory cell recruitment to heavily-parasitized parenchymal foci but an intact response to chemotherapy. In L. donovani infection in the liver, the absence of either Irgm1 or Irgm3 impairs early inflammation and initial resistance; the absence of Irgm3, but not Irgm1, also appears to impair the intracellular efficacy of Sb chemotherapy.

Carbon monoxide modulates cytochrome oxidase activity and oxidative stress in the developing murine brain during isoflurane exposure.

Commonly used anesthetics induce widespread neuronal degeneration in the developing mammalian brain via the oxidative-stress-associated mitochondrial apoptosis pathway. Dysregulation of cytochrome oxidase (CcOX), the terminal oxidase of the electron transport chain, can result in reactive oxygen species (ROS) formation. Isoflurane has previously been shown to activate this enzyme. Carbon monoxide (CO), as a modulator of CcOX, is of interest because infants and children are routinely exposed to CO during low-flow anesthesia. We have recently demonstrated that low concentrations of CO limit and prevent isoflurane-induced neurotoxicity in the forebrains of newborn mice in a dose-dependent manner. However, the effect of CO on CcOX in the context of anesthetic-induced oxidative stress is unknown. Seven-day-old male CD-1 mice underwent 1h exposure to 0 (air), 5, or 100ppm CO in air with or without isoflurane. Exposure to isoflurane or CO independently increased CcOX kinetic activity and increased ROS within forebrain mitochondria. However, exposure to CO combined with isoflurane paradoxically limited CcOX activation and oxidative stress. There were no changes seen in steady-state levels of CcOX I protein, indicating post-translational modification of CcOX as an etiology for changes in enzyme activity. CO exposure led to differential effects on CcOX subunit I tyrosine phosphorylation depending on concentration, while combined exposure to isoflurane with CO markedly increased the enzyme phosphorylation state. Phosphorylation of tyrosine 304 of CcOX subunit I has been shown to result in strong enzyme inhibition, and the relative reduction in CcOX kinetics following exposure to CO combined with isoflurane may have been due, in part, to such phosphorylation. Taken together, the data suggest that CO modulates CcOX in the developing brain during isoflurane exposure, thereby limiting oxidative stress. These CO-mediated effects could have implications for the development of low-flow anesthesia in infants and children to prevent anesthesia-induced oxidative stress.

Granzyme-mediated regulation of host defense in the liver in experimental Leishmania donovani infection.

In the livers of susceptible C57BL/6 (B6) mice infected with Leishmania donovani, CD8(+) T cell mechanisms are required for granuloma assembly, macrophage activation, intracellular parasite killing, and self-cure. Since gene expression of perforin and granzymes A and B (GzmA and GzmB), cytolytic proteins linked to CD8(+) cell effector function, was enhanced in infected liver tissue, B6 mice deficient in these granular proteins were used to gauge host defense roles. Neither perforin nor GzmA was required; however, mice deficient in GzmB (GzmB(-/-), GzmB cluster(-/-), and GzmA×B cluster double knockout [DKO] mice) showed both delayed granuloma assembly and initially impaired control of parasite replication. Since these two defects in B6 mice were limited to early-stage infection, innately resistant 129/Sv mice were also tested. In this genetic setting, expression of both innate and subsequent T (Th1) cell-dependent acquired resistance, including the self-cure phenotype, was entirely derailed in GzmA×B cluster DKO mice. These results, in susceptible B6 mice for GzmB and in resistant 129/Sv mice for GzmA and/or the GzmB cluster, point to granzyme-mediated host defense regulation in the liver in experimental visceral leishmaniasis.