Coenzyme A fuels T cell anti-tumor immunity.

Metabolic programming is intricately linked to the anti-tumor properties of T cells. To study the metabolic pathways associated with increased anti-tumor T cell function, we utilized a metabolomics approach to characterize three different CD8+ T cell subsets with varying degrees of anti-tumor activity in murine models, of which IL-22-producing Tc22 cells displayed the most robust anti-tumor activity. Tc22s demonstrated upregulation of the pantothenate/coenzyme A (CoA) pathway and a requirement for oxidative phosphorylation (OXPHOS) for differentiation. Exogenous administration of CoA reprogrammed T cells to increase OXPHOS and adopt the CD8+ Tc22 phenotype independent of polarizing conditions via the transcription factors HIF-1α and the aryl hydrocarbon receptor (AhR). In murine tumor models, treatment of mice with the CoA precursor pantothenate enhanced the efficacy of anti-PDL1 antibody therapy. In patients with melanoma, pre-treatment plasma pantothenic acid levels were positively correlated with the response to anti-PD1 therapy. Collectively, our data demonstrate that pantothenate and its metabolite CoA drive T cell polarization, bioenergetics, and anti-tumor immunity.

Hypoxia-inducible factor 1 alpha limits dendritic cell stimulation of CD8 T cell immunity.

Dendritic cells are sentinels of the immune system and represent a key cell in the activation of the adaptive immune response. Hypoxia-inducible factor 1 alpha (HIF-1α)-a crucial oxygen sensor stabilized during hypoxic conditions-has been shown to have both activating and inhibitory effects in immune cells in a context- and cell-dependent manner. Previous studies have demonstrated that in some immune cell types, HIF-1α serves a pro-inflammatory role. Genetic deletion of HIF-1α in macrophages has been reported to reduce their pro-inflammatory function. In contrast, loss of HIF-1α enhanced the pro-inflammatory activity of dendritic cells in a bacterial infection model. In this study, we aimed to further clarify the effects of HIF-1α in dendritic cells. Constitutive expression of HIF-1α resulted in diminished immunostimulatory capacity of dendritic cells in vivo, while conditional deletion of HIF-1α in dendritic cells enhanced their ability to induce a cytotoxic T cell response. HIF-1α-expressing dendritic cells demonstrated increased production of inhibitory mediators including IL-10, iNOS and VEGF, which correlated with their reduced capacity to drive effector CD8+ T cell function. Altogether, these data reveal that HIF-1α can promote the anti-inflammatory functions of dendritic cells and provides insight into dysfunctional immune responses in the context of HIF-1α activation.

Overproduction of IL-2 by Cbl-b deficient CD4+ T cells provides resistance against regulatory T cells.

Regulatory T cells are integral to the regulation of autoimmune and anti-tumor immune responses. However, several studies have suggested that changes in T cell signaling networks can result in T cells that are resistant to the suppressive effects of regulatory T cells. Here, we investigated the role of Cbl-b, an E3 ubiquitin ligase, in establishing resistance to Treg-mediated suppression. We found that the absence of Cbl-b, a negative regulator of multiple TCR signaling pathways, rendered T cells impartial to Treg suppression by regulating cytokine networks leading to improved anti-tumor immunity despite the presence of Treg cells in the tumor. Specifically, Cbl-b KO CD4+FoxP3- T cells hyper-produced IL-2 and together with IL-2 Rα upregulation served as an essential mechanism to escape suppression by Treg cells. Furthermore, we report that IL-2 serves as the central molecule required for cytokine-induced Treg resistance. Collectively our data emphasize the role of IL-2 as a key mechanism that renders CD4+ T cells resistant to the inhibitory effects of Treg cells.

Radiation and Heat Improve the Delivery and Efficacy of Nanotherapeutics by Modulating Intratumoral Fluid Dynamics.

Nanomedicine drug delivery systems are capable of transporting significant payloads to solid tumors. However, only a modest increase in antitumor efficacy relative to the standard of care has been observed. In this study, we demonstrate that a single dose of radiation or mild hyperthermia can substantially improve tumor uptake and distribution of nanotherapeutics, resulting in improved treatment efficacy. The delivery of nanomedicine was driven by a reduction in interstitial fluid pressure (IFP) and small perturbation of steady-state fluid flow. The transient effects on fluid dynamics in tumors with high IFP was also shown to dominate over immune cell endocytic capacity, another mechanism suspected of improving drug delivery. Furthermore, we demonstrate the specificity of this mechanism by showing that delivery of nanotherapeutics to low IFP tumors with high leukocyte infiltration does not benefit from pretreatment with radiation or heat. These results demonstrate that focusing on small perturbations to steady-state fluid dynamics, rather than large sustained effects or uncertain immune cell recruitment strategies, can impart a vulnerability to tumors with high IFP and enhance nanotherapeutic drug delivery and treatment efficacy.

Glycogen Synthase Kinase-3 Modulates Cbl-b and Constrains T Cell Activation.

The decision between T cell activation and tolerance is governed by the spatial and temporal integration of diverse molecular signals and events occurring downstream of TCR and costimulatory or coinhibitory receptor engagement. The PI3K-protein kinase B (PKB; also known as Akt) signaling pathway is a central axis in mediating proximal signaling events of TCR and CD28 engagement in T cells. Perturbation of the PI3K-PKB pathway, or the loss of negative regulators of T cell activation, such as the E3 ubiquitin ligase Cbl-b, have been reported to lead to increased susceptibility to autoimmunity. In this study, we further examined the molecular pathway linking PKB and Cbl-b in murine models. Our data show that the protein kinase GSK-3, one of the first targets identified for PKB, catalyzes two previously unreported phosphorylation events at Ser476 and Ser480 of Cbl-b. GSK-3 inactivation by PKB abrogates phosphorylation of Cbl-b at these two sites and results in reduced Cbl-b protein levels. We further show that constitutive activation of PKB in vivo results in a loss of tolerance that is mediated through the downregulation of Cbl-b. Altogether, these data indicate that the PI3K-PKB-GSK-3 pathway is a novel regulatory axis that is important for controlling the decision between T cell activation and tolerance via Cbl-b.

