γδ T lymphocytes represent ∼1% of human peripheral blood mononuclear cells and even more cells in most tissues of vertebrates. Although they have important anticancer functions, most current single-cell RNA sequencing (scRNA-seq) studies do not identify γδ T lymphocytes because their transcriptomes at the single-cell level are unknown. Here we show that high-resolution clustering of large scRNA-seq datasets and a combination of gene signatures allow the specific detection of human γδ T lymphocytes and identification of their T cell receptor (TCR)Vδ1 and TCRVδ2 subsets in large datasets from complex cell mixtures. In t-distributed stochastic neighbor embedding plots from blood and tumor samples, the few γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1 and TCRVδ2 subsets form close yet distinct subclusters, respectively neighboring NK and CD8 T cells because of expression of shared and distinct cytotoxic maturation genes. Similar pseudotime maturation trajectories of TCRVδ1 and TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets an unattended pool of terminally differentiated effector memory cells with preserved proliferative capacity, a finding confirmed by in vitro proliferation assays. Overall, the single-cell transcriptomes of thousands of individual γδ T lymphocytes from different CMV + and CMV − donors reflect cytotoxic maturation stages driven by the immunological history of donors. This landmark study establishes the rationale for identification, subtyping, and deep characterization of human γδ T lymphocytes in further scRNA-seq studies of complex tissues in physiological and disease conditions. γδ T lymphocyte | transcriptome | single-cell RNA-sequencing | human immunology | cancer S ingle-cell level mRNA-sequencing (scRNA-seq) of heterogeneous cell populations has become the reference tool for establishing cellular lineages and composition of tissues from the human body (1). In addition, the development of novel and open-source computational tools for processing scRNA-seq datasets enables delineation of both broad and subtle differences in rare cell subsets present in complex mixtures, such as peripheral blood mononuclear cells (PBMC) (2, 3). Such developments are expected to build more knowledge about the cellular composition and states composing tumors. Recent scRNA-seq analyses in melanoma and colorectal cancer evidenced the various tumor microenvironments and exhaustion patterns of the tumor-infiltrating lymphocytes (4). Most of such studies currently focus on the cytotoxic CD8 T lymphocytes, which represent the main target of immune checkpoint blockade therapies, and are readily detected by their scRNA-seq profile. Other subsets of cytolytic T cells are also critical in this therapeutic perspective; however, currently they have never been characterized by scRNA-seq and are therefore missing from all current tumor microenvironments mapped by these technologies.Human γδ T lymphocytes represent a peculiar lymphoid cell subset displaying hall...

The momentum of scRNA-seq datasets prompts for simple and powerful tools exploring their meaningful signatures. Here we present Single-Cell_Signature_Explorer (https://sites.google.com/site/fredsoftwares/products/single-cell-signature-explorer), the first method for qualitative and high-throughput scoring of any gene set-based signature at the single cell level and its visualization using t-SNE or UMAP. By scanning datasets for single or combined signatures, it rapidly maps any multi-gene feature, exemplified here with signatures of cell lineages, biological hallmarks and metabolic pathways in large scRNAseq datasets of human PBMC, melanoma, lung cancer and adult testis.

Most human blood γδ cells are cytolytic TCRVγ9Vδ2 lymphocytes with antitumor activity. They are currently investigated in several clinical trials of cancer immunotherapy but so far, their tumor infiltration has not been systematically explored across human cancers. Novel algorithms allowing the deconvolution of bulk tumor transcriptomes to find the relative proportions of infiltrating leucocytes, such as CIBERSORT, should be appropriate for this aim but in practice they fail to accurately recognize γδ T lymphocytes. Here, by implementing machine learning from microarray data, we first improved the computational identification of blood-derived TCRVγ9Vδ2 γδ lymphocytes and then applied this strategy to assess their abundance as tumor infiltrating lymphocytes (γδ TIL) in ∼10,000 cancer biopsies from 50 types of hematological and solid malignancies. We observed considerable inter-individual variation of TCRVγ9Vδ2γδ TIL abundance both within each type and across the spectrum of cancers tested. We report their prominence in B cell-acute lymphoblastic leukemia (B-ALL), acute promyelocytic leukemia (M3-AML) and chronic myeloid leukemia (CML) as well as in inflammatory breast, prostate, esophagus, pancreas and lung carcinoma. Across all cancers, the abundance of αβ TILs and TCRVγ9Vδ2 γδ TILs did not correlate. αβ TIL abundance paralleled the mutational load of tumors and positively correlated with inflammation, infiltration of monocytes, macrophages and dendritic cells (DC), antigen processing and presentation, and cytolytic activity, in line with an association with a favorable outcome. In contrast, the abundance of TCRVγ9Vδ2 γδ TILs did not correlate with these hallmarks and was variably associated with outcome, suggesting that distinct contexts underlie TCRVγ9Vδ2 γδ TIL and αβ TIL mobilizations in cancer.

We previously synthesized dendrogenin A and hypothesized that it could be a natural metabolite occurring in mammals. Here we explore this hypothesis and report the discovery of dendrogenin A in mammalian tissues and normal cells as an enzymatic product of the conjugation of 5,6α-epoxy-cholesterol and histamine. Dendrogenin A was not detected in cancer cell lines and was fivefold lower in human breast tumours compared with normal tissues, suggesting a deregulation of dendrogenin A metabolism during carcinogenesis. We established that dendrogenin A is a selective inhibitor of cholesterol epoxide hydrolase and it triggered tumour re-differentiation and growth control in mice and improved animal survival. The properties of dendrogenin A and its decreased level in tumours suggest a physiological function in maintaining cell integrity and differentiation. The discovery of dendrogenin A reveals a new metabolic pathway at the crossroads of cholesterol and histamine metabolism and the existence of steroidal alkaloids in mammals.