An interaction between Scribble and the NADPH oxidase complex controls M1 macrophage polarization and function.

The polarity protein Scribble (SCRIB) regulates apical-basal polarity, directional migration and tumour suppression in Drosophila and mammals. Here we report that SCRIB is an important regulator of myeloid cell functions including bacterial infection and inflammation. SCRIB interacts directly with the NADPH oxidase (NOX) complex in a PSD95/Dlg/ZO-1 (PDZ)-domain-dependent manner and is required for NOX-induced reactive oxygen species (ROS) generation in culture and in vivo. On bacterial infection, SCRIB localized to phagosomes in a leucine-rich repeat-dependent manner and promoted ROS production within phagosomes to kill bacteria. Unexpectedly, SCRIB loss promoted M1 macrophage polarization and inflammation. Thus, SCRIB uncouples ROS-dependent bacterial killing activity from M1 polarization and inflammatory functions of macrophages. Modulating the SCRIB-NOX pathway can therefore identify ways to manage infection and inflammation with implications for chronic inflammatory diseases, sepsis and cancer.

Natural killer cell activation enhances immune pathology and promotes chronic infection by limiting CD8+ T-cell immunity.

Infections with HIV, hepatitis B virus, and hepatitis C virus can turn into chronic infections, which currently affect more than 500 million patients worldwide. It is generally thought that virus-mediated T-cell exhaustion limits T-cell function, thus promoting chronic disease. Here we demonstrate that natural killer (NK) cells have a negative impact on the development of T-cell immunity by using the murine lymphocytic choriomeningitis virus. NK cell-deficient (Nfil3(-/-), E4BP4(-/-)) mice exhibited a higher virus-specific T-cell response. In addition, NK cell depletion caused enhanced T-cell immunity in WT mice, which led to rapid virus control and prevented chronic infection in lymphocytic choriomeningitis virus clone 13- and reduced viral load in DOCILE-infected animals. Further experiments showed that NKG2D triggered regulatory NK cell functions, which were mediated by perforin, and limited T-cell responses. Therefore, we identified an important role of regulatory NK cells in limiting T-cell immunity during virus infection.

In vivo microfocal computed tomography and micro-magnetic resonance imaging evaluation of antiresorptive and antiinflammatory drugs as preventive treatments of osteoarthritis in the rat.

OBJECTIVE: To determine whether treatment with an antiresorptive drug in combination with an antiinflammatory drug reduces periarticular bone and soft tissue adaptations associated with the progression of posttraumatic secondary osteoarthritis (OA). METHODS: We used in vivo microfocal computed tomography (micro-CT) to map bony adaptations and in vivo micro-magnetic resonance imaging (micro-MRI) to examine joint inflammation in a rat model of surgically induced OA secondary to knee triad injury. We examined the arthroprotective effects of the bisphosphonates alendronate and risedronate and the nonsteroidal antiinflammatory drug (NSAID) meloxicam. RESULTS: Micro-CT revealed reduced levels of periarticular trabecular bone loss in animals with knee triad injury treated with the bisphosphonate drugs alendronate or risedronate, or the NSAID meloxicam, compared with untreated animals. Alendronate treatment reduced bony osteophyte development. While risedronate as a monotherapy did not positively impact osteophytogenesis, combination therapy with risedronate and meloxicam reduced osteophyte severity somewhat. Micro-MRI revealed an increased, diffuse water signal in the epiphyses of untreated rats with knee triad injury 8 weeks after surgery, suggestive of a bone marrow lesion-like stimulus. In contrast, meloxicam-treated rats showed a significant reduction in fluid signal compared with both bisphosphonate-treated groups 8 weeks after surgery. Histologic analysis qualitatively confirmed the chondroprotective effect of both bisphosphonate treatments, showing fewer degradative changes compared with untreated rats with knee triad injury. CONCLUSION: Our findings indicate that select combinations of bisphosphonate and NSAID drug therapy in the early stages of secondary OA preserve trabecular bone mass and reduce the impact of osteophytic bony adaptations and bone marrow lesion-like stimulus. Bisphosphonate and NSAID therapy may be an effective disease-modifying drug regimen if administered early after the initial injury.

The fine-scale and complex architecture of human copy-number variation.

Despite considerable excitement over the potential functional significance of copy-number variants (CNVs), we still lack knowledge of the fine-scale architecture of the large majority of CNV regions in the human genome. In this study, we used a high-resolution array-based comparative genomic hybridization (aCGH) platform that targeted known CNV regions of the human genome at approximately 1 kb resolution to interrogate the genomic DNAs of 30 individuals from four HapMap populations. Our results revealed that 1020 of 1153 CNV loci (88%) were actually smaller in size than what is recorded in the Database of Genomic Variants based on previously published studies. A reduction in size of more than 50% was observed for 876 CNV regions (76%). We conclude that the total genomic content of currently known common human CNVs is likely smaller than previously thought. In addition, approximately 8% of the CNV regions observed in multiple individuals exhibited genomic architectural complexity in the form of smaller CNVs within larger ones and CNVs with interindividual variation in breakpoints. Future association studies that aim to capture the potential influences of CNVs on disease phenotypes will need to consider how to best ascertain this previously uncharacterized complexity.