Small phosphorylated metabolites from mycobacteria stimulate human ␥␦ T lymphocytes. Although such phosphoantigens could prove useful in the composition of vaccines involving ␥␦ T cell-mediated immunity, their very low abundance in natural sources limits such applications. Here, we describe the chemical production, purification, and bioactivity of a phosphorylated bromohydrin (BrHPP) analogue that mimics the biological properties of natural phosphoantigens. This compound can be obtained in gram amounts, is easy to detect, and is of high stability in aqueous solutions. Whereas unspecific binding of BrHPP to a wide panel of cell surface receptors is not detected even at micromolar concentrations, nanomolar concentrations specifically trigger effector responses of human ␥␦ T lymphocytes. Thus, BrHPP is a novel molecule enabling potent immunostimulation of human ␥␦ T lymphocytes.Stimulating ligands for ␣␤ T lymphocytes are usually composed of single peptides complexed at the surface of major histocompatibility complex molecules. Some small non-peptidic structures, however, may also constitute specific agonist ligands for T cells, particularly ␥␦ T lymphocytes. In human blood, about 3% of T cells initiate their physiological function upon recognition of small phosphorylated non-peptide antigens (phosphoantigens). This cognate interaction involves on the one hand phosphoantigens in the absence of major histocompatibility complex-presenting molecules, and on the other hand, highly selective receptors (TCR) 1 of ␥␦ subtype. In nature, phosphoantigens that can activate human ␥␦ T cells at nanomolar concentrations are produced by Gram-positive and Gram-negative bacteria and also by some eukaryotic parasites and plants. Synthetic analogues of natural phosphoantigens are also known, but their stimulating concentrations for the reactive cells never go below the micromolar range. Mycobacterium tuberculosis, the agent of human tuberculosis, produces four distinct phosphoantigens. These molecules share a moiety that is responsible for the potent stimulation of ␥␦ cells seen in tuberculosis patients (1). The structure of this common core is 3-formyl-1-butyl-pyrophosphate, a recently described phosphoester (2). Its metabolic production might be related to the non-mevalonate (or so-called Rohmer's) pathway for isoprenoid precursor biosynthesis (3). 3-formyl-1-butyl-pyrophosphate is produced in very small amounts in slow-growing mycobacteria such as Mycobacterium tuberculosis and only accumulates to submicromolar concentrations in culture media from fast-growing mycobacterial species (4). Getting large amounts of highly bioactive phosphoantigens by purification routes from such natural sources is therefore hard to conceive.Such molecules could prove therapeutically useful for immunotherapeutic approaches involving ␥␦ T cell-mediated immunity, such as elicitation of anti-infectious protection or antitumor immunity (5, 6). To address the need for readily available highly bioactive phosphoantigens, we have developed a synthetic reagen...

Abstract-In this paper we consider the problem of simultaneously localizing all members of a team of robots. Each robot is equipped with proprioceptive sensors and exteroceptive sensors. The latter provide relative observations between the robots. Proprioceptive and exteroceptive data are fused with an Extended Kalman Filter. We derive the equations for this estimator for the most general relative observation between two robots. Then we consider three special cases of relative observations and we present the structure of the filter for each case. Finally, we study the performance of the approach through many accurate simulations.

This phase 1 trial was aimed to determine the safety, pharmacokinetics, and preliminary clinical activity of CYL-02, a nonviral gene therapy product that sensitizes pancreatic cancer cells to chemotherapy. CYL-02 was administrated using endoscopic ultrasound in 22 patients with pancreatic cancer that concomitantly received chemotherapy (gemcitabine). The maximum-tolerated dose (MTD) exceeded the maximal feasible dose of CYL-02 and was not identified. Treatment-related toxicities were mild, without serious adverse events. Pharmacokinetic analysis revealed a dose-dependent increase in CYL-02 DNA exposure in blood and tumors, while therapeutic RNAs were detected in tumors. No objective response was observed, but nine patients showed stable disease up to 6 months following treatment and two of these patients experienced long-term survival. Panels of plasmatic microRNAs and proteins were identified as predictive of gene therapy efficacy. We demonstrate that CYL-02 nonviral gene therapy has a favorable safety profile and is well tolerated in patients. We characterize CYL-02 biodistribution and demonstrate therapeutic gene expression in tumors. Treated patients experienced stability of disease and predictive biomarkers of response to treatment were identified. These promising results warrant further evaluation in phase 2 clinical trial.

Tumor antigen–specific CD4 T cells accumulate at tumor sites, evoking their involvement in antitumor effector functions in situ. Contrary to CD8 cytotoxic T lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic, and functional approaches, we characterized CD4 T cell exhaustion in patients with head and neck, cervical, and ovarian cancer. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low, evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression, suggesting divergence with CD8 T cell exhaustion. scRNA-Seq and further phenotypic analyses uncovered similarities with the CD8 T cell exhaustion program. In particular, PD-1 hi CD39 + CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently higher tumor-specific CD8 T cell proliferation. Our data identify exhausted CD4 TILs as players in responsiveness to immune checkpoint blockade